Novel binding site for retigabine on KCNQ5
AssigneeWyeth
InventorsKun Liu, Qiang Wang, Thomas Argentieri
Life of this patent
Estimating the term…
Abstract
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.
Claims100
An isolated polynucleotide encoding all or a portion of a KCNQ5(W270L) polypeptide.
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.
An isolated polynucleotide encoding a KCNQ5 polypeptide containing an S5-S6 transmembrane domain from KCNQ1.
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).
An isolated polynucleotide encoding a KCNQ5 polypeptide containing an S5 transmembrane domain from KCNQ1.
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).
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.
An antibody which specifically binds a KCNQ5(W270L) polypeptide comprising SEQ ID NO:2.
An 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.
An isolated KCNQ5 polypeptide containing an S5-S6 transmembrane domain from KCNQ1.
An isolated KCNQ5 polypeptide containing an S5 transmembrane domain from KCNQ1.
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 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.
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 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 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.
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 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 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 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).
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 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.
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 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.
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 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.
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 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.
Dependencies are inferred from the claim text.