TITLE
A NOVEL BINDING SITE FOR RETIGABINE ON KCNQ5
FIELD OF THE INVENTION
Disclosed herein is a novel binding site for retigabine on KCNQ.5, and the
gene, nucleic acid, protein, vectors, and methods of use thereof.
BACKGROUND OF THE INVENTION
Ion channels are cellular proteins that regulate the flow of ions, including
calcium, potassium, sodium and chloride, into and out of cells. These channels
affect such processes as nerve transmission, muscle contraction and cellular
secretion. Among the ion channels, potassium channels are the most ubiquitous
and diverse, being found in a variety of animal cells such as nerve, muscular,
glandular, immune, reproductive, and epithelial tissue. These channels allow the
flow of potassium in and/or out of the cell under certain conditions. For example,
the outward flow of potassium ions upon opening of these channels makes the
interior of the cell more negative, counteracting depolarizing voltages applied to
the cell. These channels are regulated, e.g., by calcium sensitivity, voltage-
gating, second messengers, extracellular ligands, and ATP-sensitivity.
Potassium channels are membrane-spanning proteins that generally act to
hyperpolarize neurons and muscle cells. Physiological studies indicate that
potassium currents are found in most cells and are associated with a wide range
of functions, including the regulation of the electrical properties of excitable cells.
Depending on the type of potassium channel, its functional activity can be
controlled by transmembrane voltage, different ligands, protein phosphorylation,
or other second messengers (see, e.g., U.S. Patent No. 6,893,858).
The potassium channel family possesses approximately seventy members
in mammalian tissues. The recently identified KCNQ subfamily (Kv7) has been
shown to play an important functional role as determinants of cell excitability.
Recent evidence indicates that the KCNQ potassium channel sub-units form the
molecular basis for M-current activity in several tissue types. This gene family
has evolved to contain at least five major sub-units designated KCNQ1 through
25(Kv7.1-7.5). These sub-units have been shown to co-assemble to form
meric and homomeric functional ion channels.

Voltage dependant potassium channels are key regulators of the resting
membrane potential and modulate the excitability of electrically active cells, such
as neurons or myocytes. Several classes of voltage dependant potassium (K+)
channels have been cloned (see, e.g., Lerche C et al., J. Biol. Chem. 275:22395-
22400 (2000)).
Mutations in four of the five KCNQ potassium channel genes are
implicated in diverse diseases, causing cardiac LQT syndrome (KCNQ1),
epilepsy (KCNQ2, and 3), congenital deafness (KCNQ4). Because of the
importance of KCNQ5 in M-current formation demonstrated in the central
nervous system (CNS), it is presumed that the failure of this gene would result in
disorder of neuronal excitability (see, e.g., Lerche C et al., J. Biol. Chem.
275:22395-22400 (2000); Schroeder BC et al., J. Biol. Chem. 275:24089-95
(2000)).
Potassium channels are involved in a number of physiological processes,
including regulation of heartbeat, dilation of arteries, release of insulin, excitability
of nerve cells, and regulation of renal electrolyte transport.
Retigabine (N-(2-amino-4-(4-fluorobenzylamino)-phenyl)carbamic acid
ethyl ester) has been found to open certain types of KCNQ channels, including
KCNQ5. Retigabine, however, has no enhancing effect on KCNQ1, which is
homologous to KCNQ5 by 37% sequence identity. Retigabine exerts its cellular
effects by increasing the open probability of these channels (Main J, Mol.
Pharmacol. 58:253-62 (2000); Wickenden A et al., Mol. Pharmacol. 58:591-600
(2000)). This increase in the opening of individual KCNQ channels collectively
results in the hyperpolarization of cell membranes, particularly in depolarized
cells, produced by significant increases in whole-cell KCNQ-mediated
conductance.
Disclosed herein are mutants of KCNQ5 which have lost the functional
property to respond to retigabine.
SUMMARY OF THE INVENTION
One aspect is for an isolated polynucleotide encoding all or a portion of a
KCNQ5(W270L) polypeptide.
Another aspect is for an isolated polynucleotide comprising a
polynucleotide selected from the group consisting of:

(a) a nucleic acid sequence comprising SEQ ID NO:1;
(b) a polynucleotide encoding SEQ ID NO:2;
(c) a nucleic acid sequence encoding a polypeptide having at least
about 95% homology with SEQ ID NO:1, provided that a
substitution at nucleotides 808-810 is for a codon that produces a
conservative substitution for the amino acid leucine;
(d) a nucleic acid molecule which is capable of hybridizing under highly
stringent conditions to SEQ ID NO:1;
(e) a nucleic acid molecule which is complementary to (a), (b), (c), or
(d); and
(f) a variant of SEQ ID NO: 1.
Another embodiment is an isolated polynucleotide encoding a KCNQ5
polypeptide containing an S5-S6 transmembrane domain from KCNQ1.
A further aspect is for an isolated polynucleotide comprising a
polynucleotide selected from the group consisting of:
(a) a nucleic acid sequence comprising SEQ ID NO:3, wherein
nucleotides 769-1062 are substituted with SEQ ID NO:5;
(b) a polynucleotide encoding SEQ ID NO:4. wherein amino acids 257-
354 are substituted with an S5-S6 transmembrane domain from
KCNQ1;
(c) a nucleic acid molecule which is capable of hybridizing under highly
stringent conditions to the nucleic acid sequence of (a) or (b); and
(d) a nucleic acid molecule which is complementary to (a), (b), or (c).
Another aspect is for an isolated polynucleotide comprising a
polynucleotide selected from the group consisting of:
(a) a nucleic acid sequence comprising SEQ ID NO:3, wherein
nucleotides 769-873 are substituted with nucleotides 1-105 of SEQ
ID NO:5;
(b) a polynucleotide encoding SEQ ID NO:4, wherein amino acids 257-
291 of SEQ ID NO:4 are substituted with an S5 transmembrane
domain from KCNQ1;
(c) a nucleic acid molecule which is capable of hybridizing under highly
stringent conditions to the nucleic acid sequence of (a) or (b); and
(d) a nucleic acid molecule which is complementary to (a), (b), or (c).

Another embodiment is an isolated polypeptide comprising an amino acid
sequence selected from the group consisting of:
(a) an amino acid sequence of a KCNQ5(W270L) polypeptide;
(b) an amino acid sequence comprising SEQ ID NO:2;
(c) a variant of (a); and
(d) an amino acid sequence having at least 90% identity to the amino
acid sequence of SEQ ID NO:2, provided that a substitution at
amino acid 270 is a conservative substitution for the amino acid
leucine.
A further aspect is for a KCNQ dimeric channel comprising at least one
KCNQ5 subunit which is the aforementioned isolated polypeptide. Another
aspect is for a KCNQ tetrameric channel comprising at least one KCNQ5 subunit
which is the aforementioned isolated polypeptide.
A further embodiment is an antibody which specifically binds a
KCNQ5(W270L) polypeptide comprising SEQ ID NO:2.
Another aspect is for antibody which specifically binds a KCNQ5(W270L)
polypeptide fragment comprising at least 8 contiguous amino acids from SEQ ID
NO:2, wherein said fragment includes amino acid 270 of SEQ ID NO:2.
A further aspect is for an isolated KCNQ5 polypeptide containing an S5-
S6 transmembrane domain from KCNQ1.
A further aspect is for an isolated KCNQ5 polypeptide containing an S5
transmembrane domain from KCNQ1. A further aspect is for a KCNQ dimeric
channel comprising at least one KCNQ5 subunit which is the aforementioned
isolated KCNQ5 polypeptide containing an S5-S6 transmembrane domain or an
S5 transmembrane domain from KCNQ1. Another aspect is for a KCNQ
tetrameric channel comprising at least one KCNQ5 subunit which is the
aforementioned isolated KCNQ5 polypeptide containing an S5-S6
transmembrane domain or an S5 transmembrane domain from KCNQ1.
Another aspect is for a method of screening for agents, the method
comprising:
(a) contacting an agent with a KCNQ5 molecule selected from the
group consisting of:
(i) a polynucleotide encoding all or a portion of a
KCNQ5(W270L) polypeptide;

(ii) a polynucleotide encoding a KCNQ5 polypeptide containing
an S5-S6 transmembrane domain from KCNQ1;
(iii) a polynucleotide encoding a KCNQ5 polypeptide containing
an S5 transmembrane domain from KCNQ1;
(iv) a polypeptide comprising an amino acid sequence of a
KCNQ5(W270L) polypeptide;
(v) a KCNQ5 polypeptide containing an S5-S6 transmembrane
domain from KCNQ1; and
(vi) a KCNQ5 polypeptide containing an S5 transmembrane
domain from KCNQ1; and
(b) . detecting an effect of said agent on the KCNQ5 activity;
wherein detection of a decrease or an increase in KCNQ5 activity is indicative of
an agent being a modulator of KCNQ5.
A further embodiment is a method of screening for agents, the method
comprising:
(a) contacting a cell with an agent; and
(b) determining the level of expression of a KCNQ5 molecule selected
from the group consisting of:
(i) a polynucleotide encoding all or a portion of a
KCNQ5(W270L) polypeptide;
(ii) a polynucleotide encoding a KCNQ5 polypeptide containing
an S5-S6 transmembrane domain from KCNQ1;
(iii) a polynucleotide encoding a KCNQ5 polypeptide containing
an S5 transmembrane domain from KCNQ1;
(iv) a polypeptide comprising an amino acid sequence of a
KCNQ5(W270L) polypeptide;
(v) a KCNQ5 polypeptide containing an S5-S6 transmembrane
domain from KCNQ1; and
(vi) a KCNQ5 polypeptide containing an S5 transmembrane
domain from KCNQ1;
wherein detection of a decrease or an increase in KCNQ5 expression is
indicative of an agent being a modulator of KCNQ5.
Another aspect is for methods of inducing or maintaining bladder control,
treatment or prevention of urinary incontinence, or treatment or prevention of

neuropathic pain in a mammal, the method comprising administering to a
mammal in need thereof of a pharmacologically effective amount of the agent
identified by any of the aforementioned methods.
Another aspect is for a method for identifying polypeptides capable of
binding to a KCNQ5 polypeptide comprising:
(a) applying a mammalian two-hybrid procedure in which a sequence
encoding a KCNQ5 polypeptide is carried by one hybrid vector and
sequence from a cDNA or genomic DNA library is carried by the
second hybrid vector, wherein the KCNQ5 polypeptide is selected
from the group consisting of:
(i) a polypeptide comprising an amino acid sequence of a
KCNQ5(W270L) polypeptide;
(ii) a KCNQ5 polypeptide containing an S5-S6 transmembrane
domain from KCNQ1; and
(iii) a KCNQ5 polypeptide containing an S5 transmembrane
domain from KCNQ1;
(b) transforming the host cell with the vectors;
(c) isolating positive transformed cells; and
(d) extracting said second hybrid vector to obtain a sequence encoding
a polypeptide which binds to the KCNQ5 polypeptide.
A further aspect is for method for detecting a KCNQ5 polypeptide
comprising detecting binding of an antibody selected from the group consisting of
(a) an antibody which selectively binds a KCNQ5 polypeptide
comprising an amino acid sequence of a KCNQ5(W270L)
polypeptide;
(b) an antibody which selectively binds a KCNQ5 polypeptide
containing an S5-S6 transmembrane domain from KCNQ1;
(c) an antibody which selectively binds a KCNQ5 polypeptide
containing an S5 transmembrane domain from KCNQ1; and
(d) an antibody which selectively binds a KCNQ5(W270L) polypeptide
fragment comprising at least 8 contiguous amino acids from SEQ
ID NO:2, wherein said fragment includes amino acid 270 from SEQ
ID NO:2;

to a molecule in a sample suspected of containing a KCNQ5 polypeptide, a
KCNQ5(W270L) polypeptide, or a KCNQ5(W270L) polypeptide fragment,
wherein the antibody is contacted with the sample under conditions that permit
specific binding with any KCNQ5 polypeptide, KCNQ5(W270L) polypeptide, or
KCNQ5(W270L) polypeptide fragment present in the sample and binding of the
antibody to the molecule in the sample indicates the presence of a KCNQ5
polypeptide, KCNQ5(W270L) polypeptide, or KCNQ5(W270L) polypeptide
fragment.
A further embodiment is a method for detecting expression of KCNQ5
comprising detecting mRNA encoding a KCNQ5 polypeptide selected from the
group consisting of:
(i) a polypeptide comprising an amino acid sequence of a
KCNQ5(W270L) polypeptide;
(ii) a KCNQ5 polypeptide containing an S5-S6 transmembrane domain
from KCNQ1; and
(iii) a KCNQ5 polypeptide containing an S5 transmembrane domain
from KCNQ1;
in a sample from a cell or tissue suspected of expressing KCNQ5 with a probe
comprising at least 12 contiguous nucleotides from a polynucleotide selected
from the group consisting of:
(i) a polynucleotide encoding all or a portion of a KCNQ5(W270L)
polypeptide;
(ii) a polynucleotide encoding a KCNQ5 polypeptide containing an S5-
S6 transmembrane domain from KCNQ1; and
(iii) a polynucleotide encoding a KCNQ5 polypeptide containing an S5
transmembrane domain from KCNQ1.
Another embodiment is a method for determining whether a KCNQ5 gene
has been mutated or deleted comprising detecting, in a sample of cells or tissue
from a subject, the presence or absence of a genetic alteration characterized by
at least one of an alteration affecting the integrity of a gene encoding a KCNQ5
protein or the misexpression of a KCNQ5 gene, wherein the detecting step is
performed with at least one of a probe or primer comprising at least 12
contiguous nucleotides from a polynucleotide selected from the group consisting
of:

(i) a polynucleotide encoding all or a portion of a KCNQ5(W270L)
polypeptide;
(ii) a polynucleotide encoding a KCNQ5 polypeptide containing an S5-
S6 transmembrane domain from KCNQ1; and
(iii) a polynucleotide encoding a KCNQ5 polypeptide containing an S5
transmembrane domain from KCNQ1.
A further aspect is for method of identifying variants of a KCNQ5
polypeptide comprising screening a combinatorial library comprising KCNQ5
mutants for KCNQ5 polypeptide agonists or antagonists; wherein the KCNQ5
polypeptide is selected from the group consisting of:
(i) a polypeptide comprising an amino acid sequence of a
KCNQ5(W270L) polypeptide;
(ii) a KCNQ5 polypeptide containing an S5-S6 transmembrane domain
from KCNQ1; and
(iii) a KCNQ5 polypeptide containing an S5 transmembrane domain
from KCNQ1.
A further aspect is for a method of isolating a KCNQ5 polypeptide
comprising:
(a) contacting a KCNQ5 antibody with a sample suspected of
containing a KCNQ5 polypeptide selected from the group
consisting of:
(iv) a polypeptide comprising an amino acid sequence of a
KCNQ5(W270L) polypeptide;
(v) a KCNQ5 polypeptide containing an S5-S6 transmembrane
domain from KCNQ1; and
(vi) a KCNQ5 polypeptide containing an S5 transmembrane
domain from KCNQ1; and
(b) isolating a KCNQ5 antibody-KCNQ5 polypeptide complex from the
sample.
A further embodiment is a method of producing a KCNQ5 polypeptide
comprising:
(a) culturing a transformed host cell comprising an expression vector;
wherein said expression vector comprises a polynucleotide
selected from the group consisting of:

(i) a polynucleotide encoding all or a portion of a
KCNQ5(W270L) polypeptide;
(ii) a polynucleotide encoding a KCNQ5 polypeptide containing
an S5-S6 transmembrane domain from KCNQ1; and
(iii) a polynucleotide encoding a KCNQ5 polypeptide containing
an S5 transmembrane domain from KCNQ1;
in a suitable medium such that a KCNQ5 polypeptide is produced;
and
(b) optionally, recovering the KCNQ5 polypeptide of step (a).
Another embodiment is a method for the treatment of a mammal in need
of increased KCNQ5 activity comprising administering to the mammal in need
thereof a therapeutically effective amount of a KCNQ5 molecule selected from
the group consisting of:
(i) a polynucleotide encoding all or a portion of a KCNQ5(W270L)
polypeptide;
(ii) a polynucleotide encoding a KCNQ5 polypeptide containing an S5-
S6 transmembrane domain from KCNQ1;
(iii) a polynucleotide encoding a KCNQ5 polypeptide containing an S5
transmembrane domain from KCNQ1;
(iv) a polypeptide comprising an amino acid sequence of a
KCNQ5(W270L) polypeptide;
(v) a KCNQ5 polypeptide containing an S5-S6 transmembrane domain
from KCNQ1; and
(vi) a KCNQ5 polypeptide containing an S5 transmembrane domain
from KCNQ1
A further aspect is for a method for the treatment of a mammal in need of
decreased KCNQ5 activity comprising administering to the mammal in need
thereof a therapeutically effective amount of:
(a) a KCNQ5 antisense polynucleotide which is antisense to a
polynucleotide selected from the group consisting of:
(i) a polynucleotide encoding all or a portion of a
KCNQ5(W270L) polypeptide;
(ii) a polynucleotide encoding a KCNQ5 polypeptide containing
an S5-S6 transmembrane domain from KCNQ1; and

(iii) a polynucleotide encoding a KCNQ5 polypeptide containing
an S5 transmembrane domain from KCNQ1; or
(b) a KCNQ5 antibody selected from the group consisting of:
(A) an antibody which selectively binds a KCNQ5 polypeptide
comprising an amino acid sequence of a KCNQ5(W270L)
polypeptide;
(B) an antibody which selectively binds a KCNQ5 polypeptide
containing an S5-S6 transmembrane domain from KCNQ1;
(C) an antibody which selectively binds a KCNQ5 polypeptide
containing an S5 transmembrane domain from KCNQ1; and
(D) an antibody which selectively binds a KCNQ5(W270L)
polypeptide fragment comprising at least 8 contiguous amino
acids from SEQ ID NO:2, wherein said fragment includes
amino acid 270 from SEQ ID NO:2.
Another aspect is for a method for obtaining anti-KCNQ5 polypeptide
antibodies comprising:
(a) immunizing an animal with an immunogenic KCNQ5 polypeptide or
an immunogenic portion thereof unique to a KCNQ5 polypeptide,
wherein said KCNQ5 polypeptide is selected from the group
consisting of:
(i) a polypeptide comprising an amino acid sequence of a
KCNQ5(W270L) polypeptide;
(ii) a KCNQ5 polypeptide containing an S5-S6 transmembrane
domain from KCNQ1; and
(iii) a KCNQ5 polypeptide containing an S5 transmembrane
domain from KCNQ1; and
(b) isolating from the animal antibodies that specifically bind to a
KCNQ5 polypeptide.
A further aspect is for a method for assaying the ability of a KCNQ5
polypeptide to encode a functional ion channel comprising:
(a) transfecting a host cell with a polynucleotide encoding a KCNQ5
polypeptide selected from the group consisting of:
(i) a polypeptide comprising an amino acid sequence of a
KCNQ5(W270L) polypeptide;

(ii) a KCNQ5 polypeptide containing an S5-S6 transmembrane
. domain from KCNQ1; and
(iii) a KCNQ5 polypeptide containing an S5 transmembrane
domain from KCNQ1;
(b) expressing the KCNQ5 polypeptide in the host cell; and
(c) electrophysiologically measuring the ion current magnitude of the
KCNQ5 polypeptide.
Another embodiment is a method for preventing in a subject a disease or
condition that would benefit from modulation of KCNQ5 activity and/or expression
comprising administering to the subject a KCNQ5 polypeptide or agent which
modulates KCNQ5 expression or at least one KCNQ5 activity, wherein the
KCNQ5 polypeptide is selected from the group consisting of:
(i) a polypeptide comprising an amino acid sequence of a
KCNQ5(W270L) polypeptide;
(ii) a KCNQ5 polypeptide containing an S5-S6 transmembrane domain
from KCNQ1; and
(iii) a KCNQ5 polypeptide containing an S5 transmembrane domain
from KCNQ1
A further embodiment is a kit for detecting KCNQ5 polypeptide or
polynucleotide comprising:
(a) a labeled compound or agent capable of detecting a KCNQ5
polypeptide or polynucleotide in a biological sample;
(b) means for determining the amount of KCNQ5 polypeptide or
polynucleotide in the sample;
(c) means for comparing the amount of KCNQ5 polypeptide or
polynucleotide In the sample with a standard; and
(d) optionally, instructions for using the kit to detect KCNQ5
polypeptide or polynucleotide;
wherein the KCNQ5 polypeptide or polynucleotide is selected from the group
consisting of:
(i) a polynucleotide encoding all or a portion of a KCNQ5(W270L)
polypeptide;
(ii) a polynucleotide encoding a KCNQ5 polypeptide containing an S5-
S6 transmembrane domain from KCNQ1;

(iii) a polynucleotide encoding a KCNQ5 polypeptide containing an S5
transmembrane domain from KCNQ1;
(iv) a polypeptide comprising an amino acid sequence of a
KCNQ5(W270L) polypeptide;
(v) a KCNQ5 polypeptide containing an S5-S6 transmembrane domain
from KCNQ1; and
(vi) a KCNQ5 polypeptide containing an S5 transmembrane domain
from KCNQ1.
Another embodiment is a kit for identifying modulators of KCNQ5 activity
comprising:
(a) a cell or composition comprising a KCNQ5 polypeptide;
(b) means for determining KCNQ5 polypeptide activity; and
(c) optionally, instructions for using the kit to identify modulators of
KCNQ5 activity;
wherein the KCNQ5 polypeptide is selected from the group consisting of:
(i) a polypeptide comprising an amino acid sequence of a
KCNQ5(W270L) polypeptide;
(ii) a KCNQ5 polypeptide containing an S5-S6 transmembrane domain
from KCNQ1; and
(iii) a KCNQ5 polypeptide containing an S5 transmembrane domain
from KCNQ1.
A further embodiment is a kit for diagnosing a disorder associated with
aberrant KCNQ5 expression and/or activity in a subject comprising:
(a) a reagent for determining expression of KCNQ5 polypeptide or
polynucleotide;
(b) a control to which the results of the subject are compared; and
(c) optionally, instructions for using the kit for diagnostic purposes;
wherein the KCNQ5 polypeptide or polynucleotide is selected from the group
consisting of:
(i) a polynucleotide encoding all or a portion of a KCNQ5(W270L)
polypeptide;
(ii) a polynucleotide encoding a KCNQ5 polypeptide containing an S5-
S6 transmembrane domain from KCNQ1;

(iii) a polynucleotide encoding a KCNQ5 polypeptide containing an S5
transmembrane domain from KCNQ1;
(iv) a polypeptide comprising an amino acid sequence of a
KCNQ5(W270L) polypeptide;
(v) a KCNQ5 polypeptide containing an S5-S6 transmembrane domain
from KCNQ1; and
(vi) a KCNQ5 polypeptide containing an S5 transmembrane domain
from KCNQ1
Other objects and advantages will become apparent to those skilled in the
art upon reference to the detailed description that hereinafter follows.
BRIEF DESCRIPTION OF THE SEQUENCES
SEQ ID NO:1 represents KCNQ(W270L) cDNA.
SEQ ID NO:2 represents KCNQ(W270L) protein.
SEQ ID NO:3 represents wild type human KCNQ5 cDNA.
SEQ ID NO:4 represents wild type human KCNQ5 protein.
SEQ ID NO:5 represents an S5-S6 transmembrane domain from human KCNQ1
cDNA.
SEQ ID NO:6 represents an S5-S6 transmembrane domain from human KCNQ1
(translated amino acid sequence).
SEQ ID NO:7 represents wild type human KCNQ1 DNA.
SEQ ID NO:8 represents wild type human KCNQ1 protein.
BRIEF DESCRIPTION OF THE FIGURES
FIGURE 1 (A), current traces of Q5Q1P evoked by a series of depolarized
voltage pulses from —100 to 60 mV in 10 mV increments (holding potential was -
100 mV) in the absence (left panel) and the presence (right panel) of 50 uivl
retigabine. (B), the time course of retigabine effect on Q5Q1P mutants. (C),
current (/)-voltage relationship of Q5Q1P mutants in the absence (n=9) and the
presence (n=7, 5 min after the application) of 50 µM retigabine. No significant
difference is found in any one of the data points (p>0.05).
FIGURE 2 (A), current traces of Q5Q1S5 evoked by a series of depolarized
voltage pulses from —100 to 60 mV in 10 mV increments (holding potential was —

100 mV) in the absence (left panel) and the presence (right panel) of 50 uM
retigabine. (B), the time course of retigabine effect on Q5Q1P mutants. (C),
current (/)-vo!tage relationship of Q5Q1S5 mutants in the absence (n=7) and the
presence (n=7) of 50 µM retigabine. No significant difference is found in any one
of the data points (p>0.05) between these two groups.
FIGURE 3 Mutagenesis analysis of S5 domain in KCNQ5. Sequence alignment
of all five members in KCNQ family is shown on the top. The most diverse
residues between KCNQ1 and the other members are highlighted in bold case
and the corresponding residues in KCNQ5 were individually mutated to their
counterparts in KCNQ1. Then each mutant with single mutation was tested with
50 uM retigabine and the fold increase (IΔ/Io) in current amplitude was plotted in
the graph on bottom. In all mutants, n equals 6.
FIGURE 4 (A), current traces recorded from KCNQ1 wild-type in the absence
(Control) and the presence of 50 µM and 200 uM retigabine. (B), current traces
recorded from KCNQ1 L171W in the absence (Control) and the presence of 50
uM and 200 µM retigabine. (C), superimposed current traces evoked by voltage
pulse to 80 mV from KCNQ1 L171W in the absence and the presence of 50 and
200 uM retigabine. (D), current-voltage relationships of KCNQ1 L171W in the
absence (open circle) and the presence of 50 µM (open square) and 200 µM
(open triangle) retigabine. The steady state amplitudes of the currents evoked by
a series of depolarized voltage pulses from —100 to 80 mV in 10 mV increment
were normalized to the level evoked by the membrane potential of 80 mV in the
control group. The holding potential was -100 mV. Data were collected from 10
oocytes in each group. The inset graph shows the channel conductance (G)
normalized to the level at 80 mV (Gmax) in the absence (open circle) and
presence of 200 uM retigabine (open triangle), to compare the effect of retigabine
on voltage dependence of channel activation.
DETAILED DESCRIPTION OF THE INVENTION
Applicants specifically incorporate the entire contents of all cited
references in this disclosure. Further, when an amount, concentration, or other

value or parameter is given as either a range, preferred range, or a list of upper
preferable values and lower preferable values, this is to be understood as
specifically disclosing all ranges formed from any pair of any upper range limit or
preferred value and any lower range limit or preferred value, regardless of
whether ranges are separately disclosed. Where a range of numerical values is
recited herein, unless otherwise stated, the range is intended to include the
endpoints thereof, and all integers and fractions within the range. It is not
intended that the scope of the invention be limited to the specific values recited
when defining a range.
1. Definitions
In the context of this disclosure, a number of terms shall be utilized.
As used herein, the term "about" or "approximately" means within 20%,
preferably within 10%, and more preferably within 5% of a given value or range.
, "Altered levels" refers to the production of gene product(s) in organisms in
amounts or proportions that differ from that of normal or non-transformed
organisms. Overexpression of the polypeptide may be accomplished by first
constructing a chimeric gene or chimeric construct in which the coding region is
operably linked to a promoter capable of directing expression of a gene or
construct in the desired tissues at the desired stage of development. For
reasons of convenience, the chimeric gene or chimeric construct may comprise
promoter sequences and translation leader sequences derived from the same
genes. 3' noncoding sequences encoding transcription termination signals may
also be provided. The instant chimeric gene or chimeric construct can then be
constructed. The choice of plasmid vector is dependent upon the method that
will be used to transform host cells. The skilled artisan is well aware of the
genetic elements that must be present on the plasmid vector in order to
successfully transform, select, and propagate host cells containing the chimeric
gene or chimeric construct. The skilled artisan will also recognize that different
independent transformation events will result in different levels and patterns of
expression (see, e.g., De Almedia ERP et al, Mol. Genet. Genomics 218:78-86
(1989)), and thus that multiple events must be screened in order to obtain lines
displaying the desired expression level and pattern. Such screening may be
accomplished by Southern analysis of DNA, Northern analysis of mRNA

expression, Western or immunocytochemical analysis of protein expression, or
phenotypic analysis.
An "antibody" includes an immunoglobulin molecule capable of binding an
epitope present on an antigen. As used herein, the term encompasses not only
intact immunoglobulin molecules such as monoclonal and polyclonal antibodies,
but also anti-idotypic antibodies, mutants, fragments, fusion proteins, bi-specific
antibodies, humanized proteins, and modifications of the immunoglobulin
molecule that comprises an antigen recognition site of the required specificity.
The term "cDNAs" includes complementary DNA, that is mRNA molecules
present in a cell or organism made into cDNA with an enzyme such as reverse
transcriptase. A "cDNA library" includes a collection of mRNA molecules present
in a cell or organism, converted into cDNA molecules with the enzyme reverse
transcriptase, then inserted into vectors. The library can then be probed for the
specific cDNA (and thus mRNA) of interest.
The term "comprising" is intended to include embodiments encompassed
by the terms "consisting essentially of and "consisting of. Similarly, the term
"consisting essentially of is intended to include embodiments encompassed by
the term "consisting of.
A "KCNQ5 polypeptide", "KCNQ5 amino acid sequence", or "KCNQ5
protein" as used herein refers to non-wild type KCNQ5 polypeptides having at
least one amino acid modification which makes the KCNQ5 polypeptide
substantially insensitive to the K+ channel activator retigabine while retaining
KCNQ5 M-current potassium channel activity. "Wild type KCNQ5" (for example,
SEQ ID NO:4, which represents human wild type KCNQ5), on the other hand, is
responsive to retigabine (see Wickendon AD et al., Brit. J. Pharmacol. 132:381-
84 (2001)). "KCNQ5(W270L) polypeptide", "KCNQ5(W270L) amino acid
sequence", or "KCNQ5(W270L) protein" refers to a human KGNQ5 polypeptide
having a point mutation at amino acid 270 from a tryptophan residue (at amino
acid position 270 in the full-length, human wild type protein) to a leucine residue,
which imparts retigabine insensitivity to the KCNQ5(W270L) polypeptide. SEQ
ID NO:2 represents a KCNQ5(W270L) polypeptide.
KCNQ5 polypeptide, in one embodiment, is a human KCNQ5 polypeptide.
In another embodiment, KCNQ5 polypeptide is a non-human, mammalian
KCNQ5 polypeptide. Preferred non-human, mammalian KCNQ5 polypeptides

include rat KCNQ5 polypeptide (Co-owned, co-pending U.S. Provisional
Application Serial No. 60/760,249) and mouse KCNQ5 polypeptide (GenBank®
Accession No. NM_023872), both of which are sensitive to retigabine (see, e.g.,
Jensen HS et al., Brain Res. Mol. Brain Res. 139:52-62 (2005)), share high
homology with human wild type KCNQ5 (94.7% for rat and 95.2% for mouse),
and contain an amino acid which is believed to be equivalent to W270 (W269 in
rat and VV271 in mouse).
As used herein, a KCNQ5 or KCNQ5(W270L) "chimeric protein" or "fusion
protein" comprises a KCNQ5 or KCNQ5(W270L) polypeptide operably linked to a
non-KCNQ5 or non-KCNQ5(W270L) polypeptide. A "non-KCNQ5 polypeptide"
refers to a polypeptide having an amino acid sequence corresponding to a
protein which is not substantially homologous to the KCNQ5 protein, for example,
a protein which is different from the KCNQ5 protein and which is derived from the
same or a different organism. A "non-KCNQ5(W270L) polypeptide" refers to a
polypeptide having an amino acid sequence corresponding to a protein which is
not substantially homologous to the KCNQ5(W270L) protein, for example, a
protein which is different from the KCNQ5(W270L) protein and which is derived
from the same or a different organism. Within a KCNQ5 or KCNQ5(W270L)
fusion protein, the KCNQ5 or KCNQ5(W270L) polypeptide can correspond to all
or a portion of a KCNQ5 or KCNQ5(W270L) protein. In a preferred embodiment,
a KCNQ5 or KCNQ5(W270L) fusion protein comprises at least one biologically
active portion of a KCNQ5 or KCNQ5(W270L) protein. Within the fusion protein,
the term "operably linked" is intended to indicate that the KCNQ5 or
KCNQ5(W270L) polypeptide and the non-KCNQ5 or non-KCNQ5(W270L)
polypeptide are fused in-frame to each other. The non-KCNQ5 or non-
KCNQ5(W270L) polypeptide can be fused to the N-terminus or C-terminus of the
KCNQ5 or KCNQ5(W270L) polypeptide.
A "KCNQ5 polynucleotide" or "KCNQ5 nucleic acid sequence" refers to
non-wild type KCNQ5 polynucleotides which encode KCNQ5 polypeptides
having at least one amino acid modification which makes the KCNQ5 polypeptide
substantially insensitive to the K* channel activator retigabine while retaining
KCNQ5 M-current potassium channel activity. "Wild type KCNQ5 polynucleotide"
or "wild type KCNQ5 nucleic acid sequence" (for example, SEQ ID NO:3, which
represents human wild type KCNQ5 polynucleotide), on the other hand, encodes

wild type KCNQ5 which is responsive to retigabine (see Wickendon AD et al.,
Brit. J. Pharmacol. 132:381-84 (2001)).
A "coding sequence" or a sequence "encoding" an expression product,
such as a RNA, polypeptide, protein, or enzyme, is a nucleotide sequence that,
when expressed, results in the production of that RNA, polypeptide, protein, or
enzyme, i.e., the nucleotide sequence encodes an amino acid sequence for that
polypeptide, protein, or enzyme.
The term "complementary" is used to describe the relationship between
nucleotide bases that are capable to hybridizing to one another. For example,
with respect to DNA, adenosine is complementary to thymine and cytosine is
complementary to guanine.
The terms "effective amount", "therapeutically effective amount", and
"effective dosage" as used herein, refer to the amount of an effector molecule
that, when administered to a mammal in need, is effective to at least partially
ameliorate conditions related to or adverse conditions of, for example, the central
nervous system (CNS) and peripheral systems, including various types of pain
such as, for example, somatic, cutaneous, or visceral pain caused by, for
example burn, bruise, abrasion, laceration, broken bone, torn ligament, torn
tendon, torn muscle, viral, bacterial, protozoal or fungal infection, contact
dermatitis, inflammation (caused by, e.g., trauma, infection, surgery, burns, or
diseases with an inflammatory component), cancer, toothache; neuropathic pain
caused by, for example, injury to the central or peripheral nervous system due to
cancer, HIV (human immunodeficiency virus) infection, tissue trauma, infection,
autoimmune disease, diabetes, arthritis, diabetic neuropathy, trigeminal
neuralgia, or drug administration; treating anxiety caused by, for example, panic
disorder, generalized anxiety disorder, or stress disorder, particularly acute
stress disorder, affective disorders, Alzheimer's disease, ataxia, CNS damage
caused by trauma, stroke or neurodegenerative illness, cognitive deficits,
compulsive behavior, dementia, depression, Huntington's disease, mania,
memory impairment, memory disorders, memory dysfunction, motion disorders,
motor disorders, age-related memory loss, neurodegenerative diseases,
Parkinson's disease and Parkinson-like motor disorders, phobias, Pick's disease,
psychosis, schizophrenia, spinal cord damage, tremor, seizures, convulsions,
epilepsy, Stargardt-like macular dystrophy, cone-rod macular dystrophy, Salla

disease, epilepsy, muscle relaxants, fever reducers, anxiolytics, antimigraine
agents, analgesics, bipolar disorders, unipolar depression, functional bowel
disorders (e.g., dyspepsia and irritable bowl syndrome), diarrhea, constipation,
various types of urinary incontinence (e.g., urge urinary incontinence, stress
urinary incontinence, overflow urinary incontinence or unconscious urinary
incontinence, and mixed urinary incontinence), urinary urgency, bladder
instability, neurogenic bladder, hearing loss, tinnitus, glaucoma, cognitive
disorders, chronic inflammatory and neuralgic pain; for preventing and reducing
drug dependence or tolerance for treatment of, for example, cancer,
inflammation, ophthalmic diseases, and various CNS disorders.
The terms "express" and "expression" mean allowing or causing the
information in a gene or DNA sequence to become manifest, for example
producing a protein by activating the cellular functions involved in transcription
and translation of a corresponding gene or DNA sequence. A DNA sequence is
expressed in or by a cell to form an "expression product" such as a protein. The
expression product itself, e.g. the resulting protein, may also be said to be
"expressed" by the cell. An expression product can be characterized as
intracellular, extracellular, or secreted. The term "intracellular" means something
that is inside a cell. The term "extracellular" means something that is outside a
cell. A substance is "secreted" by a cell if it appears in significant measure
outside the cell, from somewhere on or inside the cell. "Antisense inhibition"
refers to the production of antisense RNA transcripts capable of suppression the
expression of the target protein. "Overexpression" refers to the production of a
gene product in an organism that exceeds levels of production in normal or non-
transformed organisms. "Suppression" refers to suppressing the expression of
foreign or endogenous genes or RNA transcripts.
The term "expression system" means a host cell and compatible vector
under suitable conditions, e.g. for the expression of a protein coded for by foreign
DNA carried by the vector and introduced to the host cell.
The term "gene" means a DNA sequence, including regulatory sequences
preceding (5' non-coding sequences) and following (3' non-coding sequences)
the coding sequence, that codes for or corresponds to a particular sequence of
amino acids which comprise all or part of one or more proteins or enzymes.
"Native gene" refers to a gene as found in nature with its own regulatory

sequences. "Chimeric gene" or "chimeric construct" refers to any gene or
construct, not a native gene, comprising regulatory and coding sequences that
are not found together in nature. Accordingly, a chimeric gene or chimeric
construct may comprise regulatory sequences and coding sequences that are
derived from different sources, or regulatory sequences and coding sequences
derived from the same source, but arranged in a manner different than that found
in nature. "Endogenous gene" refers to a native gene in its natural location in the
genome of an organism. A "foreign" gene refers to a gene not normally found in
the host organism, but which is introduced into the host organism by gene
transfer. Foreign genes can comprise native genes inserted into a non-native
organism, or chimeric genes. A "transgene" is a gene that has been introduced
into the genome by a transformation procedure.
The term "genetically modified" includes a cell containing and/or
expressing a foreign gene or nucleic acid sequence which in turn modifies the
genotype or phenotype of the cell or its progeny. This term includes any
addition, deletion, or disruption to a cell's endogenous nucleotides.
A "gene product" includes an amino acid (e.g., peptide or polypeptide)
generated when a gene is transcribed and translated.
The term "heterologous" refers to a combination of elements not naturally
occurring. For example, heterologous DNA refers to DNA not naturally located in
the cell, or in a chromosomal site of the cell. Preferably, the heterologous DNA
includes a gene foreign to the cell. A heterologous expression regulatory
element is such an element operably associated with a different gene than the
one it is operably associated with in nature.
The term "homomeric" as used herein refers to an ion channel comprising
only one type of subunit. For example, a homomeric dimer ("homodimer") KCNQ
channel could be composed of two identical KCNQ5 polypeptide subunits. A
homomeric tetramer ("homotetramer") could be composed of four identical
KCNQ5 polypeptide subunits. The term "heteromeric" as used herein refers to
an ion channel comprising at least two different subunits. For example, a
heteromeric dimer ("heterodimer") KCNQ channel could be composed of one
KCNQ5 polypeptide subunit and one KCNQ3 subunit, or a heterodimer KCNQ
channel could be composed of one KCNQ5 polypeptide subunit and a different
KCNQ5 polypeptide subunit. A heteromeric tetramer ("heterotetramer") KCNQ

channel could be composed of 1, 2, 3, or 4 KCNQ5 polypeptide subunits,
provided that if all four subunits are KCNQ5 polypeptide subunits that at least
one of the subunits is different from the other three.
"Homologous" refers to the degree of sequence similarity between two
polymers (i.e. polypeptide molecules or nucleic acid molecules). The homology
percentage figures referred to herein reflect the maximal homology possible
between the two polymers, i.e., the percent homology when the two polymers are
so aligned as to have the greatest number of matched (homologous) positions.
The terms "homologous" and "homology" also refer to the relationship between
proteins that possess a "common evolutionary origin", including proteins from
superfamilies (e.g., the immunoglobulin superfamily) and homologous proteins
from different species (e.g., myosin light chain, etc.) (see, e.g., Reeck GR et al..
Cell 50:667 (1987)). Such proteins (and their encoding genes) have sequence
homology, as reflected by their sequence similarity, whether in terms of percent
similarity or the presence of specific residues or motifs at conserved positions.
Accordingly, the term "sequence similarity" refers to the degree of identity or
correspondence between nucleic acid or amino acid sequences of proteins that
may or may not share a common evolutionary origin (see Reeck GR et ai,
supra). However, in common usage and in the instant application, the term
"homologous", when modified with an adverb such as "highly", may refer to
sequence similarity and may or may not relate to a common evolutionary origin.
To determine the percent identity of two amino acid sequences or of two
nucleic acid sequences, the sequences are aligned for optimal comparison
purposes (e.g., gaps can be introduced in one or both of a first and a second
amino acid or nucleic acid sequence for optimal alignment). In a preferred
embodiment, the length of a reference sequence aligned for comparison
purposes is at Jeast 30%, preferably at least 40%, more preferably at least 50%,
even more preferably at least 60%, and even more preferably at least 70%, 80%,
or 90% of the length of the reference sequence. The residues at corresponding
positions are then compared and when a position in one sequence is occupied
by the same residue as the corresponding position in the other sequence, then
the molecules are identical at that position. The percent identity between two
sequences, therefore, is a function of the number of identical positions shared by
two sequences (i.e., % identity=# of identical positions/total # of positions x 100).

The percent identity between the two sequences is a function of the number of
identical positions shared by the sequences, taking into account the number of
gaps, and the length of each gap, which are introduced for optimal alignment of
the two sequences.
The comparison of sequences and determination of percent identity
between two sequences can be accomplished using a mathematical algorithm.
A non-limiting example of a mathematical algorithm utilized for comparison of
sequences is the algorithm of Karlin S and Altschul SF, Proc. Natl. Acad. Sci.
USA 87:2264-68 (1990), modified as in Karlin S and Altschul SF, Proc. Natl.
Acad. Sci. USA 90:5873-77 (1993). Such an algorithm is incorporated into the
NBLAST and XBLAST programs (version 2.0) of Altschul SF et al, J. Mol. Biol.
215:403-10 (1990). BLAST nucleotide searches can be performed with the
NBLAST program score=100, wordlength=12 to obtain nucleotide sequences
homologous to the nucleic acid molecules. BLAST protein searches can be
performed with the XBLAST program, score=50, wordlength=3 to obtain amino
acid sequences homologous to the protein molecules. To obtain gapped
alignments for comparison purposes, Gapped BLAST can be utilized as
described in Altschul SF et al, Nucleic Acids Res. 25:3389-3402 (1997). When
utilizing BLAST and Gapped BLAST programs, the default parameters of the
respective programs (e.g., XBLAST and NBLAST) can be used. Another
preferred, non-limiting algorithm utilized for the comparison of sequences is the
algorithm of Myers EWand MillerW, Comput. Appl. Biosci. 4:11-17 (1988).
Such an algorithm is incorporated into the ALIGN program (version 2.0) which is
part of the GCG sequence alignment software package. When utilizing the
ALIGN program for comparing amino acid sequences, a PAM120 weight residue
table, a gap length penalty of 12, and a gap penalty of 4 can be used.
Another non-limiting example of a mathematical algorithm utilized for the
alignment of protein sequences is the Lipman-Pearson algorithm (Lipman DJ and
Pearson WR, Science 227:1435-41 (1985)). When using the Lipman-Pearson
algorithm, a PAM250 weight residue table, a gap length penalty of 12, a gap
penalty of 4, and a Kutple of 2 can be used. A preferred, non-limiting example of
a mathematical algorithm utilized for the alignment of nucleic acid sequences is
the Wilbur-Lipman algorithm (Wilbur WJ and Lipman DJ, Proc. Natl. Acad. Sci.
USA 80:726-30 (1983)). When using the Wilbur-Lipman algorithm, a window of

20, gap penalty of 3, Ktuple of 3 can be used. Both the Lipman-Pearson
algorithm and the Wilbur-Lipman algorithm are incorporated, for example, into
the MEGALIGN program (e.g., version 3.1.7) which is part of the DNASTAR
sequence analysis software package.
Additional algorithms for sequence analysis are known in the art, and
include ADVANCE and ADAM, described in Torelli A and Robotti CA, Comput.
Appl. Biosci. 10:3-5 (1994); and FASTA, described in Pearson WR and Lipman
DJ, Proc. Natl. Acad. Sci. USA 85:2444-48 (1988).
In a preferred embodiment, the percent identity between two amino acid
sequences is determined using the GAP program in the GCG software package,
using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16,14,
12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another
preferred embodiment, the percent identity between two nucleotide sequences is
determined using the GAP program in the GCG software package, using a
NWSgapdna. CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length
weight of 1, 2, 3,4, 5, or 6.
Protein alignments can also be made using the Geneworks global protein
alignment program (e.g., version 2.5.1) with the cost to open gap set at 5, the
cost to lengthen gap set at 5, the minimum diagonal length set at 4, the
maximum diagonal offset set at 130, the consensus cutoff set at 50% and
utilizing the Pam 250 matrix.
The nucleic acid and protein sequences can further be used as a "query
sequence" to perform a search against public databases to, for example, identify
other family members or related sequences. Such searches can be performed
using the NBLAST and XBLAST programs (version 2.0) of Altschul SF et a/., J.
Mol. Biol. 215:403-10 (1990). BLAST nucleotide searches can be performed with
the NBLAST program, score=100, wordlength=12 to obtain nucleotide
sequences homologous to KCNQ5 or KCNQ5(W270L) nucleic acid molecules.
BLAST protein searches can be performed with the XBLAST program, score=50,
wordlength=3 to obtain amino acid sequences homologous to KCNQ5 or
KCNQ5(W270L) protein molecules. To obtain gapped alignments for
comparison purposes, Gapped BLAST can be utilized as described in Altschul
SF et al., Nucleic Acids Res. 25:3389-3402 (1997). When utilizing BLAST and
Gapped BLAST programs, the default parameters of the respective programs

(e.g., XBLAST and NBLAST) can be used. For example, the nucleotide
sequences can be analyzed using the default Blastn matrix 1-3 with gap
penalties set at: existence 11 and extension 1. The amino acid sequences can
be analyzed using the default settings: the Blosum62 matrix with gap penalties
set at existence 11 and extension 1.
The term "host cell" means any cell of any organism that is selected,
modified, transformed, grown, or used or manipulated in any way, for the
production of a substance by the cell, for example the expression by the cell of a
gene, a DNA or RNA sequence, a protein, or an enzyme.
A nucleic acid molecule is "hybridizable" to another nucleic acid molecule,
such as a cDNA, genomic DNA, or RNA, when a single-stranded form of the
nucleic acid molecule can anneal to the other nucleic acid molecule under the
appropriate conditions of temperature and solution ionic strength (Sambrook J et
al. (eds.), Molecular Cloning: A Laboratory Manual (2d Ed. 1989) Cold Spring
Harbor Laboratory Press, NY. Vols. 1-3 (ISBN 0-87969-309-6)). The conditions
of temperature and ionic strength determine the "stringency" of the hybridization.
For preliminary screening for homologous nucleic acids, low stringency
hybridization conditions, corresponding to a Tm of 55 °C, can be used, e.g., 5x
SSC, 0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5x SSC,
0.5% SDS). Moderate stringency hybridization conditions correspond to a higher
Tm, e.g., 40% formamide, with 5x or 6x SCC. High stringency hybridization
conditions correspond to the highest Tm, e.g., 50% formamide, 5x or 6x SCC.
Hybridization requires that the two nucleic acids contain complementary
sequences although, depending on the stringency of the hybridization,
mismatches between bases are possible. The appropriate stringency for
hybridizing nucleic acids depends on the length of the nucleic acids and the
degree of complementation, variables weii known in the art. The greater the
degree of similarity or homology between two nucleotide sequences, the greater
the value of Tm for hybrids of nucleic acids having those sequences. The relative
stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in
. the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater
than 100 nucleotides in length, equations for calculating Tm have been derived
(Sambrook et a/., supra).

"Inhibitors", "activators", "openers", or "modulators" of voltage-gated
potassium channels comprising a KCNQ subunit refer to inhibitory or activating
molecules identified using in vitro and in vivo assays for KCNQ channel function.
In particular, inhibitors, activators, and modulators refer to compounds that
increase KCNQ channel function, thereby reducing pain in a subject. "Inhibitors"
are compounds that decrease, block, prevent, delay activation, inactivate,
desensitize, or down regulate the channel, or speed or enhance deactivation.
"Activators" are compounds that increase, open, activate, facilitate, enhance
activation, sensitize or up regulate channel activity, or delay or slow inactivation.
Such assays for inhibitors and activators also include, e.g., expressing
recombinant KCNQ in cells or cell membranes and then measuring flux of ions
through the channel directly or indirectly.
The term "isolated" means that the material is removed from its original or
native environment (e.g., the natural environment if it is naturally occurring).
Therefore, a naturally-occurring polynucleotide or polypeptide present in a living
animal is not isolated, but the same polynucleotide or polypeptide, separated or
modified by human intervention from some or all of the coexisting materials in the
natural system, is isolated. For example, an "isolated nucleic acid fragment" is a
polymer of RNA or DNA that is single- or double-stranded, optionally containing
synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid
fragment in the form of a polymer of DNA may be comprised of one or more
segments of cDNA, genomic DNA, or synthetic DNA. Such polynucleotides
could be part of a vector and/or such polynucleotides or polypeptides could be
part of a composition and still be isolated in that such vector or composition is not
part of the environment in which it is found in nature. Similarly, the term
"substantially purified" refers to a substance, which has been separated or
otherwise removed, through human intervention, from the immediate chemical
environment in which it occurs in nature. Substantially purified polypeptides or
nucleic acids may be obtained or produced by any of a number of techniques
and procedures generally known in the field (see, e.g., Scopes R (1987) In:
Protein purification: principles and practice, Springer-Verlag, NY. General protein
and DNA/RNA purification references: Current protocols in molecular biology,
Green publishing associates and John Wiley & Sons).

The term "mammal" refers to a human, a non-human primate, canine,
feline, bovine, ovine, porcine, murine, or other veterinary or laboratory mammal.
Those skilled in the art recognize that a therapy which reduces the severity of a
pathology in one species of mammal is predictive of the effect of the therapy on
another species of mammal.
The term "modulate" refers to the suppression, enhancement, or induction
of a function. For example, "modulation" or "regulation" of gene expression
refers to a change in the activity of a gene. Modulation of expression can
include, but is not limited to, gene activation and gene repression. "Modulate" or
"regulate" also refers to methods, conditions, or agents which increase or
decrease the biological activity of a protein, enzyme, inhibitor, signal transducer,
receptor, transcription activator, cofactor, and the like. This change in activity
can be an increase or decrease of mRNA translation, DNA transcription, and/or
mRNA or protein degradation, which may in turn correspond to an increase or
decrease in biological activity. Such enhancement or inhibition may be
contingent upon occurrence of a specific event, such as activation of a signal
transduction pathway and/or may be manifest only in particular cell types.
"Modulated activity" refers to any activity, condition, disease or phenotype
that is modulated by a biologically active form of a protein. Modulation may be
affected by affecting the concentration of biologically active protein, e.g., by
regulating expression or degradation, or by direct agonistic or antagonistic effect
as, for example, through inhibition, activation, binding, or release of substrate,
modification either chemically or structurally, or by direct or indirect interaction
which may involve additional factors.
As used herein, a "naturally-occurring" nucleic acid molecule refers to an
RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g.,
encodes a natural protein).
As used herein, "nucleic acid molecule" refers to the phosphate ester
polymeric form of ribonucleosides (adenosine, guanosine, uridine, or cytidine;
"RNA molecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine,
deoxythymidine, or deoxycytidine; "DNA molecules"), or any phosphoester
analogs thereof, such as phosphorothioates and thioesters, in either single-
stranded form, or a double-stranded helix. Double-stranded DNA-DNA, DNA-
RNA, and RNA-RNA helices are possible. The term nucleic acid molecule, and

in particular DNA or RNA molecule, refers only to the primary and secondary
structure of the molecule, and does not limit it to any particular tertiary forms.
Thus, this term includes double-stranded DNA found, inter alia, in linear (e.g.,
restriction fragments) or circular DNA molecules, plasmids, and chromosomes.
In discussing the structure of particular double-stranded DNA molecules,
sequences may be described herein according to the normal convention of giving
only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA
(i.e., the strand having a sequence homologous to the mRNA).
The term "operably linked" means that a nucleic acid molecule, i.e., DNA,
and one or more regulatory sequences (e.g., a promoter or portion thereof) are
connected in such a way as to permit transcription of mRNA from the nucleic acid
molecule or permit expression of the product (i.e., a polypeptide) of the nucleic
acid molecule when the appropriate molecules are bound to the regulatory
sequences. Within a fusion construct, the term "operably linked" is intended to
indicate that the KCNQ5 or KCNQ5(W270L) polynucleotide and a non-KCNQ5 or
non-KCNQ5(W270L) polynucleotide are fused in-frame to each other. The non-
KCNQ5 or non-KCNQ5(W270L) poiynucleotide can be fused 3' or 5' to the
KCNQ5 or KCNQ5CW270L) polynucleotide.
The term "percent homology" refers to the extent of amino acid sequence
identity between polynucleotides or poiypeptides. The homology between any
two polynucleotides or poiypeptides is a direct function of the total number of
matching nucleotides or amino acids at a given position in either sequence, e.g.,
if half of the total number of nucleotides in either of the sequences are the same
then the two sequences are said to exhibit 50% homology.
A "polynucleotide" or "nucleotide sequence" is a series of nucleotide bases
(also called "nucleotides") in a nucleic acid, such as DNA and RNA, and means
any chain of two or more nucieotides. A nucleotide sequence typically carries
genetic information, including the information used by cellular machinery to make
proteins and enzymes. These terms include double or single stranded genomic
and cDNA, RNA, any synthetic and genetically manipulated polynucleotide, and
both sense and anti-sense polynucleotide. This includes single- and double-
stranded molecules, i.e., DNA-DNA, DNA-RNA, and RNA-RNA hybrids, as well
as "protein nucleic acids" (PNA) formed by conjugating bases to an amino acid

backbone. This also includes nucleic acids containing modified bases such as,
for example, thio-uracil, thio-guanine, and fluoro-uracil.
It is contemplated that where the nucleic acid molecule is RNA, the T
(thymine) in non-RNA sequences provided herein is substituted with U (uracil).
For example, SEQ ID NO:1 is disclosed herein as a cDNA sequence. Thus, It
would be obvious to one of ordinary skill in the art that an RNA molecule
comprising sequences from this sequences, for example, would have T
substituted with U.
The term "polypeptide" includes a compound of two or more subunit amino
acids, amino acid analogs, or peptidomimetics. The subunits may be linked by
peptide bonds. In another embodiment, the subunit may be linked by other
bonds, e.g., ester, ether, etc. As used herein the term "amino acid" includes
either natural and/or unnatural or synthetic amino acids, including glycine and
both the D or L optical isomers, and amino acid analogs and peptidomimetics. A
peptide of three or more amino acids is commonly referred to as an oligopeptide.
Peptide chains of greater than three or more amino acids are referred to as a
polypeptide or a protein.
A "primer" includes a short polynucleotide, generally with a free 3'-OH
group that binds to a target or "template" present in a sample of interest by
hybridizing with the target, and thereafter promoting polymerization of a
polynucleotide complementary to the target A "polymerase chain reaction"
("PCR") is a reaction in which replicate copies are made of a target
polynucleotide using a "pair of primers" or "set of primers" consisting of an
"upstream" and a "downstream" primer, and a catalyst of polymerization, such as
a DNA polymerase, and typically a thermally-stable polymerase enzyme.
Methods for PCR are well known in the art, and are taught, for example, in
MacPherson et al, IRL Press at Oxford University Press (1991). All processes of
producing replicate copies of a polynucleotide, such as PCR or gene cloning, are
collectively referred to herein as "replication". A primer can also be used as a
probe in hybridization reactions, such as Southern or Northern blot analyses
(see, e.g., Sambrook J et a/., supra).
A "probe" when used in the context of polynucleotide manipulation
includes an oligonucleotide that is provided as a reagent to detect a target
present in a sample of interest by hybridizing with the target. Usually, a probe

will comprise a label or a means by which a label can be attached, either before
or subsequent to the hybridization reaction. Suitable labels include, but are not
limited to, radioisotopes, fluorochromes, chemiluminescent compounds, dyes,
and proteins, including enzymes.
A "promoter sequence" is a DNA regulatory region capable of binding
RNA polymerase in a cell and initiating transcription of a downstream (3'
direction) coding sequence. For purposes herein, the promoter sequence is
bounded at its 3' terminus by the transcription initiation site and extends
upstream (5' direction) to include the minimum number of bases or elements
necessary to initiate transcription at levels detectable above background. Within
the promoter sequence will be found a transcription initiation site (conveniently
defined, for example, by mapping with nuclease S1), as well as protein binding
domains (consensus sequences) responsible for the binding of RNA polymerase.
The term "purified" as used herein refers to material that has been isolated
under conditions that reduce or eliminate the presence of unrelated materials,
that is, contaminants, including native materials from which the material is
obtained. For example, a purified protein is preferably substantially free of other
proteins or nucleic acids with which it is associated in a cell; a purified nucleic
acid molecule is preferably substantially free of proteins or other unrelated
nucleic acid molecules with which it can be found within a cell. As used herein,
the term "substantially free" is used operationally, in the context of analytical
testing of the material. Preferably, purified material substantially free of
contaminants is at least 50% pure; more preferably, at least 90% pure; and more
preferably still at least 99% pure. Purity can be evaluated by chromatography,
gel electrophoresis, immunoassay, composition analysis, biological assay, and
other methods known in the art.
Methods for purification are well-known in the art. For example, nucleic
acids can be purified by precipitation, chromatography (including preparative
solid phase chromatography, oligonucleotide hybridization, and triple helix
chromatography), ultracentrifugation, and other means. Polypeptides and
proteins can be purified by various methods including, without limitation,
preparative disc-gel electrophoresis, isoelectric focusing, HPLC, reversed-phase
HPLC, gel filtration, ion exchange and partition chromatography, precipitation
and salting-out chromatography, extraction, and countercurrent distribution. For

some purposes, it is preferable to produce the polypeptide in a recombinant
system in which the protein contains an additional sequence tag that facilitates
purification, such as, but not limited to, a polyhistidine sequence, or a sequence
that specifically binds to an antibody, such as FLAG and GST. The polypeptide
can then be purified from a crude lysate of the host cell by chromatography on an
appropriate solid-phase matrix. Alternatively, antibodies produced against the
protein or against peptides derived therefrom can be used as purification
reagents. Cells can be purified by various techniques, including, for example,
centrifugation, matrix separation (e.g., nylon wool separation), panning and other
immunoselection techniques, depletion (e.g., complement depletion of
contaminating cells), and cell sorting (e.g., fluorescence activated cell sorting
(FACS)). Other purification methods are possible. A purified material may
contain less than about 50%, preferably less than about 75%, and most
preferably less than about 90%, of the cellular components with which it was
originally associated. The "substantially pure" indicates the highest degree of
purity which can be achieved using standard purification techniques known in the
art.
The term "test compound" includes compounds with known chemical
structure but not necessarily with a known function or biological activity. Test
compounds could also have unidentified structures or be mixtures of unknown
compounds, for example from crude biological samples such as plant extracts.
Large numbers of compounds could be randomly screened from "chemical
libraries" which refers to collections of purified chemical compounds or
collections of crude extracts from various sources. The chemical libraries may
contain compounds that were chemically synthesized or purified from natural
products. The compounds may comprise inorganic or organic small molecules or
larger organic compounds such as, for example, proteins, peptides,
glycoproteins, steroids, lipids, phospholipids, nucleic acids, and lipoproteins. The
amount of compound tested can very depending on the chemical library, but, for
purified (homogeneous) compound libraries, 10 µM is typically the highest initial
dose tested. Methods of introducing test compounds to cells are well known in
the art.
The term "transfection" means the introduction of a foreign nucleic acid
into a cell. The term "transformation" means the introduction of a "foreign" (i.e.

extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the
host cell will express the introduced gene or sequence to produce a desired
substance, typically a protein or enzyme coded by the introduced gene or
sequence. The introduced gene or sequence may also be called a "cloned" or
"foreign" gene or sequence, may include regulatory or control sequences, such
as start, stop, promoter, signal, secretion, or other sequences used by a cell's
genetic machinery. The gene or sequence may include nonfunctional sequences
or sequences with no known function. A host cell that receives and expresses
introduced DNA or RNA has been "transformed" and is a "transformant" or a
"clone". The DNA or RNA introduced to a host cell can come from any source,
including cells of the same genus or species as the host cell, or cells of a
different genus or species. Accordingly, a further embodiment is for a host cell
transformed with the vector described above. In one embodiment, the host cell is
a prokaryotic cell. In a further embodiment, the host cell is a eukaryotic cell. In a
preferred embodiment, the host cell is an E. coli cell.
The term "variant" may also be used to indicate a modified or altered
gene, DNA sequence, enzyme, cell, etc. Encompassed within the term
"variant(s)" are nucleotide and amino acid substitutions, additions, or deletions.
Also, encompassed within the term "variant(s)" are chemically modified natural
and synthetic KCNQ5 molecules. For example, variant may refer to polypeptides
that differ from a reference polypeptide. Generally, the differences between the
reference polypeptide and the polypeptide that differs in amino acid sequence
from reference polypeptide are limited so that the amino acid sequences of the
reference and the variant are closely similar overall and, in some regions,
identical. A variant and reference polypeptide may differ in amino acid sequence
by one or more substitutions, deletions, additions, fusions, and truncations that
may be conservative or non-conservative and may be present in any
combination. For example, variants may be those in which several, for instance
from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2,
from 2 to 1 amino acids are inserted, substituted, or deleted, in any combination.
Additionally, a variant may be a fragment of a polypeptide that differs from a
reference polypeptide sequence by being shorter than the reference sequence,
such as by a terminal or internal deletion. A variant of a polypeptide also
includes a polypeptide which retains essentially the same biological function or

activity as such polypeptide, e.g., precursor proteins which can be activated by
cleavage of the precursor portion to produce an active mature polypeptide.
These variants may be allelic variations characterized by differences in the
nucleotide sequences of the structural gene coding for the protein, or may
involve differential splicing or post-translational modification. Variants also
include a related protein having substantially the same biological activity, but
obtained from a different species. The skilled artisan can produce variants
having single or multiple amino acid substitutions, deletions, additions, or
replacements. These variants may include, inter alia: (i) one in which one or
more of the amino acid residues are substituted with a conserved or non-
conserved amino acid residue (preferably a conserved amino acid residue) and
such substituted amino acid residue may or may not be one encoded by the
genetic code, or (ii) one in which one or more amino acids are deleted from the
peptide or protein, or (iii) one in which one or more amino acids are added to the
polypeptide or protein, or (iv) one in which one or more of the amino acid
residues include a substituent group, or (v) one in which the mature polypeptide
is fused with another compound, such as a compound to increase the half-life of
the polypeptide (for example, polyethylene glycol), or (vi) one in which the
additional amino acids are fused to the mature polypeptide such as a leader or
secretory sequence or a sequence which is employed for purification of the
mature polypeptide or a precursor protein sequence. A variant of the polypeptide
may also be a naturally occurring variant such as a naturally occurring allelic
variant, or it may be a variant that is not known to occur naturally. All such
variants defined above are deemed to be within the scope of teachings in the art.
The terms "vector", "cloning vector", and "expression vector" refer to the
vehicle by which DNA can be introduced into a host cell, resulting in expression
of the introduced sequence. An "intergeneric vector" is a vector that permits
intergeneric conjugation, i.e., utilizes a system of passing DNA from E. coli to
another cell line directly. Intergeneric conjugation has fewer manipulations than
transformation.
Vectors typically comprise the DNA of a transmissible agent, into which
foreign DNA is inserted. A common way to insert one segment of DNA into
another segment of DNA involves the use of enzymes called restriction enzymes
that cleave DNA at specific sites (specific groups of nucleotides) called restriction

sites. A "cassette" refers to a DNA coding sequence or segment of DNA that
codes for an expression product that can be inserted into a vector at defined
restriction sites. The cassette restriction sites are designed to ensure insertion of
the cassette in the proper reading frame. Generally, foreign DNA is inserted at
one or more restriction sites of the vector DNA, and then is carried by the vector
into a host cell along with the transmissible vector DNA. A segment or sequence
of DNA having inserted or added DNA, such as an expression vector, can also
be called a "DNA construct". A common type of vector is a "plasmid", which
generally is a self-contained molecule of double-stranded DNA, usually of
bacterial origin, that can readily accept additional (foreign) DNA and which can
be readily introduced into a suitable host cell. A plasmid vector often contains
coding DNA and promoter DNA and has one or more restriction sites suitable for
inserting foreign DNA. Coding DNA is a DNA sequence that encodes a particular
amino acid sequence for a particular protein or enzyme. Promoter DNA is a DNA
sequence which initiates, regulates, or otherwise mediates or controls the
expression of the coding DNA. Promoter DNA and coding DNA may be from the
same gene or from different genes, and may be from the same or different
organisms. Recombinant cloning vectors will often include one or more
replication systems for cloning or expression, one or more markers for selection
in the host, e.g. antibiotic resistance, and one or more expression cassettes.
Vector constructs may be produced using standard molecular biology and
recombinant DNA techniques within the skill of the art. Such techniques are
explained fully in the literature. See, e.g., Sambrook J et al., supra; DNA
Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985); Ausubel
FM et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.
(1994). Commonly used, commercially available vectors include, for example,
pcDNA3 and pCR vectors from Inviirogen (Carlsbad, Calif.) and pGEM vectors
from Promega (Madison, Wis.).
"Voltage-gated" activity or "voltage-gating" or "voltage dependence" refers
to a characteristic of a potassium channel composed of individual polypeptide
monomers or subunits. Generally, the probability of a voltage-gated potassium
channel opening increases as a cell is depolarized. Voltage-gated potassium
channels primarily allow efflux of potassium at membrane potentials more
positive than the reversal potential for potassium (EK) in typical cells, because

they have greater probability of being open at such voltages. EK is the
membrane potential at which there is no net flow of potassium ions because the
electrical potential (i.e., voltage potential) driving potassium efflux is balanced by
the concentration gradient for potassium. The membrane potential of cells
depends primarily on their potassium channels and is typically between -60 and -
100 mV for mammalian cells. This value is also known as the "reversal potential"
or the "Nernst" potential for potassium. Some voltage-gated potassium channels
undergo inactivation, which can reduce potassium efflux at higher membrane
potentials. Potassium channels can also allow potassium influx in certain
instances when they remain open at membrane potentials negative to Ek (see,
e.g., Adams and Nonner, in Potassium Channels, pp. 40-60 (Cook, ed., 1990)).
The characteristic of voltage gating can be measured by a variety of techniques
for measuring changes in current flow and ion flux through a channel, e.g., by
changing the [K+] of the external solution and measuring the activation potential
of the channel current (see, e.g., U.S. Patent No. 5,670,335), by measuring
current with patch clamp techniques or voltage clamp under different conditions,
and by measuring ion flux with radiolabeled tracers or voltage-sensitive dyes
under different conditions.
II. Isolated Polynucleotides Encoding KCNQ5 or KCNQ5(W270L) or Portions
Thereof
In practicing the methods disclosed herein, various agents can be used to
modulate the activity and/or expression of KCNQ5 or KCNQ5(W270L) in a cell.
In one embodiment, an agent is a nucleic acid molecule encoding a KCNQ5 or
KCNQ5(W270L) polypeptide or a portion thereof. Such nucleic acid molecules
are described in more detail below.
There is a known and definite correspondence between the amino acid
sequence of a particular protein and the nucleotide sequences that can code for
the protein, as defined by the genetic code (shown below). Likewise, there is a
known and definite correspondence between the nucleotide sequence of a
particular nucleic acid molecule and the amino acid sequence encoded by that
nucleic acid molecule, as defined by the genetic code.

GENETIC CODE
Alanine (Ala, A)
Arginine (Arg, R)
Asparagine (Asn, N)
Aspartic acid (Asp, D)
Cysteine (Cys, C)
Glutamic acid (Glu, E)
Glutamine (Gln, Q)
Glycine (Gly, G)
Histidine (His, H)
Isoleucine (Ile, I)
Leucine (Leu, L)
Lysine (Lys, K)
Methionine (Met, M)
Phenylalanine (Phe, F)
Proline (Pro, P)
Serine (Ser, S)
Threonine (Thr, T)
Tryptophan (Trp, W)
Tyrosine (Tyr, Y)
Valine (Val, V)
Termination signal (end)

GCA, GCC, GCG, GCT
AGA, ACG, CGA, CGC, CGG, CGT
AAC, AAT
GAC, GAT
TGC, TGT
GAA, GAG
CAA, CAG
GGA, GGC, GGG, GGT
CAC, CAT
ATA, ATC, ATT
CTA, CTC, CTG, CTT, TTA, TTG
AAA, AAG
ATG
TTC, TTT.
CCA, CCC, CCG, CCT
AGC, AGT, TCA, TCC, TCG, TCT
ACA, ACC, ACG, ACT
TGG
TAC, TAT
GTA, GTC, GTG, GTT
TAA, TAG, TGA

An important and well known feature of the genetic code is its redundancy,
whereby, for most of the amino acids used to make proteins, more than one
coding nucleotide triplet may be employed (illustrated above). Therefore, a
number of different nucleotide sequences may code for a given amino acid
sequence. Such nucleotide sequences are considered functionally equivalent
because they result in the production of the same amino acid sequence in all
organisms (although certain organisms may translate some sequences more
efficiently than they do others). Moreover, occasionally, a methylated variant of a
purine or pyrimidine may be found in a given nucleotide sequence. Such
methylations do not affect the coding relationship between the trinucleotide
codon and the corresponding amino acid.
In view of the foregoing, the nucleotide sequence of a DNA or RNA
molecule coding for a KCNQ5 or KCNQ5(W270L) polypeptide (or a portion
thereof) can be used to derive the KCNQ5 or KCNQ5(W270L) amino acid
sequence, using the genetic code to translate the DNA or RNA molecule into an
amino acid sequence. Likewise, for any KCNQ5 or KCNQ5(W270L) amino acid
sequence, corresponding polynucleotide sequences that can encode KCNQ5 or
KCNQ5(W270L) protein can be deduced from the genetic code (which, because
of its redundancy, will produce multiple polynucleotide sequences for any given
amino acid sequence). Thus, description and/or disclosure herein of a KCNQ5
or KCNQ5(W270L) polynucleotide sequence should be considered to also

include description and/or disclosure of the amino acid sequence encoded by the
polynucleotide sequence. Similarly, description and/or disclosure of a KCNQ5 or
KCNQ5(W270L) amino acid sequence herein should be considered to also
include description and/or disclosure of all possible polynucleotide sequences
that can encode the amino acid sequence.
One aspect pertains to isolated nucleic acid molecules that encode
KCNQ5 or KCNQ5(W270L) proteins or biologically active portions thereof, as
well as nucleic acid fragments sufficient for use as hybridization probes to identify
KCNQ5- or KCNQ5(W270L)-encoding polynucleotides (e.g., KCNQ5 or
KCNQ5(W270L) mRNA) and fragments for use as PCR primers for the
amplification or mutation of KCNQ5 or KCNQ5(W270L) polynucleotides.
Biologically active portions of KCNQ5 proteins include, for example, the six
transmembrane domains, the pore region, and the conserved C-terminal region.
It will be understood that, in discussing the uses of KCNQ5 or KCNQ5(W270L)
nucleic acid molecules, fragments of such polynucleotides as well as full length
KCNQ5 or KCNQ5(W270L) polynucleotides can be used.
A polynucleotide disclosed herein, e.g., SEQ ID NO:1, or a portion thereof,
can be isolated using standard molecular biology techniques and the sequence
information provided herein. For example, using all or portion of the
polynucleotide sequence of SEQ ID NO:1 as a hybridization probe,
KCNQ5(W270L) polynucleotides can be isolated using standard hybridization
and cloning techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and
Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring
Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., 1989).
Moreover, a polynucleotide encompassing all or a portion of SEQ ID NO:1
can be isolated by PCR using synthetic oligonucleotide primers designed based
upon the sequence of, for example, SEQ ID NO:1.
A polynucleotide can be amplified using cDNA, mRNA or alternatively,
genomic DNA, as a template and appropriate oligonucleotide primers according
to standard PCR amplification techniques. The polynucleotide so amplified can
be cloned into an appropriate vector and characterized by DNA sequence
analysis. Furthermore, oligonucleotides corresponding to KCNQ5 or

KCNQ5(W270L) polynucleotide sequences can be prepared by standard
synthetic techniques, e.g., using an automated DNA synthesizer.
In a preferred embodiment, an isolated polynucleotide comprises the
polynucleotide sequence shown in SEQ ID NO:1.
In another preferred embodiment, an isolated polynucleotide comprises a
polynucleotide which is a complement of the polynucleotide sequence shown in
SEQ ID NO:1 or a portion of this polynucleotide sequence. A polynucleotide
which is complementary to the polynucleotide sequence shown in SEQ ID NO:1
is one which is sufficiently complementary to the polynucleotide sequence shown
in SEQ ID NO:1 such that it can hybridize to the polynucleotide sequence shown
in SEQ ID NO:1, thereby forming a stable duplex.
In still another preferred embodiment, an isolated polynucleotide
comprises a polynucleotide sequence which is at least about 95%, 98%, or more
homologous to the polynucleotide sequence (e.g., to the entire length of the
nucleotide sequence) shown in SEQ ID NO:1 or a portion of this nucleotide
sequence.
Moreover, a polynucleotide can comprise only a portion of the
polynucleotide sequence of SEQ ID NO:1; for example, a fragment which can be
used as a probe or primer or a fragment encoding a biologically active portion of
a KCNQ5(W270L) protein, provided that the fragment includes nucleotides 808-
810 of SEQ ID NO:1. The probe/primer typically comprises a substantially
purified oligonudeotide. In one embodiment, the oligonucleotide comprises a
region of nucleotide sequence that hybridizes under stringent conditions to at
least about 12 or 15, preferably about 20 or 25, more preferably about 30, 35,40,
45, 50, 55,60, 65, 75, or 100 consecutive polynucleotides of a sense sequence
of SEQ ID NO:1. In another embodiment, a polynucleotide comprises a
polynucleotide sequence which is at least about 100, 200, 300,400, 500,600, or
700 nucleotides in length and hybridizes under stringent hybridization conditions
to a polynucleotides sequence of SEQ ID NO:1 or the complements thereof.
In another embodiment, a polynucleotide comprises at least about 100,
200, 300, 400, 500, 600, 700, or more contiguous nucleotides of SEQ ID NO:1,
provided that the fragment includes nucleotides 808-810 of SEQ ID NO:1.
In other embodiments, a polynucleotide has at least 95% identity, and
more preferably 98% identity, with a polynucleotide comprising at least about

100, 200, 300, 400, 500, 600, 700, or more polynucleotides of SEQ ID NO:1,
provided that a substitution at nucleotides 808-810 is for a codon that produces a
conservative substitution for the amino acid leucine (for example, a substitution
which codes for alanine, glycine, isoleucine, or valine).
In another embodiment, a polynucleotide encodes a KCNQ5 polypeptide
containing an S5-S6 transmembrane domain from KCNQ1 in place of the wild
type S5-S6 transmembrane domain of KCNQ5. The human S5-S6
transmembrane domain was determined by secondary structure prediction using
the GCG program based on hydrophobicity of the amino acid sequence. S5
contains amino acids L266 to V287, and S6 contains amino acids L326 to L352.
Due to the lack of a crystal structure of KCNQ5, SEQ ID NO:6 includes an
extended region into the putative S4-S5 linker. Thus, in one embodiment, the
S5-S6 transmembrane domain of KCNQ5 includes nucleotides 769-1062 of SEQ
ID NO:3, corresponding to amino acids 257-354 of SEQ ID NO:4. Preferably, the
S5-S6 transmembrane domain is from human KCNQ1, more preferably the S5-
S6 transmembrane domain encoded by SEQ ID NO:5. In one embodiment, the
polynucleotide is a nucleic acid sequence comprising SEQ ID NO:3, wherein
nucleotides 769-1062 are substituted with SEQ ID NO:5. In another
embodiment, the polynucleotide encodes SEQ ID NO:4, wherein amino acids
257-354 are substituted with an S5-S6 transmembrane domain from KCNQ1,
preferably the S5-S6 transmembrane domain represented by SEQ ID NO:6.
In another embodiment, a polynucleotide encodes a KCNQ5 polypeptide
containing an S5 transmembrane domain from KCNQ1 in place of the wild type
S5 transmembrane domain of KCNQ5. The S5 transmembrane domain in
human KCNQ5 corresponds to polynucleotides 769-873 of SEQ ID NO:3,
encoding amino acids S257-A291 of SEQ ID NO:4. Preferably, the S5
transmembrane domain is from human KCNQ1, more preferably the S5
transmembrane domain encoded by nucleotides 1-105 of SEQ ID NO:5. In one
embodiment, the polynucleotide is a nucleic acid sequence comprising SEQ ID
NO:3, wherein nucleotides 769-873 are substituted with nucleotides 1-105 of
SEQ ID NO:5. In another embodiment, the polynucleotide encodes SEQ ID
NO:4, wherein amino acids 257-291 are substituted with an S5 transmembrane
domain from KCNQ1, preferably the S5 transmembrane domain represented by
amino acids 1-35 of SEQ ID NO:6.

Probes based on the KCNQ5 or KCNQ5(W270L) polynucleotide sequence
can be used to detect transcripts or genomic sequences encoding the same or
homologous proteins. In preferred embodiments, the probe further comprises a
label group attached thereto, for example, the label group can be a radioisotope,
a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can
be used as a part of a diagnostic test kit for identifying cells or tissues,
particularly the brain, skeletal muscle, and the urinary bladder, which misexpress
a wild-type KCNQ5 protein, such as by measuring a level of a KCNQ5- or
KCNQ5(W270L)-encoding polynucleotide in a sample of cells from a subject, for
example, detecting KCNQ5 or KCNQ5(W270L) mRNA levels or determining
whether a wild-type KCNQ5 gene has been mutated or deleted.
A nucleic acid fragment encoding a "biologically active portion of a
KCNQ5(W270L) protein" can be prepared by isolating a portion of the
polynucleotide sequence of SEQ ID NO:1, provided that the fragment includes
nucleotides 808-810 of SEQ ID NO:1, which encodes a polypeptide having a
KCNQ5(W270L) biological activity (i.e., the generation of voltage-dependent,
slowly activating K+-selective currents that are insensitive to the K+ channel
blocker TEA and display of a marked inward rectification at positive membrane
voltages), expressing the encoded portion of the KCNQ5(W270L) protein (e.g.,
by recombinant expression in vitro), and assessing the activity of the encoded
portion of the KCNQ5(W270L) protein.
Polynucleotides that differ from SEQ ID NO:1 due to degeneracy of the
genetic code, and thus encode the same KCNQ5(W270L) protein as that
encoded by SEQ ID NO:1 are encompassed by the present disclosure.
Accordingly, in another embodiment, an isolated polynucleotide has a
polynucleotide sequence encoding a protein having an amino acid sequence
shown in SEQ ID NO:2.
Nucleic acid molecules corresponding to natural allelic variants and
homologues of the KCNQ5 or KCNQ5(W270L) molecules can be isolated, for
example, based on their homology to the KCNQ5 or KCNQ5(W270L)
polynucleotides disclosed herein using the cDNAs disclosed herein, or portions
thereof, as a hybridization probe according to standard hybridization techniques.
For example, a KCNQ5(W270L) DNA can be isolated from a genomic DNA
library using all or portion of SEQ ID NO:1 as a hybridization probe and standard

hybridization techniques (e.g., as described in Sambrook J et al., supra).
Moreover, a polynucleotide encompassing all or a portion of a KCNQ5 gene can
be isolated by the polymerase chain reaction using oligonucleotide primers
designed based upon the sequence of SEQ ID NO:1. For example, mRNA can
be isolated from cells (e.g., by the guanidinium-thipcyanate extraction procedure
of Chirgwin et al., Biochemistry 18: 5294-99 (1979)) and cDNA can be prepared
using reverse transcriptase (e.g., Moloney MLV reverse transcriptase, available
from Gibco/BRL, Bethesda, Md.; or AMV reverse transcriptase, available from
Seikagaku America, Inc., St. Petersburg, Fla.). Synthetic oligonucleotide primers
for PCR amplification can be designed based upon the polynucleotide sequence
shown in SEQ ID NO:1. A polynucleotide can be amplified using cDNA or,
alternatively, genomic DNA, as a template and appropriate oligonucleotide
primers according to standard PCR amplification techniques. The polynucleotide
so amplified can be cloned into an appropriate vector and characterized by DNA
sequence analysis. Furthermore, oligonucleotides corresponding to a KCNQ5 or
KCNQ5(W270L) polynucleotide sequence can be prepared by standard synthetic
techniques, e.g., using an automated DNA synthesizer.
In another embodiment, an isolated polynucleotide can be identified based
on shared nucleotide sequence identity using a mathematical algorithm. Such
algorithms are outlined in more detail above (see, e.g., section I, infra).
In another embodiment, an isolated polynucleotide is at least 15, 20, 25,
30 or more polynucleotides in length and hybridizes under stringent conditions to
the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1 or
complements thereof. In another embodiment, the polynucleotide is at least 30,
50, 100, 150, 200, 250, 300, 3.50, 400, 450, 500, 550, or 600 nucleotides in
length. Preferably, the conditions are such that sequences at least 95%,
preferably at least about 98%, homologous to each other typically remain
hybridized to each other. Preferably, an isolated nucleic acid molecule that
hybridizes under stringent conditions to the sequence of SEQ ID NO:1 or
complements thereof corresponds to a naturally-occurring nucleic acid molecule.
In another embodiment, minor changes may be introduced by mutation
into polynucleotide sequences, for example, of SEQ ID NO:1, thereby leading to
changes in the amino acid sequence of the encoded protein, without altering the
functional activity of a KCNQ5(W270L) protein. For example, nucleotide

substitutions leading to amino acid substitutions at "non-essential" amino acid
residues may be made in the sequence of SEQ ID NO:1. A "non-essential"
amino acid residue is a residue that can be altered from the sequence of a
KCNQ5(W270L) polynucleotide (e.g., the sequence of SEQ ID NO:1) without
altering the functional activity of a KCNQ5(W270L) molecule. Exemplary
residues which are non-essential and, therefore, amenable to substitution can be
identified by one of ordinary skill in the art by performing an amino acid alignment
of KCNQ5(W270L)-related molecules and determining residues that are not
conserved. Such residues, because they have not been conserved, are more
likely amenable to substitution.
Accordingly, another aspect pertains to polynucleotides encoding KCNQ5
or KCNQ5(W270L) proteins that contain changes in amino acid residues that are
not essential for a KCNQ5 or KCNQ5(W270L) activity. Such KCNQ5(W270L)
proteins, for example, differ in.amino acid sequence from SEQ ID NO:2 yet retain
an inherent KCNQ5(W270L) activity. An isolated polynucleotide encoding a non-
natural variant of, for example, a KCNQ5(W270L) protein can be created by
introducing one or more nucleotide substitutions, additions, or deletions into the
polynucleotide sequence of SEQ ID NO:1 such that one or more amino acid
substitutions, additions, or deletions are introduced into the encoded protein,
provided that a substitution at nucleotides 808-810 is for a codon that produces a
conservative substitution for the amino acid leucine. Mutations can be
introduced into SEQ ID NO:1 by standard techniques, such as site-directed
mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino
acid substitutions are made at one or more non-essential amino acid residues. A
"conservative amino acid substitution" is one in which the amino acid residue is
replaced with an amino acid residue having a similar side chain. Families of
amino acid residues having similar side chains have been defined in the art,
including basic side chains (e.g., lysine, arginine, histidine), acidic side chains
(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine,
asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,
methionine, tryptophan), beta-branched side chains (e.g., threonine, valine,
isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,
histidine). Thus, a nonessential amino acid residue in a KCNQ5(W270L)

polypeptide is preferably replaced with another amino acid residue from the
same side chain family.
Alternatively, in another embodiment, mutations can be introduced
randomly along all or part of a KCNQ5 or KCNQ5(W270L) coding sequence,
such as by saturation mutagenesis, and the resultant mutants can be screened,
for example, for their ability to activate transcription or to identify mutants that
retain functional activity. Following mutagenesis, the KCNQ5 or KCNQ5(W270L)
mutant protein can be expressed recombinantly in a host cell and the functional
activity of the mutant protein can be determined using assays available in the art
for assessing KCNQ5 activity. The assays include, but are not limited to, patch
clamp whole cell recording using mammalian cells as hosts or two-electrode
voltage clamping using Xenopus laevis oocytes as hosts.
Yet another aspect pertains to isolated polynucleotides encoding KCNQ5
or KCNQ5(W270L) fusion proteins. Such polynucleotides, comprising at least a
first polynucleotide sequence encoding a full-length KCNQ5 or KCNQ5(W270L)
protein, polypeptide, or peptide having KCNQ5 or KCNQ5(W270L) activity
operably linked to a second polynucleotide sequence encoding a non-KCNQ5 or
non-KCNQ5(W270L) protein, polypeptide, or peptide can be prepared by
standard recombinant DNA techniques.
In a preferred embodiment, a KCNQ5 or KCNQ5(W270L) protein can be
assayed for the ability to encode functional ion channels using
electrophysiological methods as described above, for example patch clamp
whole cell recording using mammalian cells as hosts or two-electrode voltage
clamping using Xenopus laevis oocytes as hosts. In one aspect, the KCNQ5
polypeptide contains an S5-S6 transmembrane domain from KCNQ1. In another
aspect, the KCNQ5 polypeptide contains an S5 transmembrane domain from
KCNQ1.
In addition to the polynucleotides encoding KCNQ5 or KCNQ5(W270L)
proteins described above, another aspect pertains to isolated polynucleotides
which are antisense thereto. An "antisense" nucleic acid comprises a nucleotide
sequence which is complementary to a "sense" nucleic acid encoding a protein,
for example, complementary to the coding strand of a double-stranded cDNA
molecule or complementary to an mRNA sequence. Accordingly, an antisense
nucleic acid can hydrogen bond to a sense nucleic acid. The antisense nucleic

acid can be complementary to an entire KCNQ5 or KCNQ5(W270L) coding
strand, or only to a portion thereof. In one embodiment, an antisense nucleic
acid molecule is antisense to a "coding region" of the coding strand of a
nucleotide sequence encoding KCNQ5 or KCNQ5(W270L). The term "coding
region" refers to the region of the nucleotide sequence comprising codons which
are translated into amino acid residues. The term "noncoding region" refers to 51
and 3" sequences which flank the coding region that are not translated into amino
acids (i.e., also referred to as 5' and 3' untranslated regions).
Given the coding strand sequences encoding KCNQ5 or KCNQ5(W270L)
disclosed herein, antisense nucleic acids can be designed according to the rules
of Watson and Crick base pairing. The antisense polynucleotide can be
complementary to the entire coding region of KCNQ5 or KCNQ5(W270L) mRNA,
but more preferably is an oligonucleotide which is antisense to only a portion of
the coding or noncoding region of KCNQ5 or KCNQ5(W270L) mRNA. For
example, the antisense oligonucleotide can be complementary to the region
surrounding the translation start site of KCNQ5(W270L) mRNA. An antisense
oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35,40,45 or 50
nucleotides in length. An antisense polynucleotide can be constructed using
chemical synthesis and enzymatic ligation reactions using procedures known in •
the art. For example, an antisense nucleic acid (e.g., an antisense
oligonudeotide) can be Ghemicaily synthesized using naturally occurring
nucleotides or variously modified nucleotides designed to increase the biological
stability of the molecules or to increase the physical stability of the duplex formed
between the antisense and sense nucleic acids, for example, phosphorothioate
derivatives and acridine substituted nucleotides can be used. Examples of
modified nucleotides which can be used to generate the antisense nucleic acid
include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine,
xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-
carboxymethylaminomethyl-2-thiouridine, 5-carboxyrnethyIaminomethyluracil,
dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-
methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-
methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-
methylguanine, 5-methyIaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluraciI, 5-methoxyuracil, 2-

methylthio-N6-isopentenytadenine, uracil-5-oxyacetic acid (v), wybutoxosine,
pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-
thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic
acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,
and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced
biologically using an expression vector into which a nucleic acid has been
subcloned in an antisense orientation (i.e., RNA transcribed from the inserted
nucleic acid will be of an antisense orientation to a target nucleic acid of interest,
described further in the following subsection).
The antisense polynucleotides are typically administered to a subject or
generated in situ such that they hybridize with or bind to cellular mRNA and/or
genomic DNA encoding a KCNQ5 or KCNQ5(W270L) protein to thereby inhibit
expression of the protein, for example, by inhibiting transcription and/or
translation. The hybridization can be by conventional nucleotide
complementarity to form a stable duplex, or, for example, in the case of an
antisense polynucleotide which binds to DNA duplexes, through specific
interactions in the major groove of the double helix. An example of a route of
administration of antisense polynucleotides include direct injection at a tissue
site. Alternatively, antisense polynucleotides can be modified to target selected
cells and then administered systemically. For example, for systemic
administration, antisense molecules can be modified such that they specifically
bind to receptors or antigens expressed on a selected cell surface, for example,
by linking the antisense polynucleotides to peptides or antibodies which bind to
cell surface receptors or antigens. The antisense polynucleotides can also be
delivered to cells using the vectors described herein. To achieve sufficient
intracellular concentrations of the antisense molecules, vector constructs in
which the antisense polynucleotide is placed under the control of a strong pol !l
or pol III promoter are preferred.
In yet another embodiment, the antisense polynucleotide is an a-anomeric
nucleic acid molecule. An a-anomeric nucleic acid molecule forms specific
double-stranded hybrids with complementary RNA in which, contrary to the usual
(3-units, the strands run parallel to each other (Gaultier C et al, Nucleic Acids
Res. 15:6625-41 (1987)). The antisense nucleic acid molecule can also
comprise a 2'-o-methylribonucleotide (Inoue H et al., Nucleic Acids Res. 15:6131-

48 (1987)), or a chimeric RNA-DNA analogue (Inoue H et al, FEBS Lett.
215:327-30(1987)).
In still another embodiment, an antisense polynucleotide is a ribozyme.
Ribozymes are catalytic RNA molecules with ribonuclease activity which are
capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which
they have a complementary region. Thus, ribozymes (e.g., hammerhead
ribozymes (described in Haselhoff J and Gerlach WL, Nature 334:585-91(1988)))
can be used to catalytically cleave KCNQ5 or KCNQ5(W270L) mRNA transcripts
to thereby inhibit translation of KCNQ5 or KCNQ5(W270L) mRNA. A ribozyme
having specificity for a KCNQ5(W270L)-encoding nucleic acid can be designed
based upon the nucleotide sequence of SEQ ID NO:1. For example, a derivative
of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide
sequence of the active site is complementary to the nucleotide sequence to be
cleaved in a KCNQ5(W270L)-encoding mRNA (see, e.g., U.S. Patent Nos.
4,987,071 and 5,116,742). Alternatively, KCNQ5(W270L) mRNA can be used to
select a catalytic RNA having a specific ribonuclease activity from a pool of RNA
molecules (see, e.g., Bartel D and Szostak JW, Science 261:1411-18 (1993)).
Alternatively, gene expression can be inhibited by targeting nucleotide
sequences complementary to a regulatory region of KCNQ5 or KCNQ5(W270L)
(e.g., KCNQ5(W270L) promoter and/or enhancers) to form triple helical
structures that prevent transcription of a KCNQ5 or KCNQ5(W270L) gene in
target cells (see generally, Helene C, Anticancer Drug Des. 6:569-84 (1991);
Helene C et al., Ann. N. Y. Acad Sci. 660:27-36 (1992); Maher LJ, Bioassays
14:807-15(1992)).
In yet another embodiment, the KCNQ5 or KCNQ5(W270L)
polynucleotides can be modified at the base moiety, sugar moiety, or phosphate
backbone to improve, for example, the stability, hybridization, or solubility of the
molecule. For example, the deoxyribose phosphate backbone of the
polynucleotides can be modified to generate peptide nucleic acids (see Hyrup B
et al, Bioorg. Med. Chem. 4:5-23 (1996)). As used herein, the terms "peptide
nucleic acids" and "PNAs" refer to nucleic acid mimics, for example, DNA mimics,
in which the deoxyribose phosphate backbone is replaced by a pseudopeptide
backbone and only the four natural nucleobases are retained. The neutral
backbone of PNAs has been shown to allow for specific hybridization to DNA and

RNA under conditions of low ionic strength. The synthesis of PNA oligomers can
be performed using standard solid phase peptide synthesis protocols as
described in Hyrup B et al., supra; Perry-O'Keefe H et al., Proc. Natl. Acad. Sci.
USA 93:14670-75 (1996).
PNAs of KCNQ5 or KCNQ5(W270L) polynucleotides can be used in
therapeutic and diagnostic applications. For example, PNAs can be used as
antisense or antigene agents for sequence-specific modulation of gene
expression by, for example, inducing transcription or translation arrest or
inhibiting replication. PNAs of KCNQ5 or KCNQ5(W270L) nucleic acid
molecules can also be used in the analysis of single base pair mutations in a
gene (e.g., by PNA-directed PCR clamping), as "artificial restriction enzymes"
when used in combination with other enzymes, (e.g., S1 nucleases (Hyrup B ef
a/., supra), or as probes or primers for DNA sequencing or hybridization (Hyrup B
et a/., supra; Perry-O'Keefe H ef al., supra).
In another embodiment, PNAs of KCNQ5 or KCNQ5(W270L)
polynucleotides can be modified (e.g., to enhance their stability or cellular
uptake) by attaching lipophilic or other helper groups to PNA, by the formation of
PNA-DNA chimeras, or by the use of liposomes or other techniques of drug
delivery known in the art. For example, PNA-DNA chimeras of KCNQ5 or
KCNQ5(W270L) polynucleotides can be generated which may combine the
advantageous properties of PNA and DNA. Such chimeras allow DNA
recognition enzymes (e.g., RNase H and DNA polymerases) to interact with the
DNA portion while the PNA portion would provide high binding affinity and
specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths
selected in terms of base stacking, number of bonds between the nucleobases,
and orientation (Hyrup B et a/., supra). The synthesis of PNA-DNA chimeras can
be performed as described in Hyrup B et al., supra, and Finn PJ et al., Nucleic
Acids Res. 24:3357-63 (1996). For example, a DNA chain can be synthesized
on a solid support using standard phosphoramidite coupling chemistry and
modified nucleoside analogs, for example, 5'-(4-methoxytrityl)amino-5'-deoxy-
thymidine phosphoramidite, can be used as a between the PNA and the 5' end of
DNA (Mag M et al., Nucleic Acid Res. 17: 5973-88 (1989)). PNA monomers are
then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA
segment and a 3' DNA segment (Finn PJ et al., supra). Alternatively, chimeric

molecules can be synthesized with a 51 DNA segment and a 3' PNA segment
(Petersen KH et al., Bioorg. Med. Chem. Lett. 5:1119-24 (1995)).
In other embodiments, the oligonucleotide may include other appended
groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents
facilitating transport across the cell membrane (see, e.g., Letsinger RL et al.,
Proc. Natl. Acad. Sci. USA 86:6553-56 (1989); Lemaitre M et al., Proc. Natl.
Acad. Sci. USA 84:648-52 (1987); PCT Publication No. WO88/09810) or the
blood-brain barrier (see, e.g., PCT Publication No. WO89/10134). In addition,
oligonucleotides can be modified with hybridization-triggered cleavage agents
(see, e.g., van der Krol AR et al.r Biotechniques 6:958-76 (1988)) or intercalating
agents (see, e.g., Zon G, Pharm. Res. 5:539-49 (1988)). To this end, the
oligonucleotide may be conjugated to another molecule (e.g., a peptide,
hybridization triggered cross-linking agent, transport agent, or hybridization-
triggered cleavage agent).
In one embodiment, KCNQ5 or KCNQ5(W270L) polynucleotide
expression can be inhibited by short interfering RNAs (siRNA). The siRNA can
be dsRNA having 19-25 nucleotides. siRNAs can be produced endogenously by
degradation of longer dsRNA molecules by an RNase Ill-related nuclease called
Dicer. siRNAs can also be introduced into a cell exogenously, or by transcription
of an expression construct. Once formed, the siRNAs assemble with protein
components into endoribonuclease-containing complexes known as RNA-
induced silencing complexes (RISCs). An ATP-generated unwinding of the
siRNA activates the RISCs, which in turn target the complementary mRNA
transcript by Watson-Crick base-pairing, thereby cleaving and destroying the
mRNA. Cleavage of the mRNA takes place near the middle of the region bound
by the siRNA strand. This sequence specific mRNA degradation results in gene
silencing.
At least two ways can be employed to achieve siRNA-mediated gene
silencing. First, siRNAs can be synthesized in vitro and introduced into cells to
transiently suppress gene expression. Synthetic siRNA provides an easy and
efficient way to achieve RNAi. siRNA are duplexes of short .mixed
oligonucleotides which can include, for example, 19 RNAs nucleotides with
symmetric dinucleotide 31 overhangs. Using synthetic 21 bp siRNA duplexes
(e.g., 19 RNA bases followed by a UU or dTdT 3' overhang), sequence specific

gene silencing can be achieved in mammalian cells. These siRNAs can
specifically suppress targeted gene translation in mammalian cells without
activation of DNA-dependent protein kinase (PKR) by longer double-stranded
RNAs (dsRNA), which may result in non-specific repression of translation of
many proteins.
Second, siRNAs can be expressed in vivo from vectors. This approach
can be used to stably express siRNAs in cells or transgenic animals. In one
embodiment, siRNA expression vectors are engineered to drive siRNA
transcription from polymerase III (pol III) transcription units. Pol III transcription
units are suitable for hairpin siRNA expression because they deploy a short AT
rich transcription termination site that leads to the addition of 2 bp overhangs
(e.g., UU) to hairpin siRNAs—a feature that is helpful for siRNA function. The
Pol III expression vectors can also be used to create transgenic mice that
express siRNA.
In another embodiment, siRNAs can be expressed in a tissue-specific
manner. Under this approach, long dsRNAs are first expressed from a promoter
(such as CMV (pol II)) in the nuclei of selected cell lines or transgenic mice. The
long dsRNAs are processed into siRNAs in the nuclei (e.g., by Dicer). The
siRNAs exit from the nuclei and mediate gene-specific silencing. A similar
approach can be used in conjunction with tissue-specific (pol II) promoters to
create tissue-specific knockdown mice.
Any 3' dinucleotide overhang, such as UU, can be used for siRNA design.
In some cases, G residues in the overhang are avoided because of the potential
for the siRNA to be cleaved by RNase at single-stranded G residues.
With regard to the siRNA sequence itself, it has been found that siRNAs
with 30-50% GC content can be more active than those with a higher G/C
content in certain cases. Moreover, since a 4-6 nucleotide poly(T) tract may act
as a termination signal for RNA pol III, stretches of >4 Ts or As in the target
sequence may be avoided in certain cases when designing sequences to be
expressed from an RNA pol 111 promoter. In addition, some regions of mRNA
may be either highly structured or bound by regulatory proteins. Thus, it may be
helpful to select siRNA target sites at different positions along the length of the
gene sequence. Finally, the potential target sites can be compared to the
appropriate genome database (human, mouse, rat, etc.). Any target sequences

with more than 16-17 contiguous base pairs of homology to other coding
sequences may be eliminated from consideration in certain cases.
In one embodiment, siRNA can be designed to have two inverted repeats
separated by a short spacer sequence and end with a string of Ts that serve as a
transcription termination site. This design produces an RNA transcript that is
predicted to fold into a short hairpin siRNA. The selection of siRNA target
sequence, the length of the inverted repeats that encode the stem of a putative
hairpin, the order of the inverted repeats, the length and composition of the
spacer sequence that encodes the loop of the hairpin, and the presence or
absence of 5'-overhangs, can vary to achieve desirable results.
The siRNA targets can be selected by scanning an mRNA sequence for
AA dinucleotides and recording the 19 nucleotides immediately downstream of
the AA. Other methods can also been used to select the siRNA targets. In one
example, the selection of the siRNA target sequence is purely empirically
determined (see, e.g., Sui G et al., Proc. Natl. Acad. Sci. USA 99:5515-20
(2002)), as long as the target sequence starts with GG and does not share
significant sequence homology with other genes as analyzed by BLAST search.
In another example, a more elaborate method is employed to select the siRNA
target sequences. This procedure exploits an observation that any accessible
site in endogenous mRNA can be targeted for degradation by synthetic
oligodeoxyribonucleotide/RNase H method (see, e.g., Lee NS et al., Nature
Biotechnol. 20:500-05 (2002)).
In another embodiment, the hairpin siRNA expression cassette is
constructed to contain the sense strand of the target, followed by a short spacer,
the antisense strand of the target, and 5-6 Ts as transcription terminator. The
order of the sense and antisense strands within the siRNA expression constructs
can be altered without affecting the gene silencing activities of the hairpin siRNA.
In certain instances, the reversal of the order may cause partial reduction in gene
silencing activities.
The length of nucleotide sequence being used as the stem of siRNA
expression cassette can range, for instance, from 19 to 29. The loop size can
range from 3 to 23 nucleotides. Other lengths and/or loop sizes can also be
used.

In yet another embodiment, a 5' overhang in the hairpin siRNA construct
can be used, provided that the hairpin siRNA is functional in gene silencing. In
one specific example, the 5' overhang includes about 6 nucleotide residues.
In still yet another embodiment, the target sequence for RNAi is a 21-mer
sequence fragment of SEQ ID NO:1, preferably including nucleotides 808-810 of
SEQ ID NO:1. The 5' end of the target sequence has dinucleotide "NA," where
"N" can be any base and "A" represents adenine. The remaining 19-mer
sequence has a GC content of between 35% and 55%. In addition, the
remaining 19-mer sequence does not include any four consecutive A or T (i.e.,
AAAA or TTTT), three consecutive G or C (i.e., GGG or CCC), or seven "GC" in
a row.
Additional criteria can also be used for selecting RNAi target sequences.
For instance, the GC content of the remaining 19-mer sequence can be limited to
between 45% and 55%. Moreover, any 19-mer sequence having three
consecutive identical bases (i.e., GGG, CCC, TTT, or AAA) or a palindrome
sequence with 5 or more bases is excluded. Furthermore, the remaining 19-mer
sequence can be selected to have low sequence homology to other genes. In
one specific example, potential target sequences are searched by BLASTN
against NCBI's human UniGene cluster sequence database. The human
UniGene database contains non-redundant sets of gene-oriented clusters. Each
UniGene cluster includes sequences that represent a unique gene. 19-mer
sequences producing no hit to other human genes under the BLASTN search
can be selected. During the search, the e-value may be set at a stringent value
(such as "1").
The effectiveness of the siRNA sequences, as well as any other derived
RNAi sequence, can be evaluated using various methods known in the art. For
instance, an siRNA sequence can be introduced into a cell that expresses
KCNQ5 or KCNQ5(W270L). The polypeptide or mRNA level of KCNQ5 or
KCNQ5(W270L) in the cell can be detected. A substantial change in the
expression level of KCNQ5 or KCNQ5(W270L) before and after the introduction
of the siRNA sequence is indicative of the effectiveness of the siRNA sequence
in suppressing the expression of KCNQ5 or KCNQ5(W270L). In one specific
example, the expression levels of other genes are also monitored before and
after the introduction of the siRNA sequence. An siRNA sequence which has

inhibitory effect on KCNQ5 or KCNQ5(W270L) expression but does not
significantly affect the expression of other genes can be selected. In another
specific example, multiple siRNA or other RNAi sequences can be introduced
into the same target cell. These siRNA or RNAi sequences specifically inhibit
KCNQ5 or KCNQ5(W270L) expression but not the expression of other genes. In
yet another specific example, siRNA or other RNAi sequences that inhibit the
expression of KCNQ5 or KCNQ5(W270L) and other gene or genes can be used.
Antisense polynucleotides may be produced from a heterologous
expression cassette in a transfectant cell or transgenic cell. Alternatively, the
antisense polynucleotides may comprise soluble oligonucleotides that are
administered to the external milieu, either in the culture medium in vitro or in the
circulatory system or in interstitial fluid in vivo. Soluble antisense polynucleotides
present in the external milieu have been shown to gain access to the cytoplasm
and inhibit translation of specific mRNA species.
III. Isolated KCNQ5 and KCNQ5(W270L) Proteins, Fragments Thereof, and Anti-
KCNQ5 and Anti-KCNQ5(W270L) Antibodies
Another aspect pertains to isolated KCNQ5 and KCNQ5(W270L) proteins,
and biologically active portions thereof, as well as polypeptide fragments suitable
for use as immunogens to raise anti-KCNQ5 or anti-KCNQ5(W270L) antibodies.
In one embodiment, KCNQ5 and KCNQ5(W270L) proteins can be isolated from
cells or tissue sources by an appropriate purification scheme using standard
protein purification techniques. In another embodiment, KCNQ5 or
KCNQ5(W270L) proteins are produced by recombinant DNA techniques.
Alternative to recombinant expression, a KCNQ5 or KCNQ5(W270L) polypeptide
can be synthesized chemically using standard peptide synthesis techniques. It
will be understood that in discussing the uses of KCNQ5 or KCNQ5(W270L)
proteins, e.g., as shown in SEQ ID NO:2, that fragments of such proteins that are
not full length KCNQ5 or KCNQ5(W270L) polypeptides as well as full length
KCNQ5 or KCNQ5(W270L) proteins can be used.
Another aspect pertains to isolated KCNQ5 or KCNQ5(W270L) proteins.
Preferably, the KCNQ5(W270L) proteins comprise the amino acid sequence
encoded by SEQ ID NO:1 or a portion thereof. In another preferred embodiment,
the protein comprises the amino acid sequence of SEQ ID NO:2 or a portion

thereof. In other embodiments, the protein has at least at least 90%, more
preferably 95%, and even more preferably 98% amino acid identity, with the
amino acid sequence shown in SEQ ID NO:2 or a portion thereof, provided that a
substitution at amino acid 270 does not reestablish retigabine sensitivity to the
KCNQ5 polypeptide. Preferred portions of KCNQ5(W270L) polypeptide
molecules are biologically active, for example, a portion of the KCNQ5(W270L)
polypeptide having the ability to encode functional potassium-selective ion
channels in a host system, for example mammalian cell lines or Xenopus laevis
oocytes.
Biologically active portions of a KCNQ5 or KCNQ5(W270L) protein include
peptides comprising amino acid sequences sufficiently homologous to or derived
from the amino acid sequence of the KCNQ5 or KCNQ5(W270L) protein, which
include less amino acids than the full length KCNQ5 or KCNQ5(W270L) proteins,
and exhibit at least one activity of a KCNQ5 or KCNQ5(W270L) protein.
Another aspect is for KCNQ5 polypeptides containing an S5-S6
transmembrane domain from KCNQ1 in place of the wild type transmembrane
domain of KCNQ5. Preferably, the S5-S6 transmembrane domain is from human
KCNQ1. In one embodiment, the amino acid sequence of the KCNQ5
polypeptide comprises SEQ ID NO:4 with amino acids 257-354 substituted with
the S5-S6 transmembrane domain from KCNQ1. Preferably, amino acids 257-
354 of SEQ ID NO:4 are substituted with SEQ ID NO:6.
Another aspect is for KCNQ5 polypeptides containing an S5
transmembrane domain from KCNQ1 in place of the wild type transmembrane
domain of KCNQ5. Preferably, the S5 transmembrane domain is from human
KCNQ1. In one embodiment, the amino acid sequence of the KCNQ5
polypeptide comprises SEQ ID NO:4 with amino acids 257-291 substituted with
the S5 transmembrane domain from KCNQ1. Preferably, amino acids 257-291
of SEQ ID NO:4 are substituted with 1-35 SEQ ID NO:6.
Also provided are KCNQ5 or KCNQ5(W270L) chimeric or fusion proteins.
For example, in one embodiment, the fusion protein is a GST-KCNQ5(W270L)
member fusion protein in which the KCNQ5(W270L) member sequences are
fused to the C-terminus of the GST sequences. In another embodiment, the
fusion protein is a KCNQ5(W270L)-HA fusion protein in which the
KCNQ5(W270L) member nucleotide sequence is inserted in a vector such as

pCEP4-HA vector (Herrscher RF et a/., Genes Dev. 9:3067-82 (1995)) such that
the KCNQ5(W270L) member sequences are fused in frame to an influenza
hemagglutinin epitope tag. Such fusion proteins can facilitate the purification of a
recombinant KCNQ5(W270L) member.
Fusion proteins and peptides produced by recombinant techniques may
be secreted and isolated from a mixture of cells and medium containing the
protein or peptide. Alternatively, the protein or peptide may be retained
cytoplasmically and the cells harvested, lysed, and the protein isolated. A cell
culture typically includes host cells, media, and other byproducts. Suitable media
for cell culture are well known in the art. Protein and peptides can be isolated
from cell culture media, host cells, or both using techniques known in the art for
purifying proteins and peptides. Techniques for transfecting host cells and
purifying proteins and peptides are known in the art.
Preferably, a KCNQ5 or KCNQ5(W270L) fusion protein is produced by
standard recombinant DNA techniques. For example, DNA fragments coding for
the different polypeptide sequences are ligated together in-frame in accordance
with standard techniques, for example employing blunt-ended or stagger-ended
termini for ligation, restriction enzyme digestion to provide for appropriate termini,
filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to
avoid undesirable joining, and enzymatic ligation. In another embodiment, the
fusion gene can be synthesized by standard techniques including automated
DNA synthesizers. Alternatively, PCR amplification of gene fragments can be
carried out using anchor primers which give rise to complementary overhangs
between two consecutive gene fragments which can subsequently be annealed
and reamplified to generate a chimeric gene sequence (see, for example,
Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons:
1892). Moreover, many expression vectors are commercially available that
already encode a fusion moiety (e.g., a GST polypeptide or an HA epitope tag).
A KCNQ5-encoding or KCNQ5(W270L)-encoding nucleic acid can be cloned into
such an expression vector such that the fusion moiety is linked in-frame to
KCNQ5 or KCNQ5(W270L) protein.
In another embodiment, the fusion protein is a KCNQ5 or KCNQ5(W270L)
protein containing a heterologous signal sequence at its N-terminus. In certain
host cells (e.g., mammalian host cells), expression and/or secretion of KCNQ5 or

KCNQ5(W270L) can be increased through use of a heterologous signal
sequence. The KCNQ5 or KCNQ5(W270L) fusion proteins can be incorporated
into pharmaceutical compositions and administered to a subject in vivo. Use of
KCNQ5 or KCNQ5(W270L) fusion proteins may be useful therapeutically for the
treatment of disorders, for example, conditions related to urinary incontinence or
neuropathic pain. Moreover, the KCNQ5 or KCNQ5(W270L) fusion proteins can
be used as immunogens to produce anti-KCNQ5 or anti-KCNQ5(W270L)
antibodies in a subject.
As provided herein are functional potassium channels wherein at least one
of the subunits of the functional channel is a KCNQ5 or KCNQ5(W270L) protein
or polypeptide described herein. KCNQ channels are known to form
homodimers, heterodimers, homotetramers, and heterotetramers. For example,
a KCNQ5(W270L) protein can form a homodimer with itself, a heterodimer with a
KCNQ5 protein from another species, a heterodimer with a KCNQ5(W270L)
protein variant, a heterodimer with KCNQ3, a homotetramer with 3 identical
KCNQ5(W270L) subunits, a heterotetramer with at least one different KCNQ5
subunit, or a heterotetramer with at least one different KCNQ protein, for
example, KCNQ3.
Another aspect pertains to variants of the KCNQ5 or KCNQ5(W270L)
proteins which function as either KCNQ5 or KCNQ5(W270L) agonists (mimetics)
or as KCNQ5 or KCNQ5(W270L) antagonists. Variants of the KCNQ5 or
KCNQ5(W270L) proteins can be generated by mutagenesis, for example,
discrete point mutation or truncation of a KCNQ5 or KCNQ5(W270L) protein. An
agonist of the KCNQ5 or KCNQ5(W270L) proteins can retain substantially the
same, or a subset, of the biological activities of the naturally occurring form of a
KCNQ5 protein. An antagonist of a KCNQ5 or KCNQ5(W270L) protein can
inhibit one or more of the activities of the naturally occurring form of the KCNQ5
protein by, for example, competitively modulating a cellular activity of a wild-type
KCNQ5 protein. Thus, specific biological effects can be elicited by treatment with
a variant of limited function. In one embodiment, treatment of a subject with a
variant having a subset of the biological activities of the naturally occurring form
of the protein has fewer side effects in a subject relative to treatment with the
naturally occurring form of the KCNQ5 protein.

One embodiment pertains to derivatives of KCNQ5 or KCNQ5(W270L)
which may be formed by modifying at least one amino acid residue of KCNQ5 or
KCNQ5(W270L) by oxidation, reduction, or other derivatization processes known
in the art.
In one embodiment, variants of a KCNQ5 or KCNQ5(W270L) protein
which function as either KCNQ5 or KCNQ5(W270L) agonists (mimetics) or as
KCNQ5 or KCNQ5(W270L) antagonists can be identified by screening
combinatorial libraries of mutants, for example, truncation mutants, of a KCNQ5
or KCNQ5(W270L) protein for KCNQ5 or KCNQ5(W270L) protein agonist or
antagonist activity. The KCNQ5 polypeptide can contain an S5-S6
transmembrane domain from KCNQ1. Alternatively, the KCNQ5 polypeptide can
contain an S5 transmembrane domain from KCNQ1. In one embodiment, a
variegated library of KCNQ5 or KCNQ5(W270L) variants is generated by
combinatorial mutagenesis at the nucleic acid level and is encoded by a
variegated gene library. A variegated library of KCNQ5 or KCNQ5(W270L)
variants can be produced by, for example, enzymatically ligating a mixture of
synthetic oligonucleotides into gene sequences such that a degenerate set of
potential KCNQ5 or KCNQ5(W270L) sequences is expressible as individual
polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage
display) containing the set of KCNQ5 or KCNQ5(W270L) sequences therein.
There are a variety of methods which can be used to produce libraries of
potential KCNQ5 or KCNQ5(W270L) variants from a degenerate oligonucleotide
sequence. Chemical synthesis of a degenerate gene sequence can be
performed in an automatic DNA synthesizer, and the synthetic gene then ligated
into an appropriate expression vector. Use of a degenerate set of genes allows
for the provision, in one mixture, of all of the sequences encoding the desired set
of potential KCNQ5 or KCNQ5(W270L) sequences. Methods for synthesizing
degenerate oligonucleotides are known in the art (see, e.g., Narang SA,
Tetrahedron 39:3-22 (1983); Itakura K et a/., Annu. Rev. Biochem. 53:323-56
(1984); Itakura K et al., Science 198:1056-63 (1977); Ike Y et al., Nucleic Acids
Res. 11:477-88(1983)).
In addition, libraries of fragments of a KCNQ5 or KCNQ5(W270L) protein
coding sequence can be used to generate a variegated population of KCNQ5 or
KCNQ5(W270L) fragments for screening and subsequent selection of variants of

a KCNJQ5 or KCNQ5(W270L) protein. In one embodiment, a library of coding
sequence fragments can be generated by treating a double stranded PCR
fragment of a KCNQ5 or KCNQ5(W270L) coding sequence with a nuclease
under conditions wherein nicking occurs only about once per molecule,
denaturing the double stranded DNA, renaturing the DNA to form double
stranded DNA which can include sense/antisense pairs from different nicked
products, removing single stranded portions from reformed duplexes by
treatment with SI nuclease, and ligating the resulting fragment library into an
expression vector. By this method, an expression library can be derived which
encodes N-terminal, C-terminal, and internal fragments of various sizes of the
KCNQ5 or KCNQ5(W270L) protein.
Several techniques are known in the art for screening gene products of
combinatorial libraries made by point mutations or truncation, and for screening
cDNA libraries for gene products having a selected property. Such techniques
are adaptable for rapid screening of the gene libraries generated by the
combinatorial mutagenesis of KCNQ5 or KCNQ5(W270L) proteins. The most
widely used techniques, which are amenable to high through-put analysis, for
screening large gene libraries typically include cloning the gene library into
replicable expression vectors, transforming appropriate cells with the resulting
library of vectors, and expressing the combinatorial genes under conditions in
which detection of a desired activity facilitates isolation of the vector encoding the
gene whose product was detected. Recursive ensemble mutagenesis (REM), a
technique which enhances the frequency of functional mutants in the libraries,
can be used in combination with the screening assays to identify KCNQ5 or
KCNQ5(W270L) variants (Arkin AP and Youvan DC, Proc. Natl. Acad. Sci. USA
89:7811-15 (1992); Delgrave S et al., Protein Eng. 6:327-31 (1993)).
in one embodiment, cell based assays can be exploited to analyze a
variegated KCNQ5 or KCNQ5(W270L) library. For example, a library of
expression vectors can be transfected into a cell line which synthesizes
KCNQ5(W270L). The transfected cells are then cultured such that
KCNQ5(W270L) and a particular mutant KCNQ5(W270L) are synthesized and
the effect of expression of the mutant on KCNQ5(W270L) activity in cell
supernatants can be detected, for example, by any of a number of enzymatic
assays. Plasmid DNA can then be recovered from the cells which score for

inhibition, or alternatively, potentiation of KCNQ5(W270L) activity, and the
individual clones further characterized.
In addition to KCNQ5 or KCNQ5(W270L) polypeptides consisting only of
naturally-occurring amino acids, KCNQ5 or KCNQ5(W270L) peptidomimetics are
also provided. Peptide analogs are commonly used in the pharmaceutical
industry as non-peptide drugs with properties analogous to those of the template
peptide. These types of non-peptide compound are termed "peptide mimetics" or
"peptidomimetics" (Fauchere J, Adv. Drug Res. 15:29 (1986); Veber DF and
Freidinger RM, Trends Neurosci. 8:392-96 (1985); Evans BE et al., J. Med.
Chem 30:1229-39 (1987)) and are usually developed with the aid of
computerized molecular modeling. Peptide mimetics that are structurally similar
to therapeutically useful peptides may be used to produce an equivalent
therapeutic or prophylactic effect. Generally, peptidomimetics are structurally
similar to a paradigm polypeptide (i.e., a polypeptide that has a biological or
pharmacological activity), such as KCNQ5(W270L), but have one or more
peptide linkages optionally replaced by a linkage selected from the group
consisting of: -CH2NH-, -CH2S-, -CH2-CH2-, -CH=CH- (cis and trans), -
COCH2-, -CH(OH)CH2-, and -CH2SO-, by methods known in the art and
further described in the following references: Spatola AF in "Chemistry and
Biochemistry of Amino Acids, Peptides, and Proteins," B. Weinstein, ed., Marcel
Dekker, New York, p. 267 (1983); Spatola, AF, Vega Data (March 1983), Vol. 1,
Issue 3, "Peptide Backbone Modifications" (general review); Morley JS, Trends
Pharmcol. Sci. 1:463-68 (1980) (general review); Hudson D et al.. Int. J. Pept.
Prot Res. 14:177-85 (1979) (-CH2NH-, CH2CH2~); Spatola AF etal., Life Sci.
38:1243-49 (1986) (-CH2-S); Hann MM, J. Chem. Soc. Perkin Trans. 1, 307-314
(1982) (-CH-CH-, cis and trans); Almquist RG et al., J. Med. Chem. 23:1392-98
(1980) (-COCB2-); Jennings-White C et al., Tetrahedron Lett. 23:2533-34
(1982) (-COCH2-); EP 0 045665 (-CH(OH)CH2-); Holladay MW et al.,
Tetrahedron Lett., 24:4401-04 (1983) (-C(OH)CH2~); Hruby VJ, Life Sci. 31:189-
99 (1982) (-CH2-S—). A particularly preferred non-peptide linkage is -CH2NH-.
Such peptide mimetics may have significant advantages over polypeptide
embodiments, including, for example: more economical production, greater
chemical stability, enhanced pharmacological properties (half-life, absorption,
potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological

activities), reduced antigenicity, and others. Labeling of peptidomimetics usually
involves covalent attachment of one or more labels, directly or through a spacer
(e.g., an amide group), to non-interfering position(s) on the peptidomimetic that
are predicted by quantitative structure-activity data and/or molecular modeling.
Such non-interfering positions generally are positions that do not form direct
contacts with the macromolecules(s) to which the peptidomimetic binds to
produce the therapeutic effect. Derivatization (e.g., labeling) of peptidomimetics
should not substantially interfere with the desired biological or pharmacological
activity of the peptidomimetic.
Systematic substitution of one or more amino acids of a KCNQ5 or
KCNQ5(W270L) amino acid sequence with a D-amino acid of the same type
(e.g., D-lysine in place of L-lysine) may be used to generate more stable
peptides. In addition, constrained peptides comprising a KCNQ5 or
KCNQ5(W270L) amino acid sequence or a substantially identical sequence
variation may be generated by methods known in the art (Rizo J and Gierasch
LM, Ann. Rev. Biochem. 61:387-416 (1992)); for example, by adding internal
cysteine residues capable of forming intramolecular disulfide bridges which
cyclize the peptide.
The amino acid sequences of KCNQ5 or KCNQ5(W270L) polypeptides
identified herein will enable those of skill in the art to produce polypeptides
corresponding to KCNQ5 or KCNQ5(W270L) peptide sequences and sequence
variants thereof. Such polypeptides may be produced in prokaryotic or
eukaryotic host cells by expression of polynucleotides encoding a KCNQ5 or
KCNQ5(W270L) peptide sequence, frequently as part of a larger polypeptide.
Alternatively, such peptides may be synthesized by chemical methods. Methods
for expression of heterologous proteins in recombinant hosts, chemical synthesis
of polypeptides, and in vitro translation are well known in the art and are
described further in Sambrook J et a/., supra; Berger and Kimmel, Methods in
Enzymology, Volume 152, Guide to Molecular Cloning Techniques (1987),
Academic Press, Inc., San Diego, Calif.; Gutte B and Merrifield RB, J. Am.
Chem. Soc. 91:501-02 (1969); Chaiken IM, CRC Crit. Rev. Biochem. 11:255-301
(1981); Kaiser ET et at., Science 243:187-92 (1989); Merrifield B, Science
232:341-47 (1986); Kent SBH, Ann. Rev. Biochem. 57:957-89 (1988); Offord, R.
E. (1980) Semisynthetic Proteins, Wiley Publishing.

Peptides typically can be produced by direct chemical synthesis. Peptides
can be produced as modified peptides, with nonpeptide moieties attached by
covalent linkage to the N-terminus and/or C-terminus. In certain preferred
embodiments, either the carboxy-terminus or the amino-terminus, or both, are
chemically modified. The most common modifications of the terminal amino and
carboxyl groups are acetylation and amidation, respectively. Amino-terminal
modifications such as acylation (e.g., acetylation) or alkylation (e.g., methylation)
and carboxy-terminal-modifications such as amidation, as well as other terminal
modifications, including cyclization, may be incorporated into various
embodiments. Certain amino-terminal and/or carboxy-terminal modifications
and/or peptide extensions to the core sequence can provide advantageous
physical, chemical, biochemical, and pharmacological properties, such as:
enhanced stability, increased potency and/or efficacy, resistance to serum
proteases, desirable pharmacokinetic properties, and others. Peptides may be
used therapeutically to treat disease.
An isolated KCNQ5 or KCNQ5(W270L) protein, or a portion or fragment
thereof, can also be used as an immunogen to generate antibodies that bind
KCNQ5 or KCNQ5(W270L) using standard techniques for polyclonal and
monoclonal antibody preparation. A full-length KCNQ5 or KCNQ5(W270L)
protein can be used or, alternatively, another aspect provides antigenic peptide
fragments of KCNQ5 or KCNQ5(W270L) for use as immunogens. An antigenic
peptide of KCNQ5 or KCNQ5(W270L) comprises at least 8 amino acid residues
and encompasses an epitope of KCNQ5 or KCNQ5(W270L) such that an
antibody raised against the peptide forms a specific immune complex with
KCNQ5 or KCNQ5(W270L). Preferably, the antigenic peptide comprises at least
10 amino acid residues, more preferably at least 15 amino acid residues, even
more preferably at least 20 amino acid residues, and most preferably at least 30
amino acid residues. In one embodiment, the antigenic peptide includes amino
acid 270 of SEQ ID NO:2.
Preferred epitopes encompassed by the antigenic peptide are regions of a
KCNQ5 or KCNQ5(W270L) polypeptide that are located on the surface of the
protein, for example, hydrophilic regions, and that are unique to a KCNQ5 or
KCNQ5(W270L) polypeptide. In one embodiment, such epitopes can be specific
for KCNQ5 or KCNQ5(W270L) proteins from one species, such as human (i.e.,

an antigenic peptide that spans a region of a KCNQ5 or KCNQ5(W270L)
polypeptide that is not conserved across species is used as immunogen; such
non-conserved residues can be determined using an alignment such as that
provided herein). A standard hydrophobicity analysis of the protein can be
performed to identify hydrophilic regions.
A KCNQ5 or KCNQ5(W270L) immunogen typically is used to prepare
antibodies by immunizing a suitable subject (e.g., rabbit, goat, mouse, or other
mammal) with the immunogen. An appropriate immunogenic preparation can
contain, for example, a recombinantly expressed KCNQ5 or KCNQ5(W270L)
protein or a chemically synthesized KCNQ5 or KCNQ5(W270L) peptide. The
preparation can further include an adjuvant, such as Freund's complete or
incomplete adjuvant, or similar immunostimulatory agent. Immunization of a
suitable subject with an immunogenic KCNQ5 or KCNQ5(W270L) preparation
induces a polyclonal anti-KCNQ5 or anti-KCNQ5(W270L) antibody response.
Accordingly, another aspect pertains to the use of anti-KCNQ5 or anti-
KCNQ5(W270L) antibodies. Polyclonal anti-KCNQ5 or anti-KCNQ5(W270L)
antibodies can be prepared as described above by immunizing a suitable subject
with a KCNQ5 or KCNQ5(W270L) immunogen. The anti-KCNQ5 or anti-
KCNQ5(W270L) antibody titer in the immunized subject can be monitored over
time by standard techniques, such as with an enzyme linked immunosorbent
assay (ELISA) using immobilized KCNQ5 or KCNQ5(W270L) polypeptide. If
desired, the antibody molecules directed against a KCNQ5 or KCNQ5(W270L)
polypeptide can be isolated from the mammal (e.g., from the blood) and further
purified by well known techniques, such as protein A chromatography to obtain
the IgG fraction. At an appropriate time after immunization, for example, when
the anti-KCNQ5 or anti-KCNQ5(W270L) antibody titers are highest, antibody-
producing cells can be obtained from the subject and used to prepare
monoclonal antibodies by standard techniques, such as the hybridoma technique
originally described by Kohler G and Milstein C, Nature 256:495-97 (1975) (see
also, Brown JP et al., J. Immunol. 127:539-46 (1981); Brown JP et al., J. Biol.
Chem. 255:4980-83 (1980); Yeh MY et al., Proc. Natl. Acad. Sci. USA 76:2927-
31 (1979); Yeh MY et al., Int. J. Cancer 29:269-75 (1982)), the more recent
human B cell hybridoma technique (Kozbor D and Roder JC, Immunol. Today
4:72-79 (1983)), the EBV-hybridoma technique (Cole et al. (1985), Monoclonal

Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96), or trioma
techniques. The technology for producing monoclonal antibody hybridomas is
well known (see generally Kenneth RH, in Monoclonal Antibodies: A New
Dimension In Biological Analyses, Plenum Publishing Corp., New York, N.Y.
(1980); Lerner EA, Yale J. Biol. Med., 54:387-402 (1981); Gefter ML et al.,
Somatic Cell Genet. 3:231-36 (1977)). Briefly, an immortal cell line (typically a
myeloma) is fused to lymphocytes (typically splenocytes) from a mammal
immunized with a KCNQ5 or KCNQ5(W270L) immunogen as described above,
and the culture supernatants of the resulting hybridoma cells are screened to
identify a hybridoma producing a monoclonal antibody that binds specifically to a
KCNQ5 or KCNQ5(W270L) polypeptide.
Any of the many well known protocols used for fusing lymphocytes and
immortalized cell lines can be applied for the purpose of generating an anti-
KCNQ5 or anti-KCNQ5(W270L) monoclonal antibody (see, e.g., Galfre G ef a/.,
Nature 266:550-52 (1977); Geifer ML et al., supra; Lerner EA, supra; Kenneth
RH, supra). Moreover, the ordinary skilled worker will appreciate that there are
many variations of such methods which also would be useful. Typically, the
immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian
species as the lymphocytes. For example, murine hybridomas can be made by
fusing lymphocytes from a mouse immunized with an immunogenic preparation
with an immortalized mouse cell line. Preferred immortal cell lines are mouse
myeloma cell lines that are sensitive to culture medium containing hypoxanthine,
aminopterin, and thymidine ("HAT medium"). Any of a number of myeloma cell
lines may be used as a fusion partner according to standard techniques, for
example, the P3-NS1/1-Ag4-1, P3-x63-Ag8.653, or Sp2/O-Ag14 myeloma lines.
These myeloma lines are available from the American Type Culture Collection
(ATCC), Rockvilie, Md. Typically, HAT-sensitive mouse myeloma cells are fused
to mouse splenocytes using polyethylene glycol ("PEG"). Hybridoma cells
resulting from the fusion are then selected using HAT medium, which kills
unfused and unproductively fused myeloma cells (unfused splenocytes die after
several days because they are not transformed). Hybridoma cells producing a
monoclonal antibody are detected by screening the hybridoma culture
supernatants for antibodies that bind a KCNQ5 or KCNQ5(W270L) molecule, for
example, using a standard ELISA assay.

As an alternative to preparing monoclonal antibody-secreting hybridomas,
a monoclonal anti-KCNQ5 or anti-KCNQ5(W270L) antibody can be identified and
isolated by screening a recombinant combinatorial immunoglobulin library (e.g.,
an antibody phage display library) with KCNQ5 or KCNQ5(W270L) to thereby
isolate immunoglobulin library members that bind a KCNQ5 or KCNQ5(W270L)
polypeptide. Kits for generating and screening phage display libraries are
commercially available (e.g., the GE Healthcare Recombinant Phage Antibody
System, Catalog No. 27-9400-01). Additionally, examples of methods and
reagents particularly amenable for use in generating and screening antibody
display library can be found in, for example, U.S. Patent No. 5,223,409; WO
92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO
92/01047; WO 92/09690; WO 90/02809; Fuchs P et al., Biotechnology (N.Y.)
9:1370-72 (1991); Hay BN et al., Hum. Antibodies Hybridomas 3:81-85 (1992);
Huse WD etal.. Science 246:1275-81 (1989); Griffiths AD et al, EMBO J.
12:725-34 (1993); Hawkins RE et al., J. Mol. Biol. 226:889-96 (1992); Clarkson T
et al., Nature 352:624-28 (1991); Gram H etal., Proc. Natl. Acad. Sci. USA
89:3576-80 (1992); Garrard LJ et al., Biotechnology (N.Y.) 9:1373-77 (1991);
Hoogenboom HR et al., Nucleic Acids Res. 19:4133-37 (1991); Barbas CF etal.,
Proc. Natl. Acad. Sci. USA 88:7978-82 (1991); and McCafferty J et al., Nature
348:552-54 (1990).
Additionally, recombinant anti-KCNQ.5 or anti-KCNQ5(W270L) antibodies,
such as chimeric and humanized monoclonal antibodies, comprising both human
and non-human portions, which can be made using standard recombinant DNA
techniques, are within the scope of the present disclosure. Such chimeric and
humanized monoclonal antibodies can be produced by recombinant DNA
techniques known in the art, for example using methods described in WO
87/02671; EP 0 184 187; EP 0 171 496; EP 0 173 494; WO 86/01533; U.S.
Patent No. 4,816,567; EP 0 125 023; Better M et al.. Science 240:1041-43
(1988); Liu AY et al., Proc. Natl. Acad. Sci. USA 84:3439-43 (1987); Liu AY etal.,
J. Immunol. 139:3521-26 (1987); Sun LK etal., Proc. Natl. Acad. Sci. USA
84:214-18 (1987); Nishimura Y et al., Cancer Res. 47:999-1005 (1987); Wood
CR et ah, Nature 314:446-49 (1985); Shaw DR et ai, J. Natl. Cancer Inst.
80:1553-59 (1988); Morrison SL, Science 229:1202-07 (1985); U.S. Patent No.

5,225,539; Verhocyan M et al., Science 239:1534-36 (1988); and Beidler CB et
a/., J. Immunol. 141:4053-60 (1988).
In addition, humanized antibodies can be made according to standard
protocols such as those disclosed in U.S. Patent No. 5,565,332. In another
embodiment, antibody chains or specific binding pair members can be produced
by recombination between vectors comprising nucleic acid molecules encoding a
fusion of a polypeptide chain of a specific binding pair member and a component
of a replicable genetic display package and vectors containing nucleic acid
molecules encoding a second polypeptide chain of a single binding pair member
using techniques known in the art, for example, as described in U.S. Patent Nos.
5,565,332; 5,871,907; or 5,733,743.
An anti-KCNQ5 or anti-KCNQ5(W270L) antibody (e.g., monoclonal
antibody) can be used to isolate a KCNQ5 or KCNQ5(W270L) polypeptide by
standard techniques, such as affinity chromatography or immunoprecipitation.
The KCNQ5 polypeptide can contain an S5-S6 transmembrane domain from
KCNQ1. Alternatively, the KCNQ5 polypeptide can contain an S5
transmembrane domain from KCNQ1. Anti-KCNQ5 or anti-KCNQ5(W270L)
antibodies can facilitate the purification of natural KCNQ5 polypeptides from cells
and of recombinantly produced KCNQ5 or KCNQ5(W270L) polypeptides
expressed in host cells. Moreover, an anti-KCNQ5 or anti-KCNQ5(W270L)
antibody can be used to detect a KCNQ5 or KCNQ5(W270L) protein (e.g., in a
cellular lysate or cell supernatant). The KCNQ5 polypeptide can contain an S5-
S6 transmembrane domain from KCNQ1. Alternatively, the KCNQ5 polypeptide
can contain an S5 transmembrane domain from KCNQ1. Detection may be
facilitated by coupling (i.e., physically linking) the antibody to a detectable
substance. Accordingly, in one embodiment, an anti-KCNQ5 or anti-
KCNQ5(W270L) antibody is labeled with a detectable substance. Examples of
detectable substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials, and radioactive materials. Examples of
suitable enzymes include horseradish peroxidase, alkaline phosphatase, f5-
galactosidase, or acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples of suitable
fluorescent materials include umbelliferone, fluorescein, fluorescein
isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or

phycoerythrin; an example of a luminescent material includes luminol; and
examples of suitable radioactive material include 125I, 131I, 35S, or 3H.
Anti-KCNQ5 or anti-KCNQ5(W270L) antibodies are also obtainable by a
process comprising:
(a) immunizing an animal with an immunogenic KCNQ5 polypeptide or
an immunogenic portion thereof unique to a KCNQ5 polypeptide;
and
(b) isolating from the animal antibodies that specifically bind to a
KCNQ5 polypeptide.
Preferably, the KCNQ5 polypeptide is selected from the group consisting of (i) a
polypeptide comprising an amino acid sequence of a KCNQ5(W270L)
polypeptide; (ii) a KCNQ5 polypeptide containing an S5-S6 transmembrane
domain from KCNQ1; and (iii) a KCNQ5 polypeptide containing an S5
transmembrane domain from KCNQ1. More preferably, the immunogenic
KCNQ5 polypeptide is SEQ ID NO:2.
Accordingly, in one embodiment, anti-KCNQ5 or anti-KCNQ5(W270L)
antibodies can be used, e.g., intracellularly to inhibit protein activity. The use of
intracellular antibodies to inhibit protein function in a cell is known in the art (see
e.g., Carlson JR, Mol. Cell. Biol. 8:2638-46 (1988); Biocca S et al., EMBO J.
9:101-08 (1990); Werge TM et al., FEBS Lett. 274:193-98 (1990); Carlson JR,
Proc. Natl. Acad. Sci. USA 90:7427-28 (1993); Marasco WA et al., Proc. Natl.
Acad. Sci. USA 90:7889-93 (1993); Biocca S et al, Biotechnology (N.Y.) 12:396-
99 (1994); Chen S-Y et al., Hum. Gene Ther. 5:595-601 (1994); Duan L et al.,
Proc. Natl. Acad. Sci. USA 91:5075-79 (1994); Chen S-Y et al., Proc. Natl. Acad.
Sci. USA 91:5932-36 (1994); Beerli RR et al., J. Biol. Chem. 269:23931-36
(1994); Beerli RR et al., Biochem. Biophys. Res. Commun. 204:666-72 (1994);
Mhashilkar AM et al., EMBO J. 14:1542-51 (1995); Richardson JH et al., Proc.
Natl. Acad. Sci. USA 92:3137-41 (1995); WO 94/02610; and WO 95/03832).
In one embodiment, a recombinant expression vector is prepared which
encodes the antibody chains in a form such that, upon introduction of the vector
into a cell, the antibody chains are expressed as a functional antibody in an
intracellular compartment of the cell. For inhibition of KCNQ5 or KCNQ5(W270L)
activity according to the inhibitory methods disclosed herein, an intracellular
antibody that specifically binds the KCNQ5 or KCNQ5(W270L) protein is

expressed in the cytoplasm of the cell or extracellularly. To prepare an
intracellular antibody expression vector, antibody light and heavy chain cDNAs
encoding antibody chains specific for the target protein of interest, for example,
KCNQ5(W270L), are isolated, typically from a hybridoma that secretes a
monoclonal antibody specific for the KCNQ5(W270L) protein. Hybridomas
secreting anti-KCNQ5(W270L) monoclonal antibodies, or recombinant anti-
KCNQ5(W270L) monoclonal antibodies, can be prepared as described above.
Once a monoclonal antibody specific for KCNQ5(W270L) protein has been
identified (e.g., either a hybridoma-derived monoclonal antibody or a recombinant
antibody from a combinatorial library), DNAs encoding the light and heavy chains
of the monoclonal antibody are isolated by standard molecular biology
techniques. For hybridoma derived antibodies, light and heavy chain cDNAs can
be obtained, for example, by PCR amplification or cDNA library screening. For
recombinant antibodies, such as from a phage display library, cDNA encoding
the light and heavy chains can be recovered from the display package (e.g.,
phage) isolated during the library screening process. Nucleotide sequences of
antibody light and heavy chain genes from which PCR primers or cDNA library
probes can be prepared are known in the art. For example, many such
sequences are disclosed in Kabat EA et al. (1991) Sequences of Proteins of
Immunological Interest, Fifth Edition, U.S. Department of Health and Human
Services, NIH Publication No. 91-3242 and in the "Vbase" human germline
sequence database.
Once obtained, the antibody light and heavy chain sequences are cloned
into a recombinant expression vector using standard methods. To allow for
cytoplasmic expression of the light and heavy chains, the nucleotide sequences
encoding the hydrophobic leaders of the light and heavy chains are removed. An
intracellular antibody expression vector can encode an intracellular antibody in
one of several different forms. For example, in one embodiment, the vector
encodes full-length antibody light and heavy chains such that a full-length
antibody is expressed intracellularly. In another embodiment, the vector encodes
a full-length light chain but only the VH/CH1 region of the heavy chain such that a
Fab fragment is expressed intracellularly. In the most preferred embodiment, the
vector encodes a single chain antibody (scFv) wherein the variable regions of the
light and heavy chains are linked by a flexible peptide linker (e.g., (Gly4Ser)3) and

expressed as a single chain molecule. To inhibit KCNQ5 or KCNQ5(W270L)
activity in a cell, the expression vector encoding the anti-KCNQ5 or anti-
KCNQ5(W270L) intracellular antibody is introduced into the cell by standard
transfection methods, as discussed herein.
IV. Recombinant Expression Vectors and Host Cells
Another aspect pertains to vectors, preferably expression vectors,
containing a nucleic acid encoding a KCNQ5 or KCNQ5(W270L) protein (or a
portion thereof). The nucleic acid can encode a KCNQ5 polypeptide containing
an S5-S6 transmembrane domain from KCNQ1. Alternatively, the nucleic acid
can encode a KCNQ5 polypeptide containing an S5 transmembrane domain from
KCNQ1. The recombinant expression vectors comprise a nucleic acid in a form
suitable for expression of the nucleic acid in a host cell, which means that the
recombinant expression vectors include one or more regulatory sequences,
selected on the basis of the host cells to be used for expression, which is
operably linked to the nucleic acid sequence to be expressed. The term
"regulatory sequence" is intended to include promoters, enhancers, and other
expression control elements (e.g., polyadenylation signals). Such regulatory
sequences are described in, for example, Goeddel. Gene Expression
Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990). Regulatory sequences include those which direct constitutive expression
of a nucleotide sequence in many types of host cell and those which direct
expression of the nucleotide sequence only in certain host cells (e.g., tissue-
specific regulatory sequences). It will be appreciated by those skilled in the art
that the design of the expression vector can depend on such factors as the
choice of the host cell to be transformed, the level of expression of protein
desired, and the like. The expression vectors can be introduced into host ceils to
thereby produce proteins or peptides, including fusion proteins or peptides,
encoded by nucleic acids as described herein (e.g., KCNQ5(W270L) proteins,
mutant forms of KCNQ5(W270L) proteins, fusion proteins, and the like).
The recombinant expression vectors can be designed for expression of
KCNQ5 or KCNQ5(W270L) proteins or protein fragments in prokaryotic or
eukaryotic cells. For example, KCNQ5 or KCNQ5(W270L) proteins can be
expressed in bacterial cells such as E. coli, insect cells (using baculovirus

expression vectors), yeast cells, amphibian cells, or mammalian cells. Suitable
host cells are discussed further in Goeddel, supra. Alternatively, the recombinant
expression vector can be transcribed and translated in vitro, for example using
T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in E. coli
with vectors containing constitutive or inducible promoters directing the
expression of either fusion or non-fusion proteins. Fusion vectors add a number
of amino acids to a protein encoded therein, usually to the amino terminus of the
recombinant protein. Such fusion vectors typically serve three purposes: 1) to
increase expression of recombinant protein, 2) to increase the solubility of the
recombinant protein, and 3) to aid in the purification of the recombinant protein
by acting as a ligand in affinity purification. Often, in fusion expression vectors, a
proteolytic cleavage site is introduced at the junction of the fusion moiety and the
recombinant protein to enable separation of the recombinant protein from the
fusion moiety subsequent to purification of the fusion protein. Such enzymes,
and their cognate recognition sequences, include Factor Xa, thrombin, and
enterokinase. Typical fusion expression vectors include, for example, pGEX
(Pharmacia Biotech Inc; Smith DB and Johnson KS, Gene 67:31-40 (1988)) and
pMAL (New England Biolabs, Beverly, Mass.) which fuse glutathione S-
transferase (GST) or maltose E binding protein, respectively, to the target
recombinant protein.
Purified fusion proteins can be utilized, for example, in KCNQ5 or
KCNQ5(W270L) activity assays, (e.g., direct assays or competitive assays
described in detail below), or to generate antibodies specific for KCNQ5 or
KCNQ5(W270L) proteins.
Examples of suitable inducible non-fusion E. coli expression vectors
include pTrc (Amann E et al., Gene68:301-15 (1988)) and pET 11d (Studier et
al.. Gene Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990) pp. 60-89). Target gene expression from the pTrc
vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion
promoter. Target gene expression from the pET 11d vector relies on
transcription from a T7 gn10-lac fusion promoter mediated by a coexpressed viral
RNA polymerase (T7 gn1). This viral polymerase is supplied by host strains

BL21(DE3) or HMS174(DE3) from a resident prophage harboring a T7 gn1 gene
under the transcriptional control of the lacUV 5 promoter.
One strategy to maximize recombinant protein expression in E. coli is to
express the protein in a host bacteria with an impaired capacity to proteolytically
cleave the recombinant protein (Gottesman S, Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) pp. 119-
28). Another strategy is to alter the nucleic acid sequence of the nucleic acid to
be inserted into an expression vector so that the individual codons for each
amino acid are those preferentially utilized in E. coli (Wada K et al., Nucleic Acids
Res. 20(Suppl.):2111-18 (1992)). Such alteration of nucleic acid sequences can
be carried out by standard DNA synthesis techniques.
In another embodiment, the KCNQ5 or KCNQ5(W270L) expression vector
is a yeast expression vector. Examples of vectors for expression in yeast S.
cerevisiae include pYepSec1 (Baldari C et al., EMBO J. 6:229-34 (1987)), pMFa
(Kurjan J and Herskowitz I, Cell 30:933-43 (1982)), pJRY88 (Schultz LD et al.,
Gene 54:113-23 (1987)), pYES2 (Invitrogen Corporation, San Diego, Calif.), and
picZ (Invitrogen Corp, San Diego, Calif.).
Alternatively, KCNQ5 or KCNQ5(W270L) proteins or polypeptides can be
expressed in insect cells using baculovirus expression vectors. Baculovirus
vectors available for expression of proteins in cultured insect cells (e.g., Sf 9
cells) include the pAc series (Smith GE et al., Mol. Cell. Biol. 3:2156-65 (1983))
and the pVL series (Lucklow VA and Summers MD, Virology 170:31-39 (1989)).
In yet another embodiment, a nucleic acid is expressed in mammalian
cells using a mammalian expression vector. Examples of mammalian expression
vectors include pCDM8 (Seed B, Nature 329:840-41 (1987)) and pMT2PC
(Kaufman RJ et al., EMBO J. 6:187-95 (1987)). When used in mammalian cells,
the expression vector's control functions are often provided by viral regulatory
elements. For example, commonly used promoters are derived from polyoma,
Adenovirus 2, cytomegalovirus, and Simian Virus 40. For other suitable
expression systems for both prokaryotic and eukaryotic cells, see chapters 16
and 17 of Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A
Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector is
capable of directing expression of the nucleic acid preferentially in a particular
cell type (e.g., tissue-specific regulatory elements are used to express the nucleic
acid). Tissue-specific regulatory elements are known in the art. Non-limiting
examples of suitable tissue-specific promoters include the albumin promoter
(liver-specific; Pinkert CA et al., Genes Dev. 1:268-77 (1987)), lymphoid-specific
promoters (Calame K and Eaton S, Adv. Immunol. 43:235-75 (1988)), in
particular promoters of T cell receptors (Winoto A and Baltimore D, EMBO J.
8:729-33 (1989)) and immunoglobulins (Banerji J et al., Cell 33:729-40 (1983);
Queen C and Baltimore D, Cell 33:741-48 (1983)), neuron-specific promoters
(e.g., the neurofilament promoter; Byrne GW and Ruddle FH, Proc. Natl. Acad.
Sci. USA 86:5473-77 (1989)), pancreas-specific promoters (Edlund T et al.,
Science 230:912-16 (1985)), and mammary gland-specific promoters (e.g., milk
whey promoter; U.S. Patent No. 4,873,316 and EP 0 264 166). Developmentally-
regulated promoters are also encompassed, for example the murine hox
promoters (Kessel M and Gruss P, Science 249:374-79 (1990)) and the a-
fetoprotein promoter (Camper SA and Tilghman SM, Genes Dev. 3:537-46
(1989)).
Moreover, inducible regulatory systems for use in mammalian cells are
known in the art, for example systems in which gene expression is regulated by
heavy metal ions (see e.g., Mayo KE et al., Cell 29:99-108 (1982); Brinster RL et
a/., Nature 296:39-42 (1982); Searle PF et al., Mol. Cell. Biol. 5:1480-89 (1985)),
heat shock (see e.g., Nouer L et al. (1991) in Heat Shock Response, ed. Nouer
L, CRC, Boca Raton, Fla., pp. 167-220), hormones (see e.g., Lee F et al., Nature
294:228-32 (1981); Hynes NE et al.. Proc. Natl. Acad. Sci. USA 78:2038-42
(1981); Klock G et al., Nature 329:734-36 (1987); Israel Dl and Kaufman RJ,
Nucieic Acids Res. 17:2589-2604 (1989); WO 93/23431), FK506-related
molecules (see e.g., WO 94/18317) or tetracyclines (Gossen M and Bujard H,
Proc. Natl. Acad. Sci. USA 89:5547-51 (1992); Gossen M ef al., Science
268:1766-69 (1995); WO 94/29442; WO 96/01313). Accordingly, another
embodiment provides a recombinant expression vector in which a KCNQ5 or
KCNQ5(W270L) DNA is operably linked to an inducible eukaryotic promoter,
thereby allowing for inducible expression of a KCNQ5 or KCNQ5(W270L) protein
in eukaryotic cells.

Also known in the art are methods for expressing endogenous proteins
using one-arm homologous recombination (see, e.g., U.S. Published Patent
Application No. 2005/0003367; Zeh et al, Assay Drug Dev. Technol. 1:755-65
(2003); Qureshi et al., Assay Drug Dev. Technol. 1:767-76 (2003)). Briefly, an
isolated genomic construct comprising a promoter operably linked to a KCNQ5 or
KCNQ5(W270L) targeting sequence is introducing into a homogeneous
population of cells (such as, for example, a homogeneous population of a human
cell line or a homogenous population of Chinese hamster ovary (CHO) cells).
The promoter is heterologous to the KCNQ5 or KCNQ5(W270L) target gene.
Following recombination, the promoter controls transcription of an mRNA that
encodes a KCNQ5 or KCNQ5(W270L) polypeptide. The population of cells is
then incubated under conditions which cause expression of the KCNQ5 or
KCNQ5(W270L) polypeptide.
A further aspect provides a recombinant expression vector comprising a
DNA molecule cloned into the expression vector in an antisense orientation.
That is, the DNA molecule is operably linked to a regulatory sequence in a
manner which allows for expression (by transcription of the DNA molecule) of an
RNA molecule which is antisense to KCNQ5 or KCNQ5(W270L) mRNA.
Regulatory sequences operably linked to a nucleic acid cloned in the antisense
orientation can be chosen which direct the continuous expression of the
antisense RNA molecule in a variety of cell types, for instance viral promoters
and/or enhancers, or regulatory sequences can be chosen which direct
constitutive, tissue specific, or cell type specific expression of antisense RNA.
The antisense expression vector can be in the form of a recombinant plasmid,
phagemid, or attenuated virus in which antisense nucleic acids are produced
under the control of a high efficiency regulatory region, the activity of which can
be determined by the cell type into which the vector is introduced. For a
discussion of the regulation of gene expression using antisense genes, see
Weintraub H et al., Trends Genet. 1:22-25 (1985).
Another aspect pertains to host cells into which a recombinant expression
vector has been introduced. For example, a KCNQ5 or KCNQ5(W270L) protein
can be expressed in bacterial cells (such as, for example, E. coli), insect cells,
yeast cells, amphibian cells (such as, for example, Xenopus laevis oocytes), or

mammalian cells (such as, for example, CHO cells or COS cells). Other suitable
host cells are known to those skilled in the art.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via
standard transformation or transfection techniques. As used herein, the terms
"transformation" and "transfection" are intended to refer to a variety of art-
recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host
cell, including, for example, calcium phosphate of calcium chloride co-
precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation.
Suitable methods for transforming or transfecting host cells can be found in
Sambrook J et al. (supra), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending
upon the expression vector and transfection technique used, only a small fraction
of cells may integrate the foreign DNA into their genome. In order to identify and
select these integrants, a gene that encodes a selectable marker (e.g.,
resistance to antibiotics) is generally introduced into the host cells along with the
gene of interest. Preferred selectable markers include those which confer
resistance to drugs, such as G418, hygromycin, and methotrexate. Nucleic acid
encoding a selectable marker can be introduced into a host cell on the same
vector as that encoding KCNQ5 or KCNQ5(W270L) protein or can be introduced
on a separate vector. Cells stably transfected with the introduced nucleic acid
can be identified by drug selection (e.g., cells that have incorporated the
selectable marker gene will survive, while the other cells die).
In the case of E. coli which are stably transfected with KCNQ5 or
KCNQ5(W270L), such lines can be made such that the KCNQ5 or
KCNQ5(W270L) gene is inducible. For example, the regulation of the expressed
gene can be brought about by the double stable expression first of a "regulator"
plasmid, which contains the tet-controlled transactivator (tTA) and a second
"response" plasmid, which contains KCNQ5 or KCNQ5(W270L), under the
control of a promoter sequence that includes the tetracycline response element
(TRE). The commercially available regulator plasmids are in vectors engineered
for neomycin selection, necessitating that response vectors be constructed to
include a second selectable marker. Using such methods, KCNQ5 or
KCNQ5(W270L) expression can be turned off in the presence of an agent, for
example, tetracycline or a tetracycline-related compound (e.g., doxycycline) and

turned on when the agent, for example, tetracycline, is not added to the culture
medium. Construction of this type of cell line permits the stable expression of
KCNQ5 or KCNQ5(W270L) in cells in which it is normally toxic.
A host cell, such as a prokaryotic or eukaryotic host cell in culture, can be
used to produce (i.e., express) a KCNQ5 or KCNQ5(W270L) protein.
Accordingly, a further aspect provides methods for producing a KCNQ5 or
KCNQ5(W270L) protein using the host cells. In one embodiment, the method
comprises culturing the host cell (into which a recombinant expression vector
encoding a KCNQ5 or KCNQ5(W270L) protein has been introduced) in a suitable
medium such that a KCNQ5 or KCNQ5(W270L) protein is produced. In another
embodiment, the method further comprises isolating a KCNQ5 or
KCNQ5(W270L) protein from the medium or the host cell.
Certain host cells can also be used to produce non-human transgenic
animals. For example, in one embodiment, a host cell is a fertilized oocyte or an
embryonic stem cell into which KCNQ5- or KCNQ5(W270L)-coding sequences
have been introduced. Such host cells can then be used to create non-human
transgenic animals in which exogenous KCNQ5 or KCNQ5(W270L) sequences
have been introduced into their genome or homologous recombinant animals in
which endogenous KCNQ5 sequences have been altered. Such animals are
useful for studying the function and/or activity of a KCNQ5 or KCNQ5(W270L)
polypeptide and for identifying and/or evaluating modulators of KCNQ5 or
KCNQ5(W270L) activity. As used herein, a "transgenic animal" is a non-human
animal, preferably a mammal, more preferably a rodent such as a rat or mouse,
in which one or more of the cells of the animal includes a transgene. Other
examples of transgenic animals include non-human primates, sheep, dogs,
cows, goats, chickens, amphibians, and the like. A transgene is exogenous DNA
which is integrated into the genome of a ceii from which a transgenic animal
develops and which remains in the genome of the mature animal, thereby
directing the expression of an encoded gene product in one or more cell types or
tissues of the transgenic animal. As used herein, a "homologous recombinant
animal" is a non-human animal, preferably a mammal, more preferably a mouse,
in which an endogenous KCNQ5 gene has been altered by homologous
recombination between the endogenous gene and an exogenous DNA molecule

introduced into a cell of the animal, for example, an embryonic cell of the animal,
prior to development of the animal.
A transgenic animal can be created by introducing a KCNQ5- or
KCNQ5(W270L)-encoding nucleic acid into the male pronucleus of a fertilized
oocyte, e.g., by microinjection or retroviral infection, and allowing the oocyte to
develop in a pseudopregnant female foster animal. The KCNQ5(W270L)
sequence of SEQ ID NO:1 or portion thereof can be introduced as a transgene
into the genome of a non-human animal. Alternatively, a KCNQ5(W270L) gene
homologue, such as another KCNQ family member, can be isolated based on
hybridization to the KCNQ5(W270L) family cDNA sequences of SEQ ID NO:1
(described further above) and used as a transgene.
Intronic sequences and polyadenylation signals can also be included in
the transgene to increase the efficiency of expression of the transgene. A tissue-
specific regulatory sequence(s) can be operably linked to a KCNQ5 or
KCNQ5(W270L) transgene to direct expression of a KCNQ5 or KCNQ5(W270L)
protein to particular cells. Methods for generating transgenic animals via embryo
manipulation and microinjection, particularly animals such as mice, have become
standard in the art and are described, for example, in U.S. Patent Nos.
4,736,866; 4,870,009; 4,873,191; and in Hogan B, Manipulating the Mouse
Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
Similar methods are used for production of other transgenic animals. A
transgenic founder animal can be identified based upon the presence of a
KCNQ5 or KCNQ5(W270L) transgene in its genome and/or expression of
KCNQ5 or KCNQ5(W270L) mRNA in tissues or cells of the animals. A
transgenic founder animal can then be used to breed additional animals carrying
the transgene. Moreover, transgenic animals carrying a transgene encoding a
KCNQ5 or KCNQ5(W270L) protein can further be bred to other transgenic
animals carrying other transgenes.
To create a homologous recombinant animal, a vector is prepared which
contains at least a portion of a KCNQ5 gene into which a deletion, addition, or
substitution has been introduced to thereby alter, for example, functionally
disrupt, the KCNQ5 gene. In a preferred embodiment, the vector is designed
such that, upon homologous recombination, the endogenous KCNQ5 gene is
functionally disrupted (i.e., no longer encodes a functional protein; also referred

to as a "knock out" vector). Alternatively, the vector can be designed such that,
upon homologous recombination, the endogenous KCNQ5 gene is mutated or
otherwise altered but still encodes a functional protein (e.g., nucleotides 808-810
of SEQ ID NO:1 can be altered to thereby alter amino acid 270 of the
endogenous KCNQ5 protein). In the homologous recombination vector, the
altered portion of the KCNQ5 gene is flanked at its 5' and 3' ends by additional
nucleic acid sequence of the KCNQ5 gene to allow for homologous
recombination to occur between the exogenous KCNQ5 gene carried by the
vector and an endogenous KCNQ5 gene in an embryonic stem cell. The
additional flanking KCNQ5 nucleic acid sequence is of sufficient length for
successful homologous recombination with the endogenous gene. Typically,
several kilobases of flanking DNA (both at the 5' and 3' ends) are included in the
vector (see, e.g., Thomas KR and Capecchi MR, Cell 51:503-12 (1987) for a
description of homologous recombination vectors). The vector is introduced into
an embryonic stem cell line (e.g., by electroporation) and cells in which the
introduced KCNQ5 gene has homologously recombmed with the endogenous
KCNQ5 gene are selected (see, e.g., Li E et al., Cell 69:915-26 (1992)). The
selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to
form aggregation chimeras (see, e.g., Bradley A, Teratocarcinomas and
Embryonic Stem Cells: A Practical Approach, Robertson EJ, ed. (IRL, Oxford,
1987) pp. 113-152). A chimeric embryo can then be implanted into a suitable
pseudopregnant female foster animal, and the embryo brought to term. Progeny
harboring the homologously recombined DNA in their germ cells can be used to
breed animals in which all cells of the animal contain the homologously
recombined DNA by germline transmission of the transgene. Methods for
constructing homologous recombination vectors and homologous recombinant
animals are described further in, for example, Bradley A, Curr. Opin. Biotech.no!.
2:823-29 (1991); WO 90/11354; WO 91/01140; and WO 93/04169.
In addition to the foregoing, the skilled artisan will appreciate that other
approaches known in the art for homologous recombination can be applied to the
disclosure herein. Enzyme-assisted site-specific integration systems are known
in the art and can be applied to integrate a DNA molecule at a predetermined
location in a second target DNA molecule. Examples of such enzyme-assisted
integration systems include the Cre recombinase-lox target system (e.g., as

described in Baubonis W and Sauer B, Nucleic Acids Res. 21:2025-29 (1993);
and Fukushige S and Sauer B, Proc. Natl. Acad. Sci. USA 89:7905-09 (1992))
and the FLP recombinase-FRT target system (e.g., as described in Dang DT and
Perrimon N, Dev. Genet. 13:367-75 (1992); and Fiering S et al, Proc. Natl. Acad.
Sci. USA 90:8469-73 (1993)). Tetracycline-regulated inducible homologous
recombination systems, such as those described in WO 94/29442 and WO
96/01313, also can be used.
For example, in another embodiment, transgenic non-humans animals can
be produced which contain selected systems which allow for regulated
expression of the transgene. One example of such a system is the cre/loxP
recombinase system of bacteriophage P1. For a description of the cre/loxP
recombinase system, see, e.g., Lakso M et al., Proc. Natl. Acad. Sci. USA
89:6232-36 (1992). Another example of a recombinase system is the FLP
recombinase system of Saccharomyces cerevisiae (O'Gorman S et ai, Science
251:1351-55 (1991)). If a cre/loxP recombinase system is used to regulate
expression of the transgene, animals containing transgenes encoding both the
Cre recombinase and a selected protein are required. Such animals can be
provided through the construction of "double" transgenic animals, for example, by
mating two transgenic animals, one containing a transgene encoding a selected
protein and the other containing a transgene encoding a recombinase.
Clones of the non-human transgenic animals described herein can also be
produced according to the methods described in, for example, Wilmut I et al..
Nature 385:810-13 (1997); WO 97/07668; and WO 97/07669. In brief, a cell, for
example, a somatic cell, from the transgenic animal can be isolated and induced
to exit the growth cycle and enter Go phase. The quiescent cell can then be
fused, for example, through the use of electrical pulses, to an enucleated oocyte
from an animal of the same species from which the quiescent ceil is isolated.
The reconstructed oocyte is then cultured such that it develops to morula or
blastocyte and then transferred to pseudopregnant female foster animal. The
offspring borne of this female foster animal will be a clone of the animal from
which the cell, for example, the somatic cell, is isolated.

V. Uses and Methods of the Invention
Ion channels are excellent targets for drugs. The polynucleotides
disclosed herein encode mutant voltage gated potassium channels, modulators
of which would be useful for identifying compounds for diagnosis, treatment,
prevention or alleviation of diseases related to or adverse conditions of the
central nervous system (CNS) and peripheral systems, including various types of
pain such as, for example, somatic, cutaneous, or visceral pain caused by, for
example burn, bruise, abrasion, laceration, broken bone, torn ligament, torn
tendon, torn muscle, viral, bacterial, protozoal or fungal infection, contact
dermatitis, inflammation (caused by, e.g., trauma, infection, surgery, burns, or
diseases with an inflammatory component), cancer, toothache; neuropathic pain
caused by, for example, injury to the central or peripheral nervous system due to
cancer, HIV (human immunodeficiency virus) infection, tissue trauma, infection,
autoimmune disease, diabetes, arthritis, diabetic neuropathy, trigeminal
neuralgia, or drug administration; treating anxiety caused by, for example, panic
disorder, generalized anxiety disorder, or stress disorder, particularly acute
stress disorder, affective disorders, Alzheimer's disease, ataxia, CNS damage
caused by trauma, stroke or neurodegenerative illness, cognitive deficits,
compulsive behavior, dementia, depression, Huntington's disease, mania,
memory impairment, memory disorders, memory dysfunction, motion disorders,
motor disorders, age-related memory loss, neurodegenerative diseases,
Parkinson's disease and Parkinson-like motor disorders, phobias, Pick's disease,
psychosis, schizophrenia, spinal cord damage, tremor, seizures, convulsions,
epilepsy, Stargardt-like macular dystrophy, cone-rod macular dystrophy, Salla
disease, epilepsy, muscle relaxants, fever reducers, anxiolytics, antimigraine
agents, analgesics, bipolar disorders, unipolar depression, functional bowel
disorders (e.g., dyspepsia and irritable bowl syndrome), diarrhea, constipation,
various types of urinary incontinence (e.g., urge urinary incontinence, stress
urinary incontinence, overflow urinary incontinence or unconscious urinary
incontinence, and mixed urinary incontinence), urinary urgency, bladder
instability, neurogenic bladder, hearing loss, tinnitus, glaucoma, cognitive
disorders, chronic inflammatory and neuralgic pain; for preventing and reducing
drug dependence or tolerance for treatment of, for example, cancer,
inflammation, ophthalmic diseases, and various CNS disorders.

The nucleic acid molecules, proteins, protein homologues, and antibodies
described herein can be used in one or more of the following methods: a)
methods of treatment, preferably in brain, skeletal muscle, or urinary bladder; b)
screening assays; c) predictive medicine (e.g., diagnostic assays, prognostic
assays, monitoring clinical trials, or pharmacogenetics). The isolated nucleic
acid molecules can be used, for example, to express KCNQ5 or KCNQ5(W270L)
protein (e.g., via a recombinant expression vector in a host cell in gene therapy
applications), to detect KCNQ5 or KCNQ5(W270L) mRNA (e.g., in a biological
sample) or a genetic alteration in a KCNQ5 gene, and to modulate KCNQ5 or
KCNQ5(W270L) activity, as described further below. In addition, the KCNQ5 or
KCNQ5(W270L) proteins can be used to screen for naturally occurring KCNQ5
or KCNQ5(W270L) binding proteins, to screen for drugs or compounds which
modulate KCNQ5 or KCNQ5(W270L) activity, as well as to treat disorders that
would benefit from modulation of KCNQ5, for example, characterized by
insufficient or excessive production of KCNQ5 protein or production of KCNQ5
protein forms which have decreased or aberrant activity compared to KCNQ5
wild type protein. Moreover, the anti-KCNQ5 or anti-KCNQ5(W270L) antibodies
can be used to detect and isolate KCNQ5 or KCNQ5(W270L) proteins, regulate
the bioavailability of KCNQ5 or KCNQ5(W270L) proteins, and modulate KCNQ5
activity (for example, reduction of KCNQ5 activity in the brain will increase the
neuronal excitability in the CNS). In preferred embodiments the methods
disclosed herein, for example, detection, modulation of KCNQ5 or
KCNQ5(W270L), etc. are performed in the CNS, skeletal muscle, or urinary
bladder smooth muscle.
A. Methods of Modulating KCNQ5 or KCNQ5(W270L)
One aspect provides for methods of modulating KCNQ5 or
KCNQ5(W270L) in a cell, for example, for the purpose of identifying agents that
modulate KCNQ5 or KCNQ5(W270L) expression and/or activity, as well as both
prophylactic and therapeutic methods of treating a subject at risk of (or
susceptible to) a disorder or having a disorder associated with aberrant KCNQ5
expression or activity or a disorder that would benefit from modulation of KCNQ5
activity.

Yet another aspect pertains to methods of modulating KCNQ5 or
KCNQ5(W270L) expression and/or activity in a cell. The modulatory methods
involve contacting the cell with an agent that modulates KCNQ5 or
KCNQ5(W270L) expression and/or activity such that KCNQ5 or KCNQ5(W270L)
expression and/or activity in the cell is modulated. The agent may act by
modulating the activity of KCNQ5 or KCNQ5(W270L) protein in the cell or by
modulating transcription of the KCNQ5 or KCNQ5(W270L) gene or translation of
the KCNQ5 or KCNQ5(W270L) mRNA.
Accordingly, in one embodiment, the agent inhibits KCNQ5 or
KCNQ5(W270L) activity. An inhibitory agent may function, for example, by
directly inhibiting KCNQ5 or KCNQ5(W270L) activity or by modulating a signaling
pathway which negatively regulates KCNQ5 or KCNQ5(W270L). In another
embodiment, the agent stimulates KCNQ5 or KCNQ5(W270L) activity. A
stimulatory agent may function, for example, by directly stimulating KCNQ5 or
KCNQ5(W270L) activity, or by modulating a signaling pathway that jeads to
stimulation of KCNQ5 or KCNQ5(W270L) activity. Exemplary inhibitory agents
include antisense KCNQ5 or KCNQ5(W270L) nucleic acid molecules (e.g., to
inhibit translation of KCNQ5 or KCNQ5(W270L) mRNA), intracellular anti-KCNQ5
or anti-KCNQ5(W270L) antibodies (e.g., to inhibit the activity of KCNQ5 or
KCNQ5(W270L) protein), and dominant negative mutants of the KCNQ5 or
KCNQ5(W270L) protein. Other inhibitory agents that can be used to inhibit the
activity of a KCNQ5 or KCNQ5(W270L) protein are chemical compounds that
inhibit KCNQ5 or KCNQ5(W270L) activity. Such compounds can be identified
using screening assays that select for such compounds, as described herein.
Additionally or alternatively, compounds that inhibit KCNQ5 or KCNQ5(W270L)
activity can be designed using approaches known in the art.
According to another modulatory method, KCNQ5 or KCNQ5(W270L)
activity is stimulated in a cell by contacting the cell with a stimulatory agent.
Examples of such stimulatory agents include active KCNQ5 or KCNQ5(W270L)
protein and nucleic acid molecules encoding KCNQ5 or KCNQ5(W270L) that are
introduced into the cell to increase KCNQ5 or KCNQ5(W270L) activity in the cell.
A preferred stimulatory agent is a nucleic acid molecule encoding a KCNQ5 or
KCNQ5(W270L) protein, wherein the nucleic acid molecule is introduced into the
cell in a form suitable for expression of the active KCNQ5 or KCNQ5(W270L)

protein in the cell. To express a KCNQ5 or KCNQ5(W270L) protein in a cell,
typically a KCNQ5 or KCNQ5(W270L) cDNA is first introduced into a
recombinant expression vector using standard molecular biology techniques, as
described herein. A KCNQ5 or KCNQ5(W270L) cDNA can be obtained, for
example, by amplification using the PCR or by screening an appropriate cDNA
library as described herein. Following isolation or amplification of KCNQ5 or
KCNQ5(W270L) cDNA, the DNA fragment is introduced into an expression
vector and transfected into target cells by standard methods, as described
herein. Other stimulatory agents that can be used to stimulate the activity and/or
expression of a KCNQ5 or KCNQ5(W270L) protein are chemical compounds that
stimulate KCNQ5 activity and/or expression in cells, such as compounds that
enhance KCNQ5 activity. Such compounds can be identified using screening
assays that select for such compounds, as described in detail herein.
The modulatory methods can be performed in vitro (e.g., by culturing the
cell with the agent or by introducing the agent into cells in culture) or,
alternatively, in vivo (e.g., by administering the agent to a subject or by
introducing the agent into cells of a subject, such as by gene therapy). For
practicing the modulatory method in vitro, cells can be obtained from a subject by
standard methods and incubated (i.e., cultured) in vitro with a modulatory agent
to modulate KCNQ5 or KCNQ5(W270L) activity in the cells.
For stimulatory or inhibitory agents that comprise nucleic acids (including
recombinant expression vectors encoding KCNQ5 or KCNQ5(W270L) protein,
antisense RNA, intracellular antibodies, or dominant negative inhibitors), the
agents can be introduced into cells of the subject using methods known in the art
for introducing nucleic acid (e.g., DNA) into cells in vivo. Examples of such
methods encompass both non-viral and viral methods, including:
Direct Injection: Naked DNA can be introduced into cells in vivo by directly
injecting the DNA into the cells (see, e.g., Acsadi G et a/., Nature 332:815-18
(1991); Wolff JA et al., Science 247:1465-68 (1990)). For example, a delivery
apparatus (e.g., a "gene gun") for injecting DNA into cells in vivo can be used.
Such an apparatus is commercially available (e.g., from Bio-Rad Laboratories,
Hercules, Calif.).
Cationic Lipids: Naked DNA can be introduced into cells in vivo by
complexing the DNA with cationic lipids or encapsulating the DNA in cationic

WO 2007/084531 PCT/US2007/001188
liposomes. Examples of suitable cationic lipid formulations include N-[-1-(2,3-
dioleoyloxy)propyl]N,N,N-triethylammonium chloride (DOTMA) and a 1:1 molar
ratio of 1,2-dimyristyloxy-propyl-3-dimethylhydroxyethylammonium bromide
(DMRIE) and dioleoyl phosphatidylethanolamine (DOPE) (see e.g., Logan JJ et
al., Gene Ther. 2:38-49 (1995); San H et al., Hum. Gene Ther. 4:781-88 (1993)).
Receptor-Mediated DNA Uptake: Naked DNA can also be introduced into
cells in vivo by complexing the DNA to a cation, such as polylysine, which is
coupled to a ligand for a cell-surface receptor (see, e.g., Wu GY and Wu CH, J.
Biol. Chem. 263:14621-24 (1988); Wilson JM et al., J. Biol. Chem. 267:963-67
(1992); and U.S. Patent No. 5,166,320). Binding of the DNA-ligand complex to
the receptor facilitates uptake of the DNA by receptor-mediated endocytosis. A
DNA-ligand complex linked to adenovirus capsids which naturally disrupt
endosomes, thereby releasing material into the cytoplasm can be used to avoid
degradation of the complex by intracellular lysosomes (see, e.g., Curiel DT et al.,
Proc. Natl. Acad. Sci. USA 88:8850-54 (1991); Cristiano RJ et al., Proc. Natl.
Acad. Sci. USA 90:2122-26 (1993)).
Retroviruses: Defective retroviruses are well characterized for use in gene
transfer for gene therapy purposes (for a review, see Miller AD, Blood 76:271-78
(1990)). A recombinant retrovirus can be constructed having a nucleotide
sequence of interest incorporated into the retroviral genome. Additionally,
portions of the retroviral genome can be removed to render the retrovirus
replication defective. The replication defective retrovirus is then packaged into
virions which can be used to infect a target cell through the use of a helper virus
by standard techniques. Protocols for producing recombinant retroviruses and
for infecting cells in vitro or in vivo with such viruses can be found in Current
Protocols in Molecular Biology, Ausubel FM et al. (eds.) Greene Publishing
Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals.
Examples of suitable retroviruses include pLJ, pZIP, pWE, and pEM which are
well known to those skilled in the art. Examples of suitable packaging virus lines
include Crip, Cre, 2, and Am. Retroviruses have been used to introduce a
variety of genes into many different cell types, including epithelial cells,
endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in
vitro and/or in vivo (see, e.g., Eglitis MA et al.. Science 230:1395-98 (1985);
Danos O and Mulligan RC, Proc. Natl. Acad. Sci. USA 85:6460-64 (1988);

Wilson JM et al, Proc. Natl. Acad. Sci. USA 85:3014-18 (1988); Armentano D et
ai, Proc. Natl. Acad. Sci. USA 87:6141-45 (1990); Huber BE etai, Proc. Natl.
Acad. Sci. USA 88:8039-43 (1991); Ferry N et al., Proc. Natl. Acad. Sci. USA
88:8377-81 (1991); Chowdhury JR et al., Science 254:1802-05 (1991); van
Beusechem VW et al, Proc. Natl. Acad. Sci. USA 89:7640-44 (1992); Kay MA et
ai, Hum. GeneTher. 3:641-47 (1992); Dai Y et al, Proc. Natl. Acad. Sci. USA
89:10892-95 (1992); Hwu P et al, J. Immunol. 150:4104-15 (1993); U.S. Patent
No. 4,868,116; U.S. Patent No. 4,980,286; WO 89/07136; WO 89/02468; WO
89/05345; and WO 92/07573). Retroviral vectors require target cell division in
order for the retroviral genome (and foreign nucleic acid inserted into it) to be
integrated into the host genome to stably introduce nucleic acid into the cell.
Thus, it may be necessary to stimulate replication of the target cell.
Adenoviruses: The genome of an adenovirus can be manipulated such
that it encodes and expresses a gene product of interest but is inactivated in
terms of its ability to replicate in a normal lytic viral life cycle (see, e.g., Berkner
KL, Biotechniques 6:616-29 (1988); Rosenfeld MA et ai, Science 252:431-34
(1991); and Rosenfeld MA et al, Cell 68:143-55 (1992)). Suitable adenoviral
vectors derived from the adenovirus strain Ad type 5 d1324 or other strains of
adenovirus (e.g., Ad2, Ad3, Ad7, etc.) are well known to those skilled in the art.
Recombinant adenoviruses are advantageous in that they do not require dividing
cells to be effective gene delivery vehicles and can be used to infect a wide
variety of cell types, including airway epithelium (Rosenfeld MA et al, Cell
68:143-55 (1992)), endothelial cells (Lemarchand P et al, Proc. Natl. Acad. Sci.
USA 89:6482-86 (1992)), hepatocytes (Herz J and Gerard RD, Proc. Natl. Acad.
Sci. USA 90:2812-16 (1993)), and muscle cells (Quantin B et al, Proc. Natl.
Acad. Sci. USA 89:2581-84 (1992)). Additionally, introduced adenoviral DNA
(and foreign DNA contained therein) is not integrated into the genome of a host
cell but remains episomal, thereby avoiding potential problems that can occur as
a result of insertional mutagenesis in situations where introduced DNA becomes
integrated into the host genome (e.g., retroviral DNA). Moreover, the carrying
capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases)
relative to other gene delivery vectors (Berkner KL et ai, supra; Haj-Ahmad Y
and Graham FL, J. Virol. 57:267-74 (1986)). Most replication-defective

adenoviral vectors currently in use are deleted for all or parts of the viral E1 and
E3 genes but retain as much as 80% of the adenoviral genetic material.
Adeno-Associated Viruses: Adeno-associated virus (AAV) is a naturally
occurring defective virus that requires another virus, such as an adenovirus or a
herpes virus, as a helper virus for efficient replication and a productive life cycle
(for a review, see Muzyczka N, Curr. Top. Microbiol. Immunol. 158:97-129
(1992)). It is also one of the few viruses that may integrate its DNA into non-
dividing cells, and exhibits a high frequency of stable integration (see, e.g., Flotte
TR et al., Am. J. Respir. Cell. Mol. Biol. 7:349-56 (1992); Samulski RJ et al., J.
Virol. 63:3822-28 (1989); and McLaughlin SK et al., J. Virol. 62:1963-73 (1988)).
Vectors containing as little as 300 base pairs of AAV can be packaged and can
integrate. Space for exogenous DNA is limited to about 4.5 kb. An AAV vector
such as that described in Tratschin JD ef at., Mol. Cell. Biol. 5:3251-60 (1985),
can be used to introduce DNA into cells. A variety of nucleic acids have been
introduced into different cell types using AAV vectors (see, e.g., Hermonat PL
and Muzyczka N, Proc. Natl. Acad. Sci. USA 81:6466-70 (1984); Tratschin JD et
a/., Mol. Cell. Biol. 4:2072-81 (1985); Wondisford FE et al, Mol. Endocrinol. 2:32-
39 (1988); Tratschin JD et al., J. Virol. 51:611-19 (1984); and Flotte TR et al., J.
Biol. Chem. 268:3781-90 (1993)).
The efficacy of a particular expression vector system and method of
introducing nucleic acid into a cell can be assessed by standard approaches
routinely used in the art. For example, DNA introduced into a cell can be
detected by a filter hybridization technique (e.g., Southern blotting) and RNA
produced by transcription of introduced DNA can be detected, for example, by
Northern blotting, RNase protection, or reverse transcriptase-polymerase chain
reaction (RT-PCR). The gene product can be detected by an appropriate assay,
for example by immunological detection of a produced protein, such as with a
specific antibody, or by a functional assay to detect a functional activity of the
gene product.
There are a variety of pathological conditions for which KCNQ5 or
KCNQ5(W270L) modulating agents can be used in treatment (see, e.g., section
V, herein).

1. Prophylactic Methods
One aspect provides for a method for preventing in a subject, a disease or
condition that would benefit from modulation of KCNQ5 activity and/or
expression, e.g., a disorder associated with an aberrant KCNQ5 expression or
activity (such as, e.g., urinary incontinence and neuropathic pain), by
administering to the subject a KCNQ5 or KCNQ5(W270L) polypeptide or an
agent which modulates KCNQ5 polypeptide expression or at least one KCNQ5
activity. In one aspect, the KCNQ5 polypeptide can contain an S5-S6
transmembrane domain from KCNQ1. Alternatively, the KCNQ5 polypeptide can
contain an S5 transmembrane domain from KCNQ1. Subjects at risk for a
disease which is caused or contributed to by aberrant KCNQ5 expression or
activity can be identified by, for example, any or a combination of diagnostic or
prognostic assays as described herein. Administration of a prophylactic agent
can occur prior to the manifestation of symptoms characteristic of KCNQ5
aberrance, such that a disease or disorder is prevented or, alternatively, delayed
in its progression. Depending on the type of KCNQ5 aberrance or condition, for
example, a KCNQ5 or KCNQ5(W270L) polypeptide, KCNQ5 or KCNQ5(W270L)
agonist, or KCNQ5 or KCNQ5(W270L) antagonist agent can be used for treating
the subject. The appropriate agent can be determined based on screening
assays described herein.
2. Therapeutic Methods
Another aspect pertains to methods of modulating KCNQ5 expression or
activity for therapeutic purposes. Accordingly, in an exemplary embodiment, the
modulatory method involves contacting a cell with a KCNQ5(W270L) polypeptide
or agent that modulates one or more of the activities of KCNQ5 protein
associated with the ceil. An agent that modulates KCNQ5 protein activity can be
an agent as described herein, such as a nucleic acid or a protein, a naturally-
occurring target molecule of a KCNQ5 protein (e.g., a KCNQ5 binding protein), a
KCNQ5 or KCNQ5(W270L) antibody, a KCNQ5 or KCNQ5(W270L) agonist or
antagonist, a peptidomimetic of a KCNQ5 or KCNQ5(W270L) agonist or
antagonist, or other small molecule. In one embodiment, the agent stimulates
one or more KCNQ5 activities. Examples of such stimulatory agents include
active KCNQ5 or KCNQ5(W270L) protein and a nucleic acid molecule encoding

KCNQ5 or KCNQ5(W270L) polypeptide that has been introduced into the cell. In
another embodiment, the agent inhibits one or more KCNQ5 activities.
Examples of such inhibitory agents include antisense KCNQ5 or KCNQ5(W270L)
nucleic acid molecules, anti-KCNQ5 or anti-KCNQ5(W270L) antibodies, and
KCNQ5 or KCNQ5(W270L) inhibitors. These modulatory methods can be
performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in
vivo (e.g., by administering the agent to a subject). As such, a further aspect
provides methods of treating an individual afflicted with a disease or disorder that
would benefit from modulation of a KCNQ5 protein (e.g., as described in section
V, herein), or which is characterized by aberrant expression or activity of a
KCNQ5 protein or nucleic acid molecule. In one embodiment, the method
involves administering an agent (e.g., an agent identified by a screening assay
described herein), or combination of agents that modulates (e.g., upregulates or
downregulates) KCNQ5 expression or activity. In another embodiment, the
method involves administering a KCNQ5 or KCNQ5(W270L) protein or nucleic
acid molecule as therapy to compensate for reduced or aberrant KCNQ5
expression or activity.
Stimulation of KCNQ5 activity is desirable in situations in which KCNQ5 is
abnormally downregulated and/or in which increased KCNQ5 activity is likely to
have a beneficial effect. Likewise, inhibition of KCNQ5 activity is desirable in
situations in which KCNQ5 is abnormally upregulated and/or in which decreased
KCNQ5 activity is likely to have a beneficial effect. Exemplary situations in which
KCNQ5 modulation will be desirable are in the treatment of KCNQ5 associated
disorders (see, e.g., section V, herein).
Agents identified by methods disclosed herein are useful for inducing,
assisting or maintaining desirable bladder control in a mammal experiencing or
susceptible to bladder instability or urinary incontinence. These methods include
prevention, treatment or inhibition of bladder-related urinary conditions and
bladder instability, including idiopathic bladder instability, nocturnal enuresis,
nocturia, voiding dysfunction and urinary incontinence. Also treatable or
preventable with the methods of this invention is bladder instability secondary to
prostate hypertrophy. The agents identified by methods disclosed herein are
also useful in promoting the temporary delay of urination whenever desirable.
The agents may also be utilized to stabilize the bladder and treat or prevent

incontinence which urge urinary incontinence, stress urinary incontinence or a
combination of urge and stress incontinence in a mammal, which may also be
referred to as mixed urge and stress incontinence. These methods include
assistance in preventing or treating urinary incontinence associated with
secondary conditions such as prostate hypertrophy.
These methods may be utilized to allow a recipient to control the urgency
and frequency of urination. The methods of this invention include the treatment,
prevention, inhibition and amelioration of urge urinary incontinence also known
as bladder instability, neurogenic bladder, voiding dysfunction, hyperactive
bladder, detrusor overactivity, detrusor hyper-reflexia or uninhibited bladder.
As described above, useful methods include treatments, prevention,
inhibition or amelioration of hyperactive or unstable bladder, neurogenic bladder,
sensory bladder urgency, or hyperreflexic bladder. These uses include, but are
not limited to, those for bladder activities and instabilities in which the urinary
urgency is associated with prostatitis, prostatic hypertrophy, interstitial cystitis,
urinary tract infections or vaginitis. The agents may also be used to assist in
inhibition or correction of the conditions of Frequency-Urgency Syndrome, and
lazy bladder, also known as infrequent voiding syndrome.
The agents may also be used to treat, prevent, inhibit, or limit the urinary
incontinence, urinary instability or urinary urgency associated with or resulting
from administrations of other medications, including diuretics, vasopressin
antagonists, anticholinergic agents, sedatives or hypnotic agents, narcotics,
alpha-adrenergic agonists, alpha-adrenergic antagonists, or calcium channel
blockers.
The agents identified by methods disclosed herein can be useful for
inducing or assisting in urinary bladder control or preventing or treating the
maladies described herein in humans in need of such relief, including adult and
pediatric uses. However, they may also be utilized for veterinary applications,
particularly including canine and feline bladder control methods. If desired, the
methods herein may also be used with farm animals, such as ovine, bovine,
porcine and equine breeds.

B. Screening Assays:
One aspect provides a method (also referred to herein as a "screening
assay") for identifying modulators, that is, candidate or test compounds or agents
(e.g., peptides, peptidomimetics, small molecules, or other drugs) which bind to
KCNQ5 or KCNQ5(W270L) proteins, have a stimulatory or inhibitory effect on, for
example, KCNQ5 or KCNQ5(W270L) expression or KCNQ5 or KCNQ5(W270L)
activity.
The test compounds can be obtained using any of the numerous
approaches in combinatorial library methods known in the art, including:
biological libraries; spatially addressable parallel solid phase or solution phase
libraries; synthetic library methods requiring deconvolution; the "one-bead one-
compound" library method; and synthetic library methods using affinity
chromatography selection. The biological library approach is limited to peptide
libraries, while the other four approaches are applicable to peptide, non-peptide
oligomer, or small molecule libraries of compounds (Lam KS, Anticancer Drug
Des. 12:145-67(1997)).
Examples of methods for the synthesis of molecular libraries can be found
in the art, for example in: DeWitt SH et al., Proc. Natl. Acad. Sci. USA 90:6909-
13 (1993); Erb E et al., Proc. Natl. Acad. Sci. USA 91:11422-26 (1994);
Zuckermann RN et al., J. Med. Chem. 37:2678-85 (1994); Cho CYet al, Science
261:1303-05 (1993); Carrell T et al., Angew. Chem. Int. Ed. Engl. 33:2059-61
(1994); Carrell T et al., Angew. Chem. Int. Ed. Engl. 33:2061-64 (1994); and
Gallop MA et al., J. Med. Chem. 37:1233-51 (1994).
Libraries of compounds may be presented, for example, in solution (e.g.,
Houghten RA etal., Biotechniques 13:412-21 (1992)), or on beads (Lam KS et
ai, Nature 354:82-84 (1991)), chips (Fodor SPA et al., Nature 364:555-56
(1993)), bacteria (U.S. Patent No. 5,223,409), spores (U.S. Patent No.
5,223,409), plasmids (Cull MG et al., Proc. Natl. Acad. Sci. USA 89:1865-69
(1992)), or on phage (Scott JK and Smith GP, Science 249:386-90 (1990); Devlin
JJ etal., Science 249:404-06 (1990); Cwirla SE et al., Proc. Natl. Acad. Sci.
87:6378-82 (1990); Felici F et al., J. Mol. Biol. 222:301-10 (1991); U.S. Patent
No. 5,223,409).
In many drug screening programs which test libraries of modulating
agents and natural extracts, high throughput assays are desirable in order to

maximize the number of modulating agents surveyed in a given period of time.
Assays which are performed in cell-free systems, such as may be derived with
purified or semi-purified proteins, are often preferred as "primary" screens in that
they can be generated to permit rapid development and relatively easy detection
of an alteration in a molecular target which is mediated by a test modulating
agent. Moreover, the effects of cellular toxicity and/or bioavailability of the test
modulating agent can be generally ignored in the in vitro system, the assay
instead being focused primarily on the effect of the drug on the molecular target
as may be manifest in an alteration of binding affinity with upstream or
downstream elements.
In one aspect, an agent is screened for by contacting the agent with a
KCNQ5 molecule and detecting the effect of the agent on KCNQ5 activity.
Detection of an increase or a decrease in KCNQ5 activity is indicative of an
agent being a modulator of KCNQ5. The KCNQ5 molecule can be a
polynucleotide encoding all or a portion of a KCNQ5(W270L) polypeptide, a
polynucleotide encoding a KCNQ5 polypeptide containing an S5-S6
transmembrane domain from KCNQ1, or a polynucleotide encoding a KCNQ5
polypeptide containing an S5 transmembrane domain from KCNQ1.
Alternatively, the KCNQ5 molecule can be a polypeptide comprising an amino
acid sequence of a KCNQ5(W270L) polypeptide, a KCNQ5 polypeptide
containing an S5-S6 transmembrane domain from KCNQ1, or a KCNQ5
polypeptide containing an S5 transmembrane domain from KCNQ1.
In another aspect, an agent is screened for by contacting a cell with an
agent and determining the level of expression of a KCNQ5 molecule. Detection
of a decrease or an increase in KCNQ5 expression is indicative of an agent
being a modulator of KCNQ5. The KCNQ5 molecule can be a polynucleotide
encoding all or a portion of a KCNQ5(W270L) polypeptide, a polynucieotide
encoding a KCNQ5 polypeptide containing an S5-S6 transmembrane domain
from KCNQ1, or a polynucleotide encoding a KCNQ5 polypeptide containing an
S5 transmembrane domain from KCNQ1. Alternatively, the KCNQ5 molecule
can be a polypeptide comprising an amino acid sequence of a KCNQ5(W270L)
polypeptide, a KCNQ5 polypeptfde containing an S5-S6 transmembrane domain
from KCNQ1, or a KCNQ5 polypeptide containing an S5 transmembrane domain
from KCNQL

One embodiment provides assays for screening candidate or test
compounds which bind to or modulate the activity of a KCNQ5 protein or
polypeptide or biologically active portion thereof, for example, modulate the
ability of KCNQ5 polypeptide to reduce neuronal excitability for anxiety and/or
neuropathic pain, or to "quiet down" bladder smooth muscle activity for urinary
incontinence. By "quiet down" is meant to suppress abnormal contractions of
bladder smooth muscle cells in incontinence patients.
Assays can be used to screen for modulating agents, including KCNQ5 or
KCNQ5(W270L) homologs, which are either agonists or antagonists of the
normal cellular function of the subject KCNQ5 or KCNQ5(W270L) polypeptides.
For example, one aspect provides a method in which an indicator composition is
provided which has a KCNQ5 or KCNQ5(W270L) protein having a KCNQ5
activity. The indicator composition can be contacted with a test compound. The
effect of the test compound on KCNQ5 activity, as measured by a change in the
indicator composition, can then be determined to thereby identify a compound
that modulates the activity of a KCNQ5 protein. A statistically significant change,
such as a decrease or increase, in the level of KCNQ5 activity in the presence of
the test compound (relative to what is detected in the absence of the test
compound) is indicative of the test compound being a KCNQ5 modulating agent.
The indicator composition can be, for example, a cell or a cell extract.
The efficacy of the modulating agent can be assessed by generating dose
response curves from data obtained using various concentrations of the test
modulating agent. Moreover, a control assay can also be performed to provide a
baseline for comparison. In the control assay, isolated and purified KCNQ5 or
KCNQ5(W270L) protein is added to a composition containing a KCNQ5-binding
element, and the formation of a complex is quantitated in the absence of the test
modulating agent.
In yet another embodiment, an assay is a cell-free assay in which a
KCNQ5 or KCNQ5(W270L) protein or biologically active portion thereof is
contacted with a test compound and the ability of the test compound to bind to
the KCNQ5 or KCNQ5(W270L) protein or biologically active portion thereof is
determined. Binding of the test compound to the KCNQ5 or KCNQ5(W270L)
protein can be determined either directly or indirectly as described above. In one
embodiment, the assay includes contacting the KCNQ5 or KCNQ5(W270L)

protein or biologically active portion thereof with a known compound which binds
wild-type KCNQ5 to form an assay mixture, contacting the assay mixture with a
test compound, and determining the ability of the test compound to interact with a
KCNQ5 or KCNQ5(W270L) protein, wherein determining the ability of the test
compound to interact with a KCNQ5 or KCNQ5(W270L) protein comprises
determining the ability of the test compound to preferentially bind to KCNQ5 or
KCNQ5(W270l_) polypeptide or biologically active portion thereof as compared to
the known compound.
The KCNQ5 or KCNQ5(W270L) protein can be provided as a lysate of
cells that express KCNQ5 or KCNQ5(W270L), as a purified or semipurified
polypeptide, or as a recombinantly expressed polypeptide. In one embodiment,
a cell-free assay system further comprises a cell extract or isolated components
of a cell, such as mitochondria. Such cellular components can be isolated using
techniques which are known in the art. Preferably, a cell free assay system
further comprises at least one target molecule with which KCNQ5 or
KCNQ5(W270L) interacts, and the ability of the test compound to modulate the
interaction of the KCNQ5 or KCNQ5(W270L) with the target molecule(s) is
monitored to thereby identify the test compound as a modulator of KCNQ5 or
KCNQ5(W270L) activity. Determining the ability of the test compound to
modulate the activity of a KCNQ5 or KCNQ5(W270L) protein can be
accomplished, for example, by determining the ability of the KCNQ5 or
KCNQ5(W270L) protein to bind to a KCNQ5 or KCNQ5(W270L) target molecule
by one of the methods described above for determining direct binding.
Determining the ability of the KCNQ5(W270L) protein to bind to a
KCNQ5(W270L) target molecule can also be accomplished using a technology
such as real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander S
and Urbaniczky C, Anal. Chern. 63:2333-45 (1991) and Szabo A et ai, Curr.
Opin. Struct. Biof. 5:699-705 (1995)). As used herein, "BIA" is a technology for
studying biospecific interactions in real time, without labeling any of the
interactants (e.g., BIAcore). Changes in the optical phenomenon of surface
plasmon resonance (SPR) can be used as an indication of real-time reactions
between biological molecules.
In yet another embodiment, the cell-free assay involves contacting a
KCNQ5 or KCNQ5(W270L) protein or biologically active portion thereof with a

known compound which binds the wild-type KCNQ5 protein to form an assay
mixture, contacting the assay mixture with a test compound, and determining the
ability of the test compound to interact with the KCNQ5 or KCNQ5(W270L)
protein, wherein determining the ability of the test compound to interact with the
KCNQ5 or KCNQ5(W270L) protein comprises determining the ability of the
KCNQ5 or KCNQ5(W270L) protein to preferentially bind to or modulate the
activity of a KCNQ5 or KCNQ5(W270L) target molecule.
The cell-free assays are amenable to use of both soluble and/or
membrane-bound forms of proteins. In the case of cell-free assays in which a
membrane-bound form a protein is used (e.g., KCNQ5 or KCNQ5(W270L)
proteins) it may be desirable to utilize a solubilizing agent such that the
membrane-bound form of the protein is maintained in solution. Examples of such
solubilizing agents include non-ionic detergents such as n-octylglucoside, n-
dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-
methylglucamide, Triton® X-100, Triton® X-114, Thesit®, lsotridecypoly(ethylene
glycol ether)n, 3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate
(CHAPS), 3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane
sulfonate (CHAPSO), or N-dodecyl=N,N-dimethyi-3-ammonio-1 -propane
sulfonate.
A KCNQ5 or KCNQ5(W270L) target molecule can be, for example, a
protein. Suitable assays are known in the art that allow for the detection of
protein-protein interactions (e.g., immunoprecipitations, two-hybrid assays, and
the like). By performing such assays in the presence and absence of test
compounds, these assays can be used to identify compounds that modulate
(e.g., inhibit or enhance) the interaction of KCNQ5 or KCNQ5(W270L) with a
target molecule(s).
Determining the ability of the KGNQ5 or KCNQ5(W270L) protein to bind to
or interact with a ligand of a KCNQ5 or KCNQ5(W270L) molecule can be
accomplished, for example, by direct binding. In a direct binding assay, the
KCNQ5 or KCNQ5(W270L) protein could be coupled with a radioisotope or
enzymatic label such that binding of the KCNQ5 or KCNQ5(W270L) protein to a
KCNQ5 or KCNQ5(W270L) target molecule can be determined by detecting the
labeled KCNQ5 or KCNQ5(W270L) protein in a complex. For example, KCNQ5
or KCNQ5(W270L) molecules, for example, KCNQ5 or KCNQ5(W270L) proteins,

can be labeled with, for example, 125I, 35S, 14C, 32P, or 3H, either directly or
indirectly, and the radioisotope detected by direct counting of radioemmission or
by scintillation counting. Alternatively, KCNQ5 or KCNQ5(W270L) molecules can
be enzymatically labeled with, for example, horseradish peroxidase, alkaline
phosphatase, or luciferase, and the enzymatic label detected by determination of
conversion of an appropriate substrate to product.
Typically, it will be desirable to immobilize KCNQ5 or KCNQ5(W270L) or
its binding proteins to facilitate separation of complexes from uncomplexed forms
of one or both of the proteins, as well as to accommodate automation of the
assay. Binding of KCNQ5 or KCNQ5(W270L) to an upstream or downstream
binding element, in the presence and absence of a candidate agent, can be
accomplished in any vessel suitable for containing the reactants. Examples
include microtiter plates, test tubes, and micro-centrifuge tubes. In one
embodiment, a fusion protein can be provided which adds a domain that allows
the protein to be bound to a matrix. For example, glutathione-S-
transferase/KCNQ5(W270L) (GST/ KCNQ5(W270L)) fusion proteins can be
adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or
glutathione derivatized microtiter plates, which are then combined with the cell
lysates and the test modulating agent, and the mixture incubated under
conditions conducive to complex formation, for example, at physiological
conditions for salt and pH, though slightly more stringent conditions may be
desired. Following incubation, the beads are washed to remove any unbound
label, and the matrix immobilized and radiolabel determined directly (e.g., beads
placed in scintillant), or in the supernatant after the complexes are subsequently
dissociated. Alternatively, the complexes can be dissociated from the matrix,
separated by SDS-PAGE, and the level of KCNQ5(W270L)-binding protein found
in the bead fraction quantitated from the gel using standard electrophoretic
techniques.
Other techniques for immobilizing proteins on matrices are also available
for use in the subject assay. For instance, KCNQ5(W270L) or its cognate
binding protein can be immobilized utilizing conjugation of biotin and streptavidin.
For instance, biotinylated KCNQ5(W270L) molecules can be prepared from
biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g.,
biotinylation kit, Pierce Biotechnology, Rockford, III.), and immobilized in the wells

of streptavidin-coated 96 well plates (Pierce Biotechnology). Alternatively,
antibodies reactive with KCNQ5(W270L) but which do not interfere with binding
of upstream or downstream elements can be derivatized to the wells of the plate,
and KCNQ5(W270L) trapped in the wells by antibody conjugation. As above,
preparations of a KCNQ5(W270L)-binding protein (KCNQ5(W270L)-BP) and a
test modulating agent are incubated in the KCNQ5(W270L)-presenting wells of
the plate, and the amount of complex trapped in the well can be quantitated.
Exemplary methods for detecting such complexes, in addition to those described
above for the GST-immobilized complexes, include immunodetection of
complexes using antibodies reactive with the KCNQ5(W270L) binding element,
or which are reactive with KCNQ5(W270L) protein and compete with the binding
element; as well as enzyme-linked assays which rely on detecting an enzymatic
activity associated with the binding element, either intrinsic or extrinsic activity.
In the instance of the latter, the enzyme can be chemically conjugated or
provided as a fusion protein with the KCNQ5(W270L) binding protein. To
illustrate, the KCNQ5(W270L) binding protein can be chemically cross-linked or
genetically fused with horseradish peroxidase, and the amount of protein trapped
in the complex can be assessed with a chromogenic substrate of the enzyme, for
example, 3,3'-diamino-benzadine terahydrochloride or 4-chloro-1-napthol.
Likewise, a fusion protein comprising the protein and glutathione-S-transferase
can be provided, and complex formation quantitated by detecting the GST
activity using 1-chloro-2,4-dinitrobenzene (Habig WH et al, J. Biol. Chem.
249:7130-39(1974)).
For processes which rely on immunodetection for quantitating one of the
proteins trapped in the complex, antibodies against the protein, such as anti-
KCNQ5 or anti-KCNQ5(W270L) antibodies, can be used. Alternatively, the
protein to be detected in the complex can be "epitope tagged" in the form of a
fusion protein which includes, in addition to the KCNQ5 or KCNQ5(W270L)
sequence, a second protein for which antibodies are readily available (e.g. from
commercial sources). For instance, the GST fusion proteins described above
can also be used for quantification of binding using antibodies against the GST
moiety. Other useful epitope tags include myc-epitopes (see, e.g., Ellison MJ
and Hochstrasser M, J. Biol. Chem. 266:21150-57 (1991)) which includes a 10-

residue sequence from c-myc, as well as the pFLAG® system (StgmaAldrich, St.
Louis, Mo.) or the pEZZ-protein A system (GE Healthcare, Piscataway, NJ).
It is also within the scope of the present disclosure to determine the ability
of a compound to modulate the interaction between KCNQ5 or KCNQ5(W270L)
and their respective target molecules without the labeling of any of the
interactants. For example, a microphysiometer can be used to detect the
interaction of KCNQ5 or KCNQ5(W270L) with their respective target molecules
without the labeling of KCNQ5, KCNQ5(W270L), or the target molecules (see,
e.g., McConnell HM et al., Science 257:1906-12 (1992)). As used herein, a
"microphysiometer" (e.g., Cytosensor) is an analytical instrument that measures
the rate at which a cell acidifies its environment using a light-addressable
potentiometric sensor (LAPS). Changes in this acidification rate can be used as
an indicator of the interaction between compound and receptor.
In addition to cell-free assays, the disclosure herein of KCNQ5 and
KCNQ5(W270L) proteins facilitates the generation of cell-based assays for
identifying small molecule agonists/antagonists and the like. For example, cells
can be caused to express or overexpress a recombinant KCNQ5 or
KCNQ5(W270L) protein in the presence and absence of a test modulating agent
of interest, with the assay scoring for modulation in KCNQ5 or KCNQ5(W270L)
responses by the target cell mediated by the test agent. For example, as with
the cell-free assays, modulating agents which produce a statistically significant
change in KCNQ5- or KCNQ5(W270L)-dependent responses (either an increase
or decrease) can be identified.
Recombinant expression vectors that can be used for expression of
KCNQ5 or KCNQ5(W270L) are known in the art (see discussion above). In one
embodiment, within the expression vector, the KCNQ5 or KCNQ5(W270L)
coding sequences are operably linked to regulatory sequences that allow for
constitutive or inducible expression of KCNQ5 or KCNQ5(W270L) in the indicator
cell(s). Use of a recombinant expression vector that allows for constitutive or
inducible expression of KCNQ5 or KCNQ5(W270L) in a cell is preferred for
identification of compounds that enhance or inhibit the activity of KCNQ5 or
KCNQ5(W270L). In an alternate embodiment, within the expression vector, the
KCNQ5 or KCNQ5(W270L) coding sequences are operably linked to regulatory
sequences of the endogenous KCNQ5 gene (i.e., the promoter regulatory region

derived from the endogenous gene). Use of a recombinant expression vector in
which KCNQ5 or KCNQ5(W270L) expression is controlled by the endogenous
regulatory sequences is preferred. In one embodiment, an assay is a cell-based
assay comprising contacting a cell expressing a KCNQ5 or KCNQ5(W270L)
target molecule (e.g., a KCNQ5 intracellular interacting molecule) with a test
compound and determining the ability of the test compound to modulate (e.g.
stimulate or inhibit) the activity of the KCNQ5 or KCNQ5(W270L) target
molecule. Determining the ability of the test compound to modulate the activity of
a KCNQ5 or KCNQ5(W270L) target molecule can be accomplished, for example,
by determining the ability of the KCNQ5 or KCNQ5(W270L) protein to bind to or
interact with the KCNQ5 or KCNQ5(W270L) target molecule or its ligand.
In an illustrative embodiment, the expression or activity of KCNQ5(W270L)
is modulated in cells and the effects of modulating agents of interest on the
readout of interest can be measured (such as, for example, the ion current
magnitude can be measured electrophysiologically from Xenopus laevis oocytes
expressing the KCNQ5(W270L) channels).
In another embodiment, modulators of KCNQ5 or KCNQ5(W270L)
expression are identified in a method wherein a cell is contacted with a candidate
compound and the expression of KCNQ5 or KCNQ5(W270L) mRNA or protein in
the cell is determined. The level of expression of KCNQ5 or KCNQ5(W270L)
mRNA or protein in the presence of the candidate compound is compared to the
level of expression of KCNQ5 or KCNQ5(W270L) mRNA or protein in the
absence of the candidate compound. The candidate compound can then be
identified as a modulator of KCNQ5 or KCNQ5(W270L) expression based on this
comparison. For example, when expression of KCNQ5(W270L) mRNA or
protein is greater (e.g., statistically significantly greater) in the presence of the
candidate compound than in its absence, the candidate compound is identified
as a stimulator of KCNQ5(W270L) mRNA or protein expression. Alternatively,
when expression of KCNQ5(W270L) mRNA or protein is less (e.g., statistically
significantly less) in the presence of the candidate compound than in its absence,
the candidate compound is identified as an inhibitor of KCNQ5(W270L) mRNA or
protein expression. The level of KCNQ5 or KCNQ5(W270L) mRNA or protein
expression in the cells can be determined by methods described herein for
detecting KCNQ5 or KCNQ5(W270L) mRNA or protein.

In a preferred embodiment, determining the ability of the KCNQ5 or
KCNQ5(W270L) protein to bind to or interact with a KCNQ5 or KCNQ5(W270L)
target molecule can be accomplished by measuring a read out of the activity of
KCNQ5 or KCNQ5(W270L) or of the target molecule. For example, the activity
of KCNQ5 or KCNQ5(W270L) or a target molecule can be determined by
detecting induction of a cellular second messenger of the target, detecting
catalytic/enzymatic activity of the target of an appropriate substrate, detecting the
induction of a reporter gene (comprising a target-responsive regulatory element
operably linked to a nucleic acid encoding a detectable marker, e.g.,
chloramphenicol acetyl transferase), or detecting a target-regulated cellular
response, for example, Ca2+ influx induced by blocking of the KCNQ5 or
KCNQ5(W270L) channels.
In yet another aspect, KCNQ5 or KCNQ5(W270L) proteins or portions
thereof can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay
(see, e.g., U.S. Patent No. 5,283,317; Zervos AS et a/., Cell 72:223-32 (1993);
Madura K et at., J. Biol. Chem. 268:12046-54 (1993); Bartel P etal.,
Biotechniques 14:920-24 (1993); Iwabuchi K et al., Oncogene 8:1693-96 (1993);
and WO 94/10300) to identify other proteins which bind to or interact with
KCNQ5 or KCNQ5(W270L) and/or are involved in KCNQ5 or KCNQ5(W270L)
activity. Such KCNQ5-binding proteins are also likely to be involved in the
propagation of signals by the KCNQ5 proteins or KCNQ5 targets as, for example,
downstream elements of a KCNQ5-mediated signaling pathway. Alternatively,
such KCNQ5-or KCNQ5(W270L)-binding proteins may be KCNQ5 or
KCNQ5(W270L) inhibitors.
The two-hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA-binding and activation
domains. Briefly, the assay utilizes two different DN'A constructs, in one
construct, the gene that codes for a KCNQ5 or KCNQ5(W270L) protein is fused
to a gene encoding the DNA binding domain of a known transcription factor (e.g.,
GAL-4). The KCNQ5 protein can be a KCNQ5 polypeptide which contains an
S5-S6 transmembrane domain from KCNQ1. Alternatively, the KCNQ5 protein
can be a KCNQ5 polypeptide which contains an S5 transmembrane domain from
KCNQ1. In the other construct, a DNA sequence, from a library of DNA
sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a

gene that codes for the activation domain of the known transcription factor. If the
"bait" and the "prey" proteins are able to interact, in vivo, forming a KCNQ5- or
KCNQ5(W270L)-dependent complex, the DNA-binding and activation domains ol
the transcription factor are brought into close proximity. This proximity allows
transcription of a reporter gene (e.g., LacZ) which is operably linked to a
transcriptional regulatory site responsive to the transcription factor. Expression
of the reporter gene can be detected and cell colonies containing the functional
transcription factor can be isolated and used to obtain the cloned gene which
encodes the protein which interacts with the KCNQ5 or KCNQ5(W270L) protein.
In one aspect, the identified agents are novel analogs of known KCNQ
channel blockers or activators. For example, in one embodiment, the identified
agents are analogs of retigabine. In another exemplary embodiment, the
identified agents are analogs of XE991. In a further exemplary embodiment, the
agents are novel analogs of a compound of the formula:

wherein:
R1 is selected from hydrogen, C1-C6-alkyl, C2-C6-alkanoyl or the radical Ar;
R2 is selected from hydrogen or C1-C6-alkyl;
R3 is selected from C1-C6-alkoxy, NH2, C1-C6-alky!amino, C1-C6-dialkylamino,
amino substituted by the radical Ar, C1-C6-alkyl, C2-Cs-alkenyl, C2-C6-alkynyl, the
radical Ar or the radical ArO—;
R4 is selected from hydrogen, C1-C6-alkyI or the radical Ar;
R5 is selected from hydrogen or C1-C6-alkyl or the radical Ar;
Alk is a straight or branched alkylene group with 1-9 carbon atoms, which can
also be substituted by the radical Ar;
Ar is a phenyl radical substituted by the radicals R6, R7 and/or R8 where these
radicals R6, R7 and R8 are the same or different and represent H, C1-C6-alkyI, C3-
C7-cycloalkyl, hydroxy, C1-C6-alkoxy, C2-C6-alkanoyloxy, halogen, hydroxy, C1-

C6-halogenoalkyl, -CN, -NH2, -NH-C1-C6-alkyl, -N(C1-C6-alkyl)2, -CO2H, -
CO-C1-C6-alkyl, -CO-O~C1-C6-alkyl, -COAr, -CO-OAr, -CONH2, --CONH-
C1-C6 alkyl. --CON(C1-C6-alkyl)2. -CONHAr, -NH-CO-C1-C6-alkyl, -NHCO--Ar,
-NHCO-C1-C6-alkoxy, -N-H-CO-Ar, -NHCO-NH2, -NHCO-Nt-C1-C6-
alkyl)2, -NHCO--NHAr, -NH-SO2--C1-C6-alkyl, -NH-SO2Ar, -NH-SO2-
nitrophenyl, -S02-OH, -SO2-C1-C6-alkyl, -SO2-Ar, -SO2-C1-C6-alkoxy, -SO2-
OAr, -SO2-NH2, -SO2-NH--C1-C6-alkyl, -SO2-N(C1-C6-alkyl)2l -SO2-NHAr, -
S02-C2-C6-alkoxy;
n is 0 or 1.
A further aspect pertains to novel agents identified by the above-described
screening assays. Accordingly, it is within the scope of the present disclosure to
further use an agent identified as described herein in an appropriate animal
model. For example, an agent identified as described herein (e.g., a
KCNQ5(W270L) modulating agent, an antisense KCNQ5(W270L) polynucleotide,
a KCNQ5(W270L)-specific antibody, or a KCNQ5(W270L)-binding partner) can
be used in an animal model to determine the efficacy, toxicity, or side effects of
treatment with such an agent. Alternatively, an agent identified as described
herein can be used in an animal model to determine the mechanism of action of
such an agent. Furthermore, another aspect pertains to uses of novel agents
identified by the above-described screening assays for treatments as described
herein.
C. Methods of Rational Drug Design
KCNQ5 or KCNQ5(W270L) and KCNQ5 or KCNQ5(W270L) binding
polypeptides can be used for rational drug design of candidate KCNQ5-
modulating agents. The KCNQ5 or KCNQ5(W270L) polypeptides can be used
for protein X-ray crystallography or other structure analysis methods, such as the
DOCK program (see, e.g., Kuntz ID et al, J. Mol. Biol. 161: 269-88 (1982); Kuntz
ID, Science 257:1078-82 (1992)) and variants thereof. Potential therapeutic
drugs may be designed rationally on the basis of structural information thus
provided.

D. Detection Assays
Portions or fragments of the cDNA sequences identified herein (and the
corresponding complete gene sequences) can be used in numerous ways as
polynucleotide reagents. For example, these sequences can be used to: (i) map
their respective genes on a chromosome and, thus, locate gene regions
associated with genetic disease; (ii) identify an individual from a minute biological
sample (tissue typing); and (iii) aid in forensic identification of a biological
sample.
E. Predictive Medicine
Another aspect pertains to the field of predictive medicine in which
diagnostic assays, prognostic assays, and monitoring clinical trials are used for
prognostic (predictive) purposes to thereby treat an individual prophylactically.
Accordingly, one aspect relates to diagnostic assays for determining KCNQ5 or
KCNQ5(W270L) protein and/or nucleic acid expression as well as KCNQ5 or
KCNQ5(W270L) activity, in the context of a biological sample (e.g., blood, serum,
cells, tissue (preferably the brain, skeletal muscle, or urinary bladder)) to thereby
determine whether an individual is afflicted with a disease or disorder, or is at risk
of developing a disorder, associated with aberrant KCNQ5 expression or activity.
A further aspect provides for prognostic (or predictive) assays for determining
whether an individual is at risk of developing a disorder associated with KCNQ5
protein, nucleic acid expression, or activity. For example, mutations in a KCNQ5
gene can be assayed in a biological sample. Such assays can be used for
prognostic or predictive purpose to thereby prophylactically treat an individual
prior to the onset of a disorder characterized by or associated with KCNQ5
protein, nucleic acid expression, or activity.
Another aspect pertains to monitoring the influence of agents (e.g., drugs,
compounds) on the expression or activity of KCNQ5 in clinical trials.
These and other agents are described in further detail in the following
sections.
1. Diagnostic Assays
An exemplary method for detecting the presence or absence of KCNQ5 or
KCNQ5(W270L) protein or nucleic acid in a biological sample involves obtaining

a biological sample from a test subject and contacting the biological sample with
a compound or an agent capable of detecting KCNQ5 or KCNQ5(W270L) protein
or nucleic acid (e.g., mRNA, genomic DNA) that encodes KCNQ5 or
KCNQ5(W270L) protein such that the presence of KCNQ5 or KCNQ5(W270L)
protein or nucleic acid is detected in the biological sample. A preferred agent for
detecting KCNQ5 or KCNQ5(W270L) mRNA or genomic DNA is a labeled
nucleic acid probe capable of hybridizing to KCNQ5 or KCNQ5(W270L) mRNA or
genomic DNA. The nucleic acid probe can be, for example, a KCNQ5(W270L)
nucleic acid, such as the nucleic acid of SEQ ID NO:1, or a portion thereof, such
as an oligonucleotide of at least 12,15, 30, 50, 100, 250, 500 or more
nucleotides in length and sufficient to specifically hybridize under stringent
conditions to KCNQ5 or KCNQ5(W270L) mRNA or genomic DNA. Other suitable
probes for use in the diagnostic assays are described herein.
A preferred agent for detecting KCNQ5 or KCNQ5(W270L) protein is an
antibody capable of binding to KCNQ5 or KCNQ5(W270L) protein, preferably an
antibody with a detectable label. Antibodies can be polyclonal, or more
preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or
F(ab')2) can be used. The term "labeled", with regard to the probe or antibody, is
intended to encompass direct labeling of the probe or antibody by coupling (i.e.,
physically linking) a detectable substance to the probe or antibody, as well as
indirect labeling of the probe or antibody by reactivity with another reagent that is
directly labeled. Examples of indirect labeling include detection of a primary
antibody using a fluorescently labeled secondary antibody and end-labeling of a
DNA probe with biotin such that it can be detected with fluorescently labeled
streptavidin. The term "biological sample" is intended to include tissues, cells,
and biological fluids isolated from a subject, as well as tissues, cells (preferably
brain, skeletal muscle, or urinary bladder), and fluids present within a subject;
that is, the detection method can be used to detect KCNQ5 or KCNQ5(W270L)
mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo.
For example, in vitro techniques for detection of KCNQ5 or KCNQ5(W270L)
mRNA include Northern hybridizations and in situ hybridizations. In vitro
techniques for detection of KCNQ5 or KCNQ5(W270L) protein include ELISAs,
Western blots, immunoprecipitation, and immunofluorescence. In vitro
techniques for detection of KCNQ5 or KCNQ5(W270L) genomic DNA include

Southern hybridizations. Furthermore, in vivo techniques for detection of KCNQ5
or KCNQ5(W270L) protein include introducing into a subject a labeled anti-
KCNQ5 or anti-KCNQ5(W270L) antibody. For example, the antibody can be
labeled with a radioactive marker whose presence and location in a subject can
be detected by standard imaging techniques.
In one embodiment, the biological sample contains protein molecules from
the test subject. Alternatively, the biological sample can contain mRNA
molecules from the test subject or genomic DNA molecules from the test subject.
A preferred biological sample is a brain or urinary bladder sample isolated by
standard means from a subject.
In another embodiment, the methods further involve obtaining a control
biological sample from a control subject, contacting the control sample with a
compound or agent capable of detecting KCNQ5 or KCNQ5(W270L) protein,
mRNA, or genomic DNA, such that the presence of KCNQ5 or KCNQ5(W270L)
protein, mRNA, or genomic DNA is detected in the biological sample, and
comparing the presence of KCNQ5 or KCNQ5(W270L) protein, mRNA, or
genomic DNA in the control sample with the presence of KCNQ5 or
KCNQ5(W270L) protein, mRNA, or genomic DNA in the test sample.
An aspect also encompasses kits for detecting the presence of KCNQ5 or
KCNQ5(W270L) in a biological sample. For example, the kit can comprise a
labeled compound or agent capable of detecting KCNQ5 or KCNQ5(W270L)
protein or mRNA in a biological sample; means for determining the amount of
KCNQ5 or KCNQ5(W270L) in the sample; and means for comparing the amount
of KCNQ5 or KCNQ5(W270L) in the sample with a standard. The compound or
agent can be packaged in a suitable container. The kit can further comprise
instructions for using the kit to detect KCNQ5 or KCNQ5(W270L) protein or
nucleic acid.
2. Prognostic Assays
The diagnostic methods described herein can furthermore be utilized to
identify subjects having or at risk of developing a disease or disorder associated
with aberrant KCNQ5 expression or activity. For example, the assays described
herein, such as the preceding diagnostic assays or the following assays, can be
utilized to identify a subject having or at risk of developing a disorder associated

with KCNQ5 protein, nucleic acid expression, or activity. Thus, a further aspect
provides a method for identifying a disease or disorder associated with aberrant
KCNQ5 expression or activity in which a test sample is obtained from a subject
and KCNQ5 or KCNQ5(W270L) protein or nucleic acid (e.g., mRNA, genomic
DNA) is detected, wherein the presence of KCNQ5 or KCNQ5(W270L) protein or
nucleic acid is diagnostic for a subject having or at risk of developing a disease
or disorder associated with aberrant KCNQ5 expression or activity. As used
herein, a "test sample" refers to a biological sample obtained from a subject of
interest. For example, a test sample can be a biological fluid (e.g., serum), cell
sample, or tissue.
Furthermore, the prognostic assays described herein can be used to
determine whether a subject can be administered an agent (e.g., an agonist,
antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or
other drug candidate) to treat a disease or disorder associated with aberrant
KCNQ5 expression or activity. Thus, another aspect provides methods for
determining whether a subject can be effectively treated with an agent for a
disorder associated with aberrant KCNQ5 expression or activity in which a test
sample is obtained and KCNQ5 or KCNQ5(W270L) protein or nucleic acid
expression or activity is detected (e.g., wherein the abundance of KCNQ5 or
KCNQ5(W270L) protein or nucleic acid expression or activity is diagnostic for a
subject that can be administered the agent to treat a disorder associated with
aberrant KCNQ5 expression or activity).
The methods can also be used to detect genetic alterations in a KCNQ5
gene, thereby determining if a subject with the altered gene is at risk for a
disorder associated with the KCNQ5 gene. In preferred embodiments, the
methods include detecting, in a sample of cells from the subject, the presence or
absence of a genetic alteration characterized by at least one of an alteration
affecting the integrity of a gene encoding a KCNQ5 protein or the mis-expression
of the KCNQ5 gene. For example, such genetic alterations can be detected by
ascertaining the existence of at least one of 1) a deletion of one or more
nucleotides from a KCNQ5 gene; 2) an addition of one or more nucleotides to a
KCNQ5 gene; 3) a substitution of one or more nucleotides of a KCNQ5 gene; 4)
a chromosomal rearrangement of a KCNQ5 gene; 5) an alteration in the level of
a messenger RNA transcript of a KCNQ5 gene; 6) aberrant modification of a

KCNQ5 gene, such as of the methylation pattern of the genomic DNA; 7) the
presence of a non-wild type splicing pattern of a messenger RNA transcript of a
KCNQ5 gene; 8) a non-wild type level of a KCNQ5 protein; 9) allelic loss of a
KCNQ5 gene; and 10) inappropriate post-translational modification of a KCNQ5
protein. As described herein, there are a large number of assay techniques
known in the art which can be used for detecting alterations in a KCNQ5 gene. A
preferred biological sample is a tissue sample isolated by standard means from a
subject, for example, a brain or urinary bladder sample. The detection can be
performed with at least one of a probe or a primer comprising at least 12
contiguous nucleotides from a KCNQ5 polynucleotide. Preferably, the KCNQ5
polynucleotide encodes all or a portion of a KCNQ5(W270L) polypeptide,
encodes a KCNQ5 polypeptide containing an S5-S6 transmembrane domain
from KCNQ1, or encodes a KCNQ5 polypeptide containing an S5
transmembrane domain from KCNQ1. More preferably, the probe or primer
comprises at least 12 contiguous nucleotides from SEQ ID NO:1 including
nucleotides 808-810.
In certain embodiments, detection of the alteration involves the use of a
probe/primer in PCR (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such
as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR)
(see, e.g., Landegren U et al., Science 241:1077-80 (1988); Nakazawa H et al.,
Proc. Natl. Acad. Sci. USA 91:360-64 (1994)), the latter of which can be
particularly useful for detecting point mutations in the KCNQ5 gene (see, e.g.,
Abravaya K et al., Nucleic Acids Res. 23:675-82 (1995)). This method can
include the steps of collecting a sample of cells from a patient, isolating nucleic
acid (e.g., genomic, mRNA, or both) from the cells of the sample, contacting the
nucleic acid sample with one or more primers which specifically hybridize to a
KCNQ5 gene under conditions such that hybridization and amplification of the
KCNQ5 gene (if present) occurs, and detecting the presence or absence of an
amplification product, or detecting the size of the amplification product and
comparing the length to a control sample. It is anticipated that PCR and/or LCR
may be desirable to use as a preliminary amplification step in conjunction with
any of the techniques used for detecting mutations described herein.
Alternative amplification methods include, for example, self-sustained
sequence replication (Guatelli JC et al., Proc. Natl. Acad. Sci. USA 87:1874-78

(1990)), transcriptional amplification system (Kwoh DY et al., Proc. Natl. Acad.
Sci. USA 86:1173-77 (1989)), Q-Beta Replicase (Lizardi PM et al., Biotechnology
(N.Y.) 6:1197 (1988)), or any other nucleic acid amplification method, followed by
the detection of the amplified molecules using techniques well known to those of
skill in the art. These detection schemes are especially useful for the detection of
nucleic acid molecules if such molecules are present in very low numbers.
In an alternate embodiment, mutations in a KCNQ5 gene from a sample
cell can be identified by alterations in restriction enzyme cleavage patterns. For
example, sample and control DNA is isolated, amplified (optionally), digested
with one or more restriction endonucleases, and fragment length sizes are
determined by gel electrophoresis and compared. Differences in fragment length
sizes between sample and control DNA indicates mutations in the sample DNA.
Moreover, the use of sequence specific ribozymes (see, for example, U.S. Patent
No. 5,498,531) can be used to score for the presence of specific mutations by
development or loss of a ribozyme cleavage site.
In other embodiments, genetic mutations in KCNQ5 can be identified by
hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density
arrays containing hundreds or thousands of oligonucleotides probes (Cronin MT
etal., Hum. Mutat. 7: 244-55 (1996); Kozal MJ etal., Nat. Med. 2:753-59 (1996)).
For example, genetic mutations in KCNQ5 can be identified in two dimensional
arrays containing light-generated DNA probes as described in Cronin MT et al.
{supra). Briefly, a first hybridization array of probes can be used to scan through
long stretches of DNA in a sample and control to identify base changes between
the sequences by making linear arrays of sequential overlapping probes. This
step allows the identification of point mutations. This step is followed by a
second hybridization array that allows the characterization of specific mutations
by using smaller, specialized probe arrays complementary to all variants or
mutations detected. Each mutation array is composed of parallel probe sets, one
complementary to the wild-type gene and the other complementary to the mutant
gene.
In yet another embodiment, any of a variety of sequencing reactions
known in the art can be used to directly sequence the KCNQ5 gene and detect
mutations by comparing the sequence of the sample KCNQ5 or KCNQ5(W270L)
with the corresponding wild-type (control) sequence. Examples of sequencing

reactions include those based on techniques developed by Maxam AM and
Gilbert W, Proc. Natl. Acad. Sci. USA 74:560-64 (1977) or Sanger F et al, Proc.
Natl. Acad. Sci. USA 74:5463-67 (1977). It is also contemplated that any of a
variety of automated sequencing procedures can be utilized when performing the
diagnostic assays (see, e.g., Naeve CW et al., Biotechniques 19:448-53 (1995)),
including sequencing by mass spectrometry (see, e.g., WO 94/16101; Cohen AS
et al., Adv. Chromatogr. 36:127-62 (1996); and Griffin HG and Griffin AM, Appl.
Biochem. Biotechnol. 38:147-59 (1993)).
Other methods for detecting mutations in the KCNQ5 gene include
methods in which protection from cleavage agents is used to detect mismatched
bases in RNA/RNAor RNA/DNA heteroduplexes (Myers RM et al., Science
230:1242-46 (1985)). In general, the art technique of "mismatch cleavage" starts
by providing heteroduplexes formed by hybridizing (labeled) RNA or DNA
containing the wild-type KCNQ5 sequence with potentially mutant RNA or DNA
obtained from a tissue sample. The double-stranded duplexes are treated with
an agent which cleaves single-stranded regions of the duplex such as which will
exist due to basepair mismatches between the control and sample strands. For
instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids
treated with S1 nuclease to enzymatically digest the mismatched regions. In
other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with
hydroxylamine or osmium tetroxide and with piperidine in order to digest
mismatched regions. After digestion of the mismatched regions, the resulting
material is then separated by size on denaturing polyaery lam ide gels to
determine the site of mutation (see, e.g., Cotton RGH et a/., Proc. Natl. Acad.
Sci. USA 85:4397-4401 (1988); Saleeba JAand Cotton RGH, Meth. Enzymol.
217:286-95 (1993)). In a preferred embodiment, the control DNA or RNA can be
labeled for detection. In still another embodiment, the mismatch cleavage
reaction employs one or more proteins that recognize mismatched base pairs in
double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined
systems for detecting and mapping point mutations in KCNQ5 obtained from
samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A
mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at
G/T mismatches (Hsu IC etal., Carcinogenesis 15:1657-62 (1994)). According
to an exemplary embodiment, a probe based on a KCNQ5(W270L) sequence, for

example, SEQ ID NO:1, is hybridized to a cDNA or other DNA product from a
test cell(s). The duplex is treated with a DNA mismatch repair enzyme, and the
cleavage products, if any, can be detected from electrophoresis protocols or the
like (see, e.g., U.S. Patent No. 5,459,039).
In other embodiments, alterations in electrophoretic mobility will be used
to identify mutations in KCNQ5 genes. For example, single strand conformation
polymorphism (SSCP) may be used to detect differences in electrophoretic
mobility between mutant and wild type nucleic acids (Orita M et a/., Proc Natl.
Acad. Sci USA: 86:2766-70 (1989); see also, Cotton RGH, Mutat. Res. 285:125-
44 (1993); Hayashi K, Genet. Anal. Tech. Appl. 9:73-79 (1992)). Single-stranded
DNA fragments of sample KCNQ5 or.KCNQ5(W270L) and control KCNQ5
nucleic acids will be denatured and allowed to renature. The secondary structure
of single-stranded nucleic acids varies according to sequence; the resulting
alteration in electrophoretic mobility enables the detection of even a single base
change. The DNA fragments may be labeled or detected with labeled probes.
The sensitivity of the assay may be enhanced by using RNA (rather than DNA),
in which the secondary structure is more sensitive to a change in sequence. In a
preferred embodiment, the subject method utilizes heteroduplex analysis to
separate double stranded heteroduplex molecules on the basis of changes in
electrophoretic mobility (Keen J et al., Trends Genet. 7:5 (1991)).
In yet another embodiment, the movement of mutant or wild-type
fragments in polyacrylamide gels containing a gradient of denaturant is assayed
using denaturing gradient gel electrophoresis (DGGE) (Myers RM et al, Nature
313:495-98 (1985)). When DGGE is used as the method of analysis, DNA will
be modified to insure that it does not completely denature, for example by adding
a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a
further embodiment, a temperature gradient is used in place of a denaturing
gradient to identify differences in the mobility of control and sample DNA
(Rosenbaum V and Riesner D, Biophys. Chem. 26:235-46 (1987)).
Examples of other techniques for detecting point mutations include, but
are not limited to, selective oligonucleotide hybridization, selective amplification,
or selective primer extension. For example, oligonucleotide primers may be
prepared in which the known mutation is placed centrally and then hybridized to
target DNA under conditions which permit hybridization only if a perfect match is

found (Saiki RK et a/., Nature 324:163-66 (1986); Saiki RK et al., Proc. Natl.
Acad. Sci. USA 86:6230-34 (1989)). Such allele specific otigonucleotides are
hybridized to PCR amplified target DNA or a number of different mutations when
the oligonucleotides are attached to the hybridizing membrane and hybridized
with labeled target DNA.
Alternatively, allele specific amplification technology which depends on
selective PCR amplification may be used in conjunction with the disclosed
compositions and methods. Oligonucleotides used as primers for specific
amplification may carry the mutation of interest in the center of the molecule (so
that amplification depends on differential hybridization) (Gibbs RA et al.. Nucleic
Acids Res. 17:2437-48 (1989)) or at the extreme 3' end of one primer where,
under appropriate conditions, mismatch can prevent, or reduce polymerase
extension (Prosser J, Trends Biotechnol. 11:238-46 (1993)). In addition, it may
be desirable to introduce a novel restriction site in the region of the mutation to
create cleavage-based detection (Gasparini P et al., Mol. Cell. Probes 6:1-7
(1992)). It is anticipated that in certain embodiments amplification may also be
performed using Taq ligase for amplification (Barany F, Proc. Natl. Acad. Sci
USA 88:189-93 (1991)). In such cases, ligation will occur only if there is a
perfect match at the 3' end of the 5' sequence making it possible to detect the
presence of a known mutation at a specific site by looking for the presence or
absence of amplification.
The methods described herein may be performed, for example, by utilizing
pre-packaged diagnostic kits comprising at least one probe nucleic acid or
antibody reagent described herein, which may be conveniently used, for
example, in clinical settings to diagnose patients exhibiting symptoms or family
history of a disease or illness involving a KCNQ5 gene.
Furthermore, any cell type or tissue in which KCNQ5 is expressed may be
utilized in the prognostic assays described herein.
VI. Administration of KCNQ5 Modulating Agents
KCNQ5 or KCNQ5(W270L) modulating agents are administered to
subjects in a biologically compatible form suitable for pharmaceutical
administration in vivo to treat, for example, conditions described in section V,
supra. By "biologically compatible form suitable for administration in vivo" is

meant a form of the protein to be administered in which any toxic effects are
outweighed by the therapeutic effects of the protein. The term "subject" is
intended to include living organisms in which an immune response can be
elicited, for example, mammals. Administration of an agent as described herein
can be in any pharmacological form including a therapeutically active amount of
an agent alone or in combination with a pharmaceutically acceptable carrier.
Administration of a therapeutically active amount of the therapeutic
compositions is defined as an amount effective, at dosages and for periods of
time necessary to achieve the desired result For example, a therapeutically
active amount of a KCNQ5 or KCNQ5(W270L) modulating agent may vary
according to factors such as the disease state, age, sex, and weight of the
individual, and the ability of peptide to elicit a desired response in the individual.
Dosage regima may be adjusted to provide the optimum therapeutic response.
For example, several divided doses may be administered daily, or the dose may
be proportionally reduced as indicated by the exigencies of the therapeutic
situation.
The therapeutic or pharmaceutical compositions can be administered by .
any suitable route known in the art including, for example, intravenous,
subcutaneous, intramuscular, transdermal, intrathecal, or intracerebral or
administration to cells in ex vivo treatment protocols. Administration can be
either rapid as by injection or over a period of time as by slow infusion or
administration of slow release formulation.
KCNQ5 or KCNQ5(W270L) can also be linked or conjugated with agents
that provide desirable pharmaceutical or pharmacodynamic properties. For
example, KCNQ5 or KCNQ5(W270L) can be coupled to any substance known in
the art to promote penetration or transport across the blood-brain barrier such as
an antibody to the transferrin receptor, and administered by intravenous injection
(see, e.g., Friden PM et al, Science 259:373-77 (1993)). Furthermore, KCNQ5
or KCNQ5(W270L) can be stably linked to a polymer such as polyethylene glycol
to obtain desirable properties of solubility, stability, half-life, and other
pharmaceutically advantageous properties (see, e.g., Davis et al., Enzyme Eng.
4:169-73 (1978); Bumham NL, Am. J. Hosp. Pharm. 51:210-18 (1994)).
Furthermore, a KCNQ5 or KCNQ5(W270L) polypeptide can be in a
composition which aids in delivery into the cytosol of a cell. For example, the

peptide may be conjugated with a carrier moiety such as a liposome that is
capable of delivering the peptide into the cytosol of a cell. Such methods are
well known in the art (see, e.g., Amselem S et al., Chem. Phys. Lipids 64:219-37
(1993)). Alternatively, a KCNQ5 or KCNQ5(W270L) polypeptide can be modified
to include specific transit peptides or fused to such transit peptides which are
capable of delivering their KCNQ5 or KCNQ5(W270L) polypeptide into a cell. In
addition, the polypeptide can be delivered directly into a cell by microinjection.
The compositions are usually employed in the form of pharmaceutical
preparations. Such preparations are made in a manner well known in the
pharmaceutical art. One preferred preparation utilizes a vehicle of physiological
saline solution, but it is contemplated that other pharmaceutically acceptable
carriers such as physiological concentrations of other non-toxic salts, five percent
aqueous glucose solution, sterile water, or the like may also be used. As used
herein, "pharmaceutically acceptable carrier" includes any and all solvents,
dispersion media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except insofar as
any standard media or agent is incompatible with the active compound, use
thereof in the therapeutic compositions is contemplated. Supplementary active
compounds can also be incorporated into the compositions. It may also be
desirable that a suitable buffer be present in the composition. Such solutions
can, if desired, be lyophilized and stored in a sterile ampoule ready for
reconstitution by the addition of sterile water for ready injection. The primary
solvent can be aqueous or alternatively non-aqueous. KCNQ5 or
KCNQ5(W270L) can also be incorporated into a solid or semi-solid biologically
compatible matrix which can be implanted into tissues requiring treatment.
The carrier can also contain other pharmaceuticaHy-acceptable excipients
for modifying or maintaining the pH, osmolarity, viscosity, clarity, color, sterility,
stability, rate of dissolution, or odor of the formulation. Similarly, the carrier may
contain still other pharmaceuticaHy-acceptable excipients for modifying or
maintaining release or absorption or penetration across the blood-brain barrier.
Such excipients are those substances usually and customarily employed to
formulate dosages for parenteral administration in either unit dosage or multi-
dose form or for direct infusion by continuous or periodic infusion.

Dose administration can be repeated depending upon the
pharmacokinetic parameters of the dosage formulation and the route of
administration used.
It is also provided that certain formulations containing a KCNQ5 or
KCNQ5(W270L) polypeptide or fragment thereof are to be administered orally.
Such formulations are preferably encapsulated and formulated with suitable
carriers in solid dosage forms. Some examples of suitable carriers, excipients,
and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum
acacia, calcium phosphate, alginates, calcium silicate, microcrystalline cellulose,
polyvinylpyrrolidone, cellulose, gelatin, syrup, methyl cellulose, methyl- and
propylhydroxybenzoates, talc, magnesium, stearate, water, mineral oil, and the
like. The formulations can additionally include lubricating agents, wetting agents,
emulsifying and suspending agents, preserving agents, sweetening agents, or
flavoring agents. The compositions may be formulated so as to provide rapid,
sustained, or delayed release of the active ingredients after administration to the
patient by employing procedures well known in the art. The formulations can
also contain substances that diminish proteolytic degradation and/or substances
which promote absorption such as, for example, surface active agents.
It is especially advantageous to formulate parenteral compositions in
dosage unit form for ease of administration and uniformity of dosage. Dosage
unit form as used herein refers to physically discrete units suited as unitary
dosages for the mammalian subjects to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the desired
therapeutic effect in association with the required pharmaceutical carrier. The
specification for the dosage unit forms are dictated by and directly dependent on
(a) the unique characteristics of the active compound and the particular
therapeutic effect to be achieved and (b) the limitations inherent in the art of
compounding such an active compound for the treatment of sensitivity in
individuals. The specific dose can be readily calculated by one of ordinary skill in
the art, e.g., according to the approximate body weight or body surface area of
the patient or the volume of body space to be occupied. The dose will also be
calculated dependent upon the particular route of administration selected.
Further refinement of the calculations necessary to determine the appropriate
dosage for treatment is routinely made by those of ordinary skill in the art. Such

calculations can be made without undue experimentation by one skilled in the art
in light of the activity disclosed herein in assay preparations of target cells. Exact
dosages are determined in conjunction with standard dose-response studies. It
will be understood that the amount of the composition actually administered will
be determined by a practitioner, in the light of the relevant circumstances
including the condition or conditions to be treated; the choice of composition to
be administered; the age, weight, and response of the individual patient; the
severity of the patient's symptoms; and the chosen route of administration.
Toxicity and therapeutic efficacy of such compounds can be determined
by standard pharmaceutical procedures in cell cultures or experimental animals,
for example, for determining the LD50 (the dose lethal to 50% of the population)
and the ED50 (the dose therapeutically effective in 50% of the population). The
dose ratio between toxic and therapeutic effects is the therapeutic index and it
can be expressed as the ratio LD50/ED50. Compounds which exhibit large
therapeutic indices are preferred. While compounds that exhibit toxic side effects
may be used, care should be taken to design a delivery system that targets such
compounds to the site of affected tissue in order to minimize potential damage to
uninfected cells and, thereby, reduce side effects.
The data obtained from the cell culture assays and animal studies can be
used in formulating a range of dosage for use in humans. The dosage of such
compounds lies preferably within a range of circulating concentrations that
include the ED50 with little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of administration
utilized. For any compound used in the methods disclosed herein, the
therapeutically effective dose can be estimated initially from cell culture assays.
A dose may be formulated in animal models to achieve a circulating plasma
concentration range that includes the IC50 (i.e., the concentration of the test
compound which achieves a half-maximal inhibition of symptoms) as determined
in cell culture. Such information can be used to more accurately determine
useful doses in humans. Levels in plasma may be measured, for example, by
high performance liquid chromatography.
In one embodiment, a KCNQ5 or KCNQ5(W270L) polypeptide may be
therapeutically administered by implanting into patients vectors or ceils capable
of producing a biologically-active form of KCNQ5 or KCNQ5(W270L) or a

precursor of KCNQ5 or KCNQ5(W270L), that is, a molecule that can be readily
converted to a biological-active form of KCNQ5 or KCNQ5(W270L) by the body.
In one approach, cells that secrete KCNQ5 or KCNQ5(W270L) may be
encapsulated into semipermeable membranes for implantation into a patient.
The cells can be cells that normally express KCNQ5 or a precursor thereof or the
cells can be transformed to express KCNQ5 or KCNQ5(W270L) or a biologically
active fragment thereof or a precursor thereof. It is preferred that the cell be of
human origin. However, the formulations and methods herein can be used for
veterinary as well as human applications and the term "patient" or "subject" as
used herein is intended to include human and veterinary patients.
Monitoring the influence of agents (e.g., drugs or compounds) on the
expression or activity of a KCNQ5 or KCNQ5(W270L) protein can be applied not
only in basic drug screening, but also in clinical trials. For example, the
effectiveness of an agent determined by a screening assay as described herein
to increase KCNQ5 gene expression, protein levels, or upregulate KCNQ5
activity can be monitored in clinical trials of subjects exhibiting decreased
KCNQ5 gene expression, protein levels, or downregulated KCNQ5 activity.
Alternatively, the effectiveness of an agent determined by a screening assay to
decrease KCNQ5 gene expression, protein levels, or downregulate KCNQ5
activity can be monitored in clinical trials of subjects exhibiting increased KCNQ5
gene expression, protein levels, or upregulated KCNQ5 activity. In such clinical
trials, the expression or activity of a KCNQ5 gene, and preferably, other genes
that have been implicated in a disorder, can be used as a "read out" or markers
of the phenotype of a particular cell.
For example, and not by way of limitation, genes, including KCNQ5, that
are modulated in cells by treatment with an agent (e.g., compound, drug, or small
molecule) which modulates KCNQ5 activity (e.g., identified in a screening assay
as described herein) can be identified. Thus, to study the effect of agents on a
KCNQ5 associated disorder, for example, in a clinical trial, cells can be isolated
and RNA prepared and analyzed for the levels of expression of KCNQ5 and
other genes implicated in the KCNQ5 associated disorder, respectively. The
levels of gene expression (i.e., a gene expression pattern) can be quantified by
Northern blot analysis or RT-PCR, as described herein, or alternatively by
measuring the amount of protein produced, by one of the methods as described

way, the gene expression pattern can serve as a marker, indicative of the
physiological response of the cells to the agent. Accordingly, this response state
may be determined before, and at various points during, treatment of the
individual with the agent.
A preferred embodiment provides a method for monitoring the
effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist,
peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug
candidate identified by the screening assays described herein) comprising the
steps of (i) obtaining a pre-administration sample from a subject prior to
administration of the agent; (ii) detecting the level of expression of a KCNQ5 or
KCNQ5(W270L) protein, mRNA, or genomic DNA in the pre-administration
sample; (iii) obtaining one or more post-administration samples from the subject;
(iv) detecting the level of expression or activity of the KCNQ5 or KCNQ5(W270L)
protein, mRNA, or genomic DNA in the post-administration samples; (v)
comparing the level of expression or activity of the KCNQ5 or KCNQ5(W270L)
protein, mRNA, or genomic DNA in the pre-administration sample with the
KCNQ5 or KCNQ5(W270L) protein, mRNA, or genomic DNA in the post
administration sample or samples; and (vi) altering the administration of the
agent to the subject accordingly. For example, increased administration of the
agent may be desirable to increase the expression or activity of KCNQ5 or
KCNQ5(W270L) to higher levels than detected, that is, to increase the
effectiveness of the agent. Alternatively, decreased administration of the agent
may be desirable to decrease expression or activity of KCNQ5 or
KCNQ5(W270L) to lower levels than detected, that is, to decrease the
effectiveness of the agent. According to such an embodiment, KCNQ5 or
KCNQ5(W270L) expression or activity may be used as an indicator of the
effectiveness of an agent, even in the absence of an observable phenotypic
response.
In a preferred embodiment, the ability of a KCNQ5 or KCNQ5(W270L)
modulating agent to modulate, for example, conditions described in section V
(supra) in a subject that would benefit from modulation of the expression and/or
activity of KCNQ5 or KCNQ5(W270L) can be measured by detecting an
improvement in the condition of the patient after the administration of the agent.

Such improvement can be readily measured by one of ordinary skill in the art
using indicators appropriate for the specific condition of the patient. Monitoring
the response of the patient by measuring changes in the condition of the patient
is preferred in situations where the collection of biopsy materials would pose an
increased risk and/or detriment to the patient.
It is likely that the level of KCNQ5 or KCNQ5(W270L) may be altered in a
variety of conditions and that quantification of KCNQ5 or KCNQ5(W270L) levels
would provide clinically useful information.
Furthermore, in the treatment of disease conditions, compositions
containing KCNQ5 or KCNQ5(W270L) can be administered exogenously, and it
would likely be desirable to achieve certain target levels of KCNQ5 or
KCNQ5(W270L) polypeptide in sera, in any desired tissue compartment, or in the
affected tissue. It would, therefore, be advantageous to be able to monitor the
levels of KCNQ5 or KCNQ5(W270L) polypeptide in a patient or in a biological
sample including a tissue biopsy sample obtained from a patient and, in some
cases, also monitoring the levels of native KCNQ5. Accordingly, another aspect
provides methods for detecting the presence of KCNQ5 or KCNQ5(W270L) in a
sample from a patient.
VII. Kits of the Invention
Another aspect pertains to kits for carrying out the screening assays,
modulatory methods, or diagnostic assays. For example, a kit for carrying out a
screening assay can include a cell comprising a KCNQ5 or KCNQ5(W270L)
polypeptide, means for determining KCNQ5 or KCNQ5(W270L) polypeptide
activity, and, optionally, instructions for using the kit to identify modulators of
KCNQ5 or KCNQ5(W270L) activity. In another embodiment, a kit for carrying out
a screening assay can include an composition comprising a KCNQ5 or
KCNQ5(W270L) polypeptide, means for determining KCNQ5 or KCNQ5(W270L)
activity, and, optionally, instructions for using the kit to identify modulators of
KCNQ5 or KCNQ5(W270L) activity.
Another embodiment provides a kit for carrying out a modulatory method.
The kit can include, for example, a modulatory agent (e.g., a KCNQ5 or
KCNQ5(W270L) inhibitory or stimulatory agent) in a suitable carrier and

packaged in a suitable container optionally with instructions for use of the
modulator to modulate KCNQ5 or KCNQ5(W270L) activity.
Another aspect pertains to a kit for diagnosing a disorder associated with
aberrant KCNQ5 expression and/or activity in a subject. The kit can include a
reagent for determining expression of KCNQ5 or KCNQ5(W270L) (e.g., a nucleic
acid probe(s) for detecting KCNQ5 or KCNQ5(W270L) mRNA or one or more
antibodies for detection of KCNQ5 or KCNQ5(W270L) proteins), a control to
which the results of the subject are compared, and, optionally, instructions for
using the kit for diagnostic purposes.
The practice of the methods disclosed herein will employ, unless
otherwise indicated, standard techniques of cell biology, cell culture, molecular
biology, transgenic biology, microbiology, recombinant DNA, and immunology,
which are within the skill of the art. Such techniques are explained fully in the
literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed.,
ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press:
1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide
Synthesis (M. J. Gaited., 1984); U.S. Patent No. 4,683,195; Nucleic Acid
Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And
Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R.
I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL
Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the
treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer
Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold
Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et
al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and
Walker, eds., Academic Press, London, 1987); Handbook Of Experimental
Immunology, Volumes l-IV (D. M. Weir and C. C. Blackwell, eds., 1986);
Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., 1986).
Examples
The disclosure herein is further defined in the following Examples. It
should be understood that these Examples, while indicating preferred
embodiments, are given by way of illustration only. From the above discussion

and these Examples, one skilled in the art can ascertain the preferred features,
and without departing from the spirit and scope thereof, can make various
changes and modifications to adapt it to various uses and conditions.
GENERAL PROCEDURES
Expression of KCNQ5 and KCNQ1 channels into Xenopus oocytes—Human KCNQ5
and KCNQ1 genes were cloned into a pKSM vector that has been engineered
specifically for oocyte expression. The gene sequences were confirmed by DNA
sequencing. The gene bank accession numbers are: KCNQ5, NM_019842 (SEQ ID
NO:3); KCNQ1, U89364.1 (SEQ ID NO:7). The constructs were linearized by Xbal
restriction enzyme (NEB, Beverly, MA). In vitro transcription was performed with an
mMESSAGE mMACHINE® kit (Ambion, Austin, TX) and the cRNA was purified by
phenol extraction. Xenopus oocytes were injected with 46 nl solution containing
approximately 9.2 ng cRNA using a Drumond Nanojet oocyte injector (Drummond
Scientific Co., Broomall, PA). Electrophysiological recording was performed 3-7 days
after injection.
Electrophysiology—All experiments were performed at room temperature (22-23°C)
using conventional oocyte two-electrode voltage clamp recording (TEVC). The
recording electrodes were pulled from 1.5 mm glass pipette using a Sutter P-97
micropipette puller (Sutter Instrument, Novato, CA) and filled with 3M KCI. The pipette
tip was carefully broken off to acquire the electrode resistance at 0.5 — 1 Mohm. A
Dagan CA-1 amplifier (Dagan Corporation, Minneapolis MN) was used and the holding
potential was clamped at —100 mV. To test voltage dependence of channel activation, a
series of depolarized voltage pulses was applied. To observe the time course of
retigabine's effect, a single (3 sec) voltage pulse at 40 mV was applied repeatedly at 15
sec intervals. A HEKA computer interface (HEKA Elektronik, Lambrecht, Germany) with
Pulse 8.5 software was used to control the amplifier and digitized the data for analysis.
Solutions and Chemicals—ND96 solution containing (in mM) NaCI, 96, KCI, 2, CaCI2,
1.8, MgCI2, 1, HEPES, 10 was used for oocyte incubation and recording. Retigabine
was made as a stock solution in DMSO at 50 mM and diluted into desired
concentrations just before application in experiments. DMSO was tested on KCNQ5

channels and no effect was observed up to 1%. In all experiments, DMSO was used
less than 0.4%.
Compounds were applied to the cells via a fast bath perfusion system (ALA
Scientific Instruments, Westbury, NY), and a bath solution change was completed within
10 seconds.
Data analysis—Current amplitude was measured online using HEKA Pulsefit 8.5
software. To acquire channel conductance from macroscopic current, the K+
reversal potential was approximated to be -80 mV in ND96 solution and the
following equation was applied: G=l/(V+80), where G is the whole oocyte
conductance, I is the whole oocyte current, and V is the voltage used to induce
the current Data were expressed as mean ± SE, and the paired student t test
was performed. Differences were considered to be significant at P<0.05.
Example 1
To make the KCNQ5 polynucleotide containing an S5-S6 region from KCNQ1
(encoding SEQ ID NO:6, amino acids S253-V355 of SEQ ID NO:8), a pair of
oligoprimers flanking the S5-S6 region of KCNQ1 gene was used to obtain the DNA
fragment from KCNQ1. Both primers contain introduced DNA sequences in each end
that overlap the KCNQ5 gene sequence in corresponding junction areas upstream of S5
and downstream of S6. The PCR product using these primers plus the KCNQ1
construct as a template was purified and used as site-directed mutagenesis primers for
second round PCR with the KCNQ5 construct as a template. Site-directed mutagenesis
using the QuickChange protocol was then applied (Stratagene, La Jolla, CA). All
mutations were confirmed by DNA sequencing.
The effect of retigabine on the functional chimera (KCNQ5 containing an
S5-S6 transmembrane domain from KCNQ1) was tested. As shown in Figure 1A
and 1B, 50 µM retigabine no longer augmented the channel activities. During a 5
min application period, neither current amplitude nor voltage dependent
activation changed significantly (Figure 1C). The dramatic disruption of
retigabine action on this chimeric channel suggested that the action site of
retigabine in KCNQ5 was most likely located in the region of S5-S6
transmembrane domain.

Example 2
To further dissect the action site of retigabine, the S5-S6 transmembrane
domain from KCNQ1 was divided into two parts, the S5 transmembrane domain
and the S6 transmembrane domain (with linker), and the corresponding chimeric
channels of KCNQ5 contained S5 transmembrane domain or S6 transmembrane
domain from KCNQ1. The chimeric channel with swapped S6 transmembrane
domain showed no current in the membrane potential ranging from -100 to 100
mV. The other chimera containing the S5 domain from KCNQ1 functioned as an
outward rectifier with a very unique activation property. As shown in Figure 2A
left panel, this mutant appeared to inactivate when membrane potential was
higher than 20 mV. When channel was activated by membrane depolarization, a
transient outward current (150 — 200 msec) was visualized, making the channel
activation significantly two-component alike. When membrane potential was
lower than 20 mV, the channel activation pattern was similar to the wild type and
no clear inactivation could be observed.
The effect of retigabine on KCNQ5 containing an S5 transmembrane
domain from KCNQ1 was then tested. Retigabine was applied to KCNQ5
containing an S5 transmembrane domain from KCNQ1-expressing oocytes in
which the steady-state current amplitude had been stabilized at the maximal
level. As shown in Figure 2B, 50 uM retigabine no longer had effect on this
mutant. In 5 min of bath application, channel current induced by depolarization
to 40 mV remained unchanged. Figure 2C shows the I-V curves before and after
the treatment of retigabine. Neither current amplitude nor voltage dependence of
the currents was modified by retigabine (Figure 2A right panel, Figure 2C). This
finding indicates that the molecular dependence of retigabine action resides
within the S5 domain.
Example 3
The S5 domains of all five KCNQ members were sequence aligned and
searched for unique amino acid residues that might account for the lack of
KCNQI's response to retigabine. As shown in Figure 3 upper panel, there are
ten unmatched residues between KCNQ1 and KCNQ5. Seven of them
highlighted in Figure 3 may be unique for KCNQ1. Therefore, mutations in
KCNQ5 were generated for each of 7 residues modified individually to the

corresponding in KCNQ1. One mutant, KCNQ5(F282Y) lost its functionality
when expressed in oocytes. The other six mutants were functional and showed
adequate level of currents in the experimental range of membrane potentials.
Retigabine's effect on each of these mutants was then tested. As shown in
Figure 3 bottom panel, two mutants, KCNQ5(A269T) and KCNQ5(W270L) (SEQ
ID NO:1, encoding SEQ ID NO:2) had significantly less response to retigabine.
Similar to the S5 domain swapped mutant in Example 2, KCNQ5(W270L)
completely lost the response to retigabine, suggesting that this tryptophan
residue is critical for retigabine action.
Example 4
To further investigate the functional dependence of retigabine effect on
this particular tryptophan residue in S5, the reversal mutation of the
corresponding residue in KCNQ1 S5 domain was made, presuming that this
mutation might make KCNQ1 capable of being activated by retigabine.
Surprisingly, this mutant, KCNQ1(L171W) indeed became sensitive to retigabine.
However, instead of being activated, KCNQ1(L171W) was blocked by retigabine.
As shown in Figure 4A, the wild type KCNQ1 channels had no significant
response to 50 µM retigabine, but a slightly inhibitory response to 200 uM
retigabine. In contrast, 50 uM retigabine clearly reduced steady state current
amplitude of KCNQ1(L171W) and 200 pM retigabine inhibited the steady-state
current by 62.5% at 80 mV (Figure 4B). Perhaps the most significant change
induced by retigabine was on channel activation. As shown in Figure 4C,
retigabine modified the activation kinetics of the current evoked at 80 mV from a
single to a double exponential activation time course, which can be described by
the modification of inactivation kinetics. Retigabine not only reduced the steady-
state current level of KCNQ1(L171W) but also modified the voltage dependence
of channel activation (Figure 4D and the inset). The highly enhanced sensitivity
to retigabine strongly suggests that the retigabine molecules are able to get
access to the tryptophan residue in S5 domain of KCNQ5 or KCNQ1(L171W),
resulting in the up or down regulation of the voltage dependent activation.

Examples 1-4 highlight the importance of the S5 helix in KCNQ5 channel
gating and provide a molecular explanation for the action of retigabine on KCNQ
potassium channels.

CLAIMS
1. An isolated polynucleotide comprising a polynucleotide selected from the
group consisting of:
(a) a nucleic acid sequence comprising SEQ ID NO:1;
(b) a polynucleotide encoding SEQ ID NO:2;
(c) a nucleic acid sequence encoding a polypeptide having at least
about 95% homology with SEQ ID NO:1, provided that a
substitution at nucleotides 808-810 is for a codon that produces a
conservative substitution for the amino acid leucine;
(d) a nucleic acid molecule which is capable of hybridizing under highly
stringent conditions to SEQ ID NO:1;
(e) a nucleic acid molecule which is complementary to (a), (b), (c), or
(d); and
(f) a variant of SEQ ID NO:1.

2. An isolated polynucleotide fragment comprising at least 12, 15, 20, 25, 30, 35,
40, 45, 50, 55, 60, 65, 75, 100, 200, 300, 400, 500, 600, or 700 contiguous
nucleotides of the sense sequence of the isolated polynucleotide of claim 1,
wherein said fragment includes nucleotides 808-810 of SEQ ID NO:1.
3. A primer or probe comprising the isolated polynucleotide fragment of claim 2.
4. A vector comprising the isolated polynucleotide of claim 1.
5. A host cell transformed with the vector of any of claim 4.

6. An isolated antisense polynucleotide which is antisense to the isolated
polynucleotide of claim 1.
7. An isolated polynucleotide encoding a KCNQ5 polypeptide containing an S5-
S6 transmembrane domain from KCNQ1.

8. The isolated polynucleotide of claim 7, wherein the S5-S6 transmembrane
domain is from human KCNQ1.
9. An isolated polynucleotide comprising a polynucleotide selected from the
group consisting of:

(a) a nucleic acid sequence comprising SEQ ID NO:3, wherein
nucleotides 769-1062 are substituted with SEQ ID NO:5;
(b) a polynucleotide encoding SEQ ID NO:4, wherein amino acids 257-
354 are substituted with an S5-S6 transmembrane domain from
KCNQ1;
(c) a nucleic acid molecule which is capable of hybridizing under highly
stringent conditions to the nucleic acid sequence of (a) or (b); and
(d) a nucleic acid molecule which is complementary to (a), (b), or (c).

10. The isolated polynucleotide of claim 9, wherein the S5-S6 transmembrane
domain of (b) is SEQ ID NO:6.
11. An isolated polynucleotide encoding a KCNQ5 polypeptide containing an S5
transmembrane domain from KCNQ1.
12. The isolated polynucleotide of claim 11, wherein the S5 transmembrane
domain is from human KCNQ1.
13. An isolated polynucleotide comprising a polynucleotide selected from the
group consisting of:

(a) a nucleic acid sequence comprising SEQ ID NO:3, wherein
nucleotides 769-873 are substituted with nucleotides 1-105 of SEQ
ID NO:5;
(b) a polynucleotide encoding SEQ ID NO:4, wherein amino acids 257-
291 of SEQ ID NO:4 are substituted with an S5 transmembrane
domain from KCNQ1;
(c) a nucleic acid molecule which is capable of hybridizing under highly
stringent conditions to the nucleic acid sequence of (a) or (b); and

(d) a nucleic acid molecule which is complementary to (a), (b), or (c).
14. The isolated polynucleotide of claim 13, wherein the S5 transmembrane
domain of (b) is amino acids 1-35 of SEQ ID NO:6.
15. An isolated polypeptide comprising an amino acid sequence selected from
the group consisting of:

(a) an amino acid sequence of a KCNQ5(W270L) polypeptide;
(b) an amino acid sequence comprising SEQ ID NO:2;
(c) a variant of (a); and
(d) an amino acid sequence having at least 90% identity to the amino
acid sequence of SEQ ID NO:2, provided that a substitution at
amino acid 270 is a conservative substitution for the amino acid
leucine.

16. An antibody which specifically binds a KCNQ5(W270L) polypeptide
comprising SEQ ID NO:2.
17. An isolated KCNQ5 polypeptide containing an S5-S6 transmembrane
domain from KCNQ1.
18. The isolated KCNQ5 polypeptide of claim 17, wherein the S5-S6
transmembrane domain is from human KCNQ1.
19. The isolated KCNQ5 polypeptide of claim 17, wherein the amino acid
sequence of the KCNQ5 polypeptide comprises SEQ ID NO:4 with amino acids
257-354 substituted with the S5-S6 transmembrane domain from KCNQ1.

20. The isolated KCNQ5 polypeptide of claim 19, wherein amino acids 257-354
of SEQ ID NO:4 are substituted with SEQ ID NO:6.
21. An isolated KCNQ5 polypeptide containing an S5 transmembrane domain
from KCNQ1

22. The isolated KCNQ5 polypeptide of claim 21, wherein the S5
transmembrane domain is from human KCNQ1.
23. The isolated KCNQ5 polypeptide of claim 21, wherein the amino acid
sequence of the KCNQ5 polypeptide comprises SEQ ID NO:4 with amino acids
257-291 substituted with the S5 transmembrane domain from KCNQ1.
24. The isolated KCNQ5 polypeptide of claim 23, wherein amino acids 257-291
of SEQ ID NO:4 are substituted with amino acids 1-35 of SEQ ID NO:6.
25. A KCNQ dimeric channel comprising at least one KCNQ5 subunit which is
the isolated polypeptide of claim 15, 17, or 21.
26. A KCNQ tetrameric channel comprising at least one KCNQ5 subunit which is
the isolated polypeptide of claim 15, 17, or 21.
27. A method of screening for agents, the method comprising:
(a) contacting an agent with a KCNQ5 molecule selected from the
group consisting of:
(i) a polynucleotide encoding all or a portion of a
KCNQ5(W270L) polypeptide;
(ii) a polynucleotide encoding a KCNQ5 polypeptide containing
an S5-S6 transmembrane domain from KCNQ1;
(iii) a polynucleotide encoding a KCNQ5 polypeptide containing
an S5 transmembrane domain from KCNQ1;
(iv) a polypeptide comprising an amino acid sequence of a
KCNQ5(W270L) polypeptide;
(v) a KCNQ5 polypeptide containing an S5-S6 transmembrane
domain from KCNQ1; and
(vi) a KCNQ5 polypeptide containing an S5 transmembrane
domain from KCNQ1; and
(b) detecting an effect of said agent on the KCNQ5 activity;

wherein detection of a decrease or an increase in KCNQ5 activity is indicative of
an agent being a modulator of KCNQ5.
28. The method of claim 27, wherein KCNQ5 activity is the reduction of neuronal
excitability or the quieting down of urinary bladder smooth muscles.
29. A method of screening for agents, the method comprising:

(a) contacting a cell with an agent; and
(b) determining the level of expression of a KCNQ5 molecule selected
from the group consisting of:
(i) a polynucleotide encoding all or a portion of a
KCNQ5(W270L) polypeptide;
(ii) a polynucleotide encoding a KCNQ5 polypeptide containing
an S5-S6 transmembrane domain from KCNQ1;
(iii) a polynucleotide encoding a KCNQ5 polypeptide containing
an S5 transmembrane domain from KCNQ1;
(iv) a polypeptide comprising an amino acid sequence of a
KCNQ5(W270L) polypeptide;
(v) a KCNQ5 polypeptide containing an S5-S6 transmembrane
domain from KCNQ1; and
(vi) a KCNQ5 polypeptide containing an S5 transmembrane
domain from KCNQ1;
wherein detection of a decrease or an increase in KCNQ5 expression is
indicative of an agent being a modulator of KCNQ5.
30. A method for identifying polypeptides capable of binding to a KCNQ5
polypeptide comprising:
(a) applying a mammalian two-hybrid procedure in which a sequence
encoding a KCNQ5 polypeptide is carried by one hybrid vector and
sequence from a cDNA or genomic DNA library is carried by the
second hybrid vector, wherein the KCNQ5 polypeptide is selected
from the group consisting of:

(i) a polypeptide comprising an amino acid sequence of a
KCNQ5(W270L) polypeptide;
(ii) a KCNQ5 polypeptide containing an S5-S6 transmembrane
domain from KCNQ1; and
(iii) a KCNQ5 polypeptide containing an S5 transmembrane
domain from KCNQ1;
(b) transforming the host cell with the vectors;
(c) isolating positive transformed cells; and
(d) extracting said second hybrid vector to obtain a sequence encoding
a polypeptide which binds to the KCNQ5 polypeptide.
31. A method for detecting a KCNQ5 polypeptide comprising detecting binding
of an antibody selected from the group consisting 6f
(a) an antibody which selectively binds a KCNQ5 polypeptide
comprising an amino acid sequence of a KCNQ5(W270L)
polypeptide;
(b) an antibody which selectively binds a KCNQ5 polypeptide
containing an S5-S6 transmembrane domain from KCNQ1;
(c) an antibody which selectively binds a KCNQ5 polypeptide
containing an S5 transmembrane domain from KCNQ1; and
(d) an antibody which selectively binds a KCNQ5(W270L) polypeptide
fragment comprising at least 8 contiguous amino acids from SEQ
ID NO:2, wherein said fragment includes amino acid 270 from SEQ
ID NO:2;
to a molecule in a sample suspected of containing a KCNQ5 polypeptide, a
KCNQ5(W270L) polypeptide, or a KCNQ5(W270L) polypeptide fragment,
wherein the antibody is contacted with the sample under conditions that permit
specific binding with any KCNQ5 polypeptide, KCNQ5(W270L) polypeptide, or
KCNQ5(W270L) polypeptide fragment present in the sample and binding of the
antibody to the molecule in the sample indicates the presence of a KCNQ5
polypeptide, KCNQ5(W270L) polypeptide, or KCNQ5(W270L) polypeptide
fragment.

32. A method for detecting expression of KCNQ5 comprising detecting mRNA
encoding a KCNQ5 polypeptide selected from the group consisting of:
(i) a polypeptide comprising an amino acid sequence of a
KCNQ5(W270L) polypeptide;
(ii) a KCNQ5 polypeptide containing an S5-S6 transmembrane domain
from KCNQ1; and
(iii) a KCNQ5 polypeptide containing an S5 transmembrane domain
from KCNQ1;
in a sample from a cell or tissue suspected of expressing KCNQ5 with a probe
comprising at least 12 contiguous nucleotides from a polynucleotide selected
from the group consisting of:
(i) a polynucleotide encoding all or a portion of a KCNQ5(W270L)
polypeptide;
(ii) a polynucleotide encoding a KCNQ5 polypeptide containing an S5-
S6 transmembrane domain from KCNQ1; and
(iii) a polynucleotide encoding a KCNQ5 polypeptide containing an S5
transmembrane domain from KCNQ1.
33. A method for determining whether a KCNQ5 gene has been mutated or
deleted comprising detecting, in a sample of cells or tissue from a subject, the
presence or absence of a genetic alteration characterized by at least one of an
alteration affecting the integrity of a gene encoding a KCNQ5 protein or the
misexpression of a KCNQ5 gene, wherein the detecting step is performed with at
least one of a probe or primer comprising at least 12 contiguous nucleotides from
a polynucleotide selected from the group consisting of:
(i) a polynucleotide encoding all or a portion of a KCNQ5(W270L)
polypeptide;
(ii) a polynucleotide encoding a KCNQ5 polypeptide containing an S5-
S6 transmembrane domain from KCNQ1; and
(iii) a polynucleotide encoding a KCNQ5 polypeptide containing an S5
transmembrane domain from KCNQ1.

34. A method of identifying variants of a KCNQ5 polypeptide comprising
screening a combinatorial library comprising KCNQ5 mutants for KCNQ5
polypeptide agonists or antagonists; wherein the KCNQ5 polypeptide is selected
from the group consisting of:
(i) a polypeptide comprising an amino acid sequence of a
KCNQ5(W270L) polypeptide;
(ii) a KCNQ5 polypeptide containing an S5-S6 transmembrane domain
from KCNQ1; and
(iii) a KCNQ5 polypeptide containing an S5 transmembrane domain
from KCNQ1.
35. A method of isolating a KCNQ5 polypeptide comprising:
(a) contacting a KCNQ5 antibody with a sample suspected of
containing a KCNQ5 polypeptide selected from the group
consisting of:
(iv) a polypeptide comprising an amino acid sequence of a
KCNQ5(W270L) polypeptide;
(v) a KCNQ5 polypeptide containing an S5-S6 transmembrane
domain from KCNQ1; and
(vi) a KCNQ5 polypeptide containing an S5 transmembrane
domain from KCNQ1; and
(b) isolating a KCNQ5 antibody-KCNQ5 polypeptide complex from the
sample.
36. A method for obtaining anti-KCNQ5 polypeptide antibodies comprising:
(a) immunizing an animal with an immunogenic KCNQ5 polypeptide or
an immunogenic portion thereof unique to a KCNQ5 polypeptide,
wherein said KCNQ5 polypeptide is selected from the group
consisting of:
(i) a polypeptide comprising an amino acid sequence of a
KCNQ5(W270L) polypeptide;
(ii) a KCNQ5 polypeptide containing an S5-S6 transmembrane
domain from KCNQ1; and

(iii) a KCNQ5 polypeptide containing an S5 transmembrane
domain from KCNQ1; and
(b) isolating from the animal antibodies that specifically bind to a
KCNQ5 polypeptide.
37. A method for assaying the ability of a KCNQ5 polypeptide to encode a
functional ion channel comprising:
(a) transfecting a host cell with a polynucleotide encoding a KCNQ5
polypeptide selected from the group consisting of:
(i) a polypeptide comprising an amino acid sequence of a
KCNQ5(W270L) polypeptide;
(ii) a KCNQ5 polypeptide containing an S5-S6 transmembrane
domain from KCNQ1; and
(iii) a KCNQ5 polypeptide containing an S5 transmembrane
domain from KCNQ1;
(b) expressing the KCNQ5 polypeptide in the host cell; and
(c) electrophysiologically measuring the ion current magnitude of the
KCNQ5 polypeptide.
38. A kit for detecting KCNQ5 polypeptide or polynucleotide comprising:
(a) a labeled compound or agent capable of detecting a KCNQ5
polypeptide or polynucleotide in a biological sample;
(b) means for determining the amount of KCNQ5 polypeptide or
polynucleotide in the sample;
(c) means for comparing the amount of KCNQ5 polypeptide or
polynucleotide in the sample with a standard; and
(d) optionally, instructions for using the kit to detect KCNQ5
polypeptide or polynucleotide;
wherein the KCNQ5 polypeptide or polynucleotide is selected from the group
consisting of:
(i) a polynucleotide encoding all or a portion of a KCNQ5(W270L)
polypeptide;

(ii) a polynucleotide encoding a KCNQ5 polypeptide containing an S5-
S6 transmembrane domain from KCNQ1;
(iii) a polynucleotide encoding a KCNQ5 polypeptide containing an S5
ransmembrane domain from KCNQ1;
(iv) a polypeptide comprising an amino acid sequence of a
KCNQ5(W270L) polypeptide;
(v) a KCNQ5 polypeptide containing an S5-S6 transmembrane domain
from KCNQ1; and
(vi) a KCNQ5 polypeptide containing an S5 transmembrane domain
from KCNQ1.
39. A kit for identifying modulators of KCNQ5 activity comprising:
(a) a cell or composition comprising a KCNQ5 polypeptide;
(b) means for determining KCNQ5 polypeptide activity; and
(c) optionally, instructions for using the kit to identify modulators of
KCNQ5 activity;
wherein the KCNQ5 polypeptide is selected from the group consisting of:
(i) a polypeptide comprising an amino acid sequence of a
KCNQ5(W270L) polypeptide;
(ii) a KCNQ5 polypeptide containing an S5-S6 transmembrane domain
from KCNQ1; and
(iii) a KCNQ5 polypeptide containing an S5 transmembrane domain
from KCNQ1
40. A kit for diagnosing a disorder associated with aberrant KCNQ5
expression and/or activity in a subject comprising:
(a) a reagent for determining expression of KCNQ5 poiypeptide or
polynucleotide;
(b) a control to which the results of the subject are compared; and
(c) optionally, instructions for using the kit for diagnostic purposes;
wherein the KCNQ5 polypeptide or polynucleotide is selected from the group
consisting of:

(i) a polynucleotide encoding all or a portion of a KCNQ5(W270L)
polypeptide;
(ii) a polynucleotide encoding a KCNQ5 polypeptide containing an S5-
S6 transmembrane domain from KCNQ1;
(iii) a polynucleotide encoding a KCNQ5 polypeptide containing an S5
transmembrane domain from KCNQ1;
(iv) a polypeptide comprising an amino acid sequence of a
KCNQ5(W270L) polypeptide;
(v) a KCNQ5 polypeptide containing an S5-S6 transmembrane domain
from KCNQ1;and
(vi) a KCNQ5 polypeptide containing an S5 transmembrane domain
from KCNQ1
41. Use of a KCNQ5 molecule in the manufacture of a medicament for
the treatment of urininary incontinence or neuropathic pain, wherein
the KCNQ5 molecule is selected from the group consisting of:
(i) a polynucleotide encoding all or a portion of a KCNQ5(W270L)
polypeptide;
(ii) a polynucleotide encoding a KCNQ5 polypeptide containing an
S5-S6 transmembrane domain from KCNQ1;
(iii) a polynucleotide encoding a KCNQ5 polypeptide containing an
S5 transmembrane domain from KCNQ1;
(iv) a polypeptide comprising an amino acid sequence of a
KCNQ5(W270L) polypeptide;
(iv) a KCNQ5 polypeptide containing an S5-S6 transmembrane
domain from KCNQ1; and
(vi) a KCNQ5 polypeptide containing an S5 transmembrane domain
from KCNQ1.

Disclosed herein are nucleic acid and polypeptide sequences of mutated KCNQ5 potassium channels which lack
responsiveness to the potassium channel activator retigabine. Also disclosed herein are methods and kits related to the use of the
aforementioned mutated KCNQ5 potassium channels.