Novel rat voltage-gated potassium channel
AssigneeWyeth
InventorsQiang Wang, Thomas Argentieri
Life of this patent
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Abstract
Disclosed herein are nucleic acid and polypeptide sequences of a novel rat voltage-gated potassium channel, KCNQ5. Also disclosed herein are methods related to the use of the aforementioned potassium channel.
Claims95
An isolated polynucleotide encoding all or a portion of an rKCNQ5 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 non-human polypeptide having at least about 95% homology with SEQ ID NO:1;
- (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 polypeptide comprising an amino acid sequence selected from the group consisting of:
- (a) an amino acid sequence of an rKCNQ5 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.
An antibody which specifically binds a KCNQ5 polypeptide comprising SEQ ID NO:2.
An antibody which specifically binds a KCNQ5 polypeptide fragment comprising at least 8 contiguous amino acids from SEQ ID NO:2.
A method of screening for agents, the method comprising:
- (a) contacting an agent with an rKCNQ5 molecule;
- and
- (b) detecting an effect of said agent on rKCNQ5 activity;
- wherein detection of a decrease or an increase in rKCNQ5 activity is indicative of an agent being a modulator of rKCNQ5.
A method of screening for agents, the method comprising:
- (a) contacting a cell with an agent;
- and
- (b) determining the level of expression of an rKCNQ5 molecule;
- wherein detection of a decrease or an increase in rKCNQ5 expression is indicative of an agent being a modulator of rKCNQ5.
A method for identifying polypeptides capable of binding to rKCNQ5 comprising:
- (a) applying a mammalian two-hybrid procedure in which a sequence encoding a rKCNQ5 polypeptide is carried by one hybrid vector and sequence from a cDNA or genomic DNA library is carried by the second hybrid vector;
- (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 rKCNQ5 polypeptide.
A method of screening a subject for KCNQ5 mutation comprising:
- (a) measuring KCNQ5 activity;
- (b) measuring rKCNQ5 activity;
- and
- (c) comparing the measured activity in steps (a) and (b), wherein an increase or decrease is indicative of a mutation.
A method for detecting KCNQ5 polypeptide comprising detecting binding of antibody selected from the group consisting of
- (a) an antibody which selectively binds a KCNQ5 polypeptide comprising SEQ ID NO:2;
- and
- (b) an antibody which selectively binds a KCNQ5 polypeptide fragment comprising at least 8 contiguous amino acids from SEQ ID NO:2;
- to a molecule in a sample suspected of containing a KCNQ5 polypeptide,
- wherein the antibody is contacted with the sample under conditions that permit specific binding with any KCNQ5 polypeptide present in the sample and binding of the antibody to the molecule in the sample indicates the presence of KCNQ5.
A method for detecting expression of KCNQ5 comprising detecting mRNA encoding KCNQ5 in a sample from a cell or tissue suspected of expressing KCNQ5 with a probe comprising at least 12 contiguous nucleotides from nucleotides 257-2008 of SEQ ID NO:1.
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 nucleotides 257-2008 of SEQ ID NO:1.
A method of identifying KCNQ5 variants comprising screening a combinatorial library comprising rKCNQ5 mutants for KCNQ5 agonists or antagonists.
A method of isolating a KCNQ5 polypeptide comprising:
- (a) contacting an rKCNQ5 antibody with a sample suspected of containing a KCNQ5 polypeptide;
- and
- (b) isolating an rKCNQ5 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 comprising an isolated polynucleotide selected from the group consisting of:
- (i) a polynucleotide encoding a polypeptide having at least 90% identity with the amino acid sequence set forth in SEQ ID NO:2;
- (ii) a polynucleotide which hybridizes to (i) under highly stringent conditions;
- and
- (iii) a polynucleotide complementary to (i) or (ii);
- 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 an rKCNQ5 polynucleotide or polypeptide.
A method of the treatment of a mammal in need of decreased KCNQ5 activity comprising administering to the mammal in need thereof a therapeutically effective amount of an rKCNQ5 antisense polynucleotide or an rKCNQ5 antibody.
A method for obtaining anti-rKCNQ5 antibodies comprising:
- (a) immunizing an animal with an immunogenic rKCNQ5 polypeptide or an immunogenic portion thereof unique to an rKCNQ5 polypeptide;
- and
- (b) isolating from the animal antibodies that specifically bind to an rKCNQ5 polypeptide.
A method of developing a sensor cell for determining the activity of a gene comprising:
- (a) providing a homogenous population of cells, wherein each of the cells comprises a signal transduction system;
- (b) introducing into the population of cells an isolated genomic construct comprising an rKCNQ5 promoter operably linked to a targeting sequence, wherein:
- (i) the targeting sequence comprises a region of homology to a target gene sufficient to promote homologous recombination of the isolated genomic construct following introduction into the cells;
- (ii) the rKCNQ5 promoter is heterologous to the target gene;
- (iii) following recombination the promoter controls transcription of an mRNA that encodes a polypeptide comprising an activatable domain;
- and
- (iv) the polypeptide is capable, upon activation of the activatable domain, of altering the signal detected from the signal transduction system;
- (c) incubating the population of cells under conditions which cause expression of the protein;
- (d) incubating the population of cells under conditions which cause activation of the activatable domain of the polypeptide;
- and
- (e) selecting cells that have altered the signal detected from the signal transduction system.
A method for the production of a KCNQ5 polypeptide comprising:
- (a) providing a homogenous population of cells;
- (b) introducing into the population of cells an isolated genomic construct comprising a promoter operably linked to an rKCNQ5 targeting sequence, wherein
- (i) the rKCNQ5 targeting sequence comprises a region of homology to a KCNQ5 target gene sufficient to promote homologous recombination of the isolated genomic construct following introduction into the cells;
- (ii) the promoter is heterologous to the KCNQ5 target gene;
- and
- (iii) following recombination the promoter controls transcription of an mRNA that encodes a KCNQ5 polypeptide;
- and
- (c) incubating the population of cells under conditions which cause expression of the KCNQ5 polypeptide.
A method for assaying the ability of a mutant rKCNQ5 polypeptide to encode a functional ion channel comprising:
- (a) transfecting a host cell with a polynucleotide encoding the mutant rKCNQ5 polypeptide;
- (b) expressing the mutant KCNQ5 polypeptide in the host cell;
- and
- (c) electrophysiologically measuring the ion current magnitude of the mutant rKCNQ5 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 an rKCNQ5 polypeptide or agent which modulates at least one KCNQ5 polypeptide expression or at least one KCNQ5 activity.
A kit for detecting KCNQ5 polypeptide or polynucleotide comprising:
- (a) a labeled compound or agent capable of detecting an rKCNQ5 polypeptide or polynucleotide in a biological sample;
- (b) means for determining the amount of rKCNQ5 polypeptide or polynucleotide in the sample;
- (c) means for comparing the amount of rKCNQ5 polypeptide or polynucleotide in the sample with a standard;
- and
- (d) optionally, instructions for using the kit to detect rKCNQ5 polypeptide or polynucleotide.
A kit for identifying modulators of rKCNQ5 activity comprising:
- (a) a cell or composition comprising an rKCNQ5 polypeptide;
- (b) means for determining rKCNQ5 polypeptide activity;
- and
- (c) optionally, instructions for using the kit to identify modulators of KCNQ5 activity.
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 rKCNQ5 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.
Dependencies are inferred from the claim text.