EP1224208A1

EP1224208A1 - Human inwardly rectifying potassium channel subunit

Info

Publication number: EP1224208A1
Application number: EP00968530A
Authority: EP
Inventors: Richard J. Swanson; Yuan Liu; Edward Mckenna
Original assignee: Merck and Co Inc
Current assignee: Merck and Co Inc
Priority date: 1999-10-04
Filing date: 2000-09-29
Publication date: 2002-07-24
Also published as: WO2001025258A1; JP2003529332A; EP1224208A4; CA2386236A1

Abstract

The present invention is directed to novel human DNA sequences encoding Kir5.1, a potassium channel subunit, the protein encoded by the DNA sequences, vectors comprising the DNA sequences, host cells containing the vectors, and methods of identifying inhibitors and agonists of potassium channels containing the human Kir5.1 subunit.

Description

TITLE OF THE INVENTION

HUMAN INWARDLY RECTIFYING POTASSIUM CHANNEL SUBUNIT

CROSS-REFERENCE TO RELATED APPLICATIONS Not applicable.

STATEMENT REGARDING FEDERALLY-SPONSORED R&D

Not applicable.

REFERENCE TO MICROFICHE APPENDIX Not applicable.

FIELD OF THE INVENTION

The present invention is directed to a novel human DNA sequence encoding a potassium channel subunit, protein encoded by the DNA sequence, methods of expressing the protein in recombinant cells, and methods of identifying activators and inhibitors of potassium channels comprising the subunit.

BACKGROUND OF THE INVENTION Potassium channels are ion channels found in nearly all cells where they form transmembrane pores that selectively allow potassium ions to pass through the membrane. Potassium channels can be classified into various types based on their molecular architecture and the functions they perform.

One particular type of potassium channel is the inwardly rectifying potassium channel. A typical inwardly rectifying potassium channel consists of two membrane-spanning domains and a single pore domain and is believed to assemble into homo- or hetero-tetrameric ion channels. As their name suggests, inwardly rectifying potassium channels preferentially conduct potassium currents in the inward rather than the outward direction. By virtue of this property, inwardly rectifying potassium channels stabilize cellular membrane potentials near the equilibrium potential for potassium ions. They are especially important in regulating the excitability of certain cardiovascular and neurological cells and tissues.

Inwardly rectifying potassium channels, often abbreviated as "Kirs," form a family of related proteins of which at least about twenty are currently known, with more expected to be discovered. Kirs have been divided into seven subfamilies based on deduced amino acid sequences (Doupnik et al., 1995, Curr. Opin. Neurobiol. 5:268-277, Coetzee et al., 1999 Ann. N.Y. Acad. Sci 868: 233-285). Members of the different subfamilies show different functional characteristics. For example, Kir3 channels are modulated by the βγ subunits of G-proteins (Reuveny et al., 1994, Nature 370:143-146); Kir2 channels are especially strong inward rectifiers that are subject to modulation by a variety of factors, e.g., extracellular pH (Coulter et al.,

1995, Neuron 15:1157-1168), intracellular ATP concentration (Collins et al., 1996, J. Neurosci. 16:1-9), protein kinase C (Henry et al., 1996, J. Physiol. 495:681-688), G- proteins (Cohen et al., 1996, J. Biol. Chem. 271:32301-32305), and Mg2+ concentration (Chuang et al., 1997, Cell 89:1121-1132); Kir6 channels are modulated by intracellular ATP concentration (Inagaki et al., 1995, Science 270: 1166-1170); Kirl and Kir4 channels are modulated by intracelluar pH (Tsai et al., 1995, Am. J. Physiol. 268:0173-1178; Doi et al., 1996, J. Biol. Chem. 271:17261-17266; Fakler et al., 1996, EMBO J. 15:4093-4099; Schuck et al., 1997, J. Biol. Chem. 272:586- 593).

Kir5.1 has been isolated from rat (Bond et al., 1994, Recept. Channels 2:183-191 and GenBank accession no. X83581) and mouse (Mouri et al., 1998, Genomics 54: 181-182 and GenBank accession no. AB016197). Rat Kir5.1 does not form functional ion channels when expressed by itself in Xenopus laevis oocytes but instead co-assembles with other Kir channel subunits such as Kir4.1 (Pessia et al.,

1996, EMBO J. 15:2980-2987) or Kir4.2 (Pearson et al., 1999, J. Physiol. 514.3:639- 653) and modifies their functional properties. In the rat, Kir5.1 is expressed in an age-dependent fashion in all proliferating and differentiating cells of the testis (Salvatore et al., 1999, FEBS Lett. 449:146-152). Another species variant, eKir5.0 (Suzuki et al., 1999, J. Biol. Chem. 274: 11376-11382 and GenBank accession no. AB009669), is found in the gills and kidney of the eel and is highly induced in chloride cells of the gills in the saltwater species compared to the freshwater species, suggesting an important role in osmoregulation. It is desirable to discover as wide a variety as possible of other, novel potassium channel subunits related to known subunits, especially those from humans and those exhibiting restricted tissue expression. Such novel subunits would be attractive targets for drug discovery and would be valuable research tools for understanding more about ion channel biology. SUMMARY OF THE INVENTION

The present invention is directed to a novel human DNA sequence encoding Kir5.1, an inwardly rectifying potassium channel subunit. The present invention includes DNA comprising the nucleotide sequence shown as SEQ.ID.NO.:l as well as DNA comprising the coding region, positions 1 to 1254, of SEQ.ID.NO.:l. Also provided is the deduced protein sequence encoded by the novel DNA sequences. The human Kir5.1 proteins of the present invention comprise the amino acid sequence shown as SEQ.ID.NO.:2 as well as fragments thereof. Methods of expressing the novel Kir5.1 potassium channel subunit protein in recombinant systems are provided as well as methods of identifying activators and inhibitors of potassium channels comprising the subunit.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a DNA sequence encoding human Kir5.1

(SEQ.ID.NO.:l) and the corresponding amino acid sequence (SEQ.ID.NO.:2). The start ATG codon is at position 1-3; the stop codon is at position 1255-1257.

Figure 2 shows an amino acid sequence alignment of human Kir5.1 (SEQ.ID.NO.:2), mouse Kir5.1 (SEQ.ID.NO.:3), rat Kir5.1 (SEQ.ID.NO.:4), and eel Kir5.0 (SEQ.ID.NO.:5), as well as a consensus sequence (SEQ.ID.NO.:6).

Figure 3A-I shows in situ hybridization studies of human Kir5.1 in kidney and pancreas. Figure 3A shows a cross section through kidney cortex, using an antisense probe. The circular areas lighting up demonstrate that Kir5.1 is expressed in the tubules. Figure 3B shows a cross section through kidney cortex, using an sense probe. Figure 3C shows a cross section through the glomerulus, with surrounding tubules, using an antisense probe. Figure 3D shows a higher magnification of Figure 3C. Figure 3E shows hematoxylin and eosin fluorescence, demonstrating the structure of the glomerulus. Figure 3F shows hematoxylin and eosin stain, showing the structure of tubules. Figure 3G-I shows in situ hybridization of Kir5.1 in the pancreas.

Figure 4A-B shows a cDNA sequence (SEQ.ID.NO.:7) encoding the rat Kir5.1 protein (SEQ.ID.NO.:4). DETAILED DESCRIPTION OF THE INVENTION

For the purposes of this invention:

"Substantially free from other proteins" means at least 90%, preferably 95%, more preferably 99%, and even more preferably 99.9%, free of other proteins. Thus, a human Kir5.1 subunit protein preparation that is substantially free from other proteins will contain, as a percent of its total protein, no more than 10%, preferably no more than 5%, more preferably no more than 1%, and even more preferably no more than 0.1%, of proteins that are not human Kir5.1 subunit proteins. Whether a given human Kir5.1 subunit protein preparation is substantially free from other proteins can be determined by conventional techniques of assessing protein purity such as, e.g., sodium dodecyl sulfate polyacryl amide gel electrophoresis (SDS-PAGE) combined with appropriate detection methods, e.g., silver staining or immunoblotting.

"Substantially free from other nucleic acids" means at least 90%, preferably 95%, more preferably 99%, and even more preferably 99.9%, free of other nucleic acids. Thus, a human Kir5.1 subunit DNA preparation that is substantially free from other nucleic acids will contain, as a percent of its total nucleic acid, no more than 10%, preferably no more than 5%, more preferably no more than 1%, and even more preferably no more than 0.1%, of nucleic acids that are not human Kir5.1 subunit nucleic acids. Whether a given human Kir5.1 subunit DNA preparation is substantially free from other nucleic acids can be determined by conventional techniques of assessing nucleic acid purity such as, e.g., agarose gel electrophoresis combined with appropriate staining methods, e.g., ethidium bromide staining, or by sequencing.

A "conservative amino acid substitution" refers to the replacement of one amino acid residue by another, chemically similar, amino acid residue. Examples of such conservative substitutions are: substitution of one hydrophobic residue (isoleucine, leucine, valine, or methionine) for another; substitution of one polar residue for another polar residue of the same charge (e.g., arginine for lysine; glutamic acid for aspartic acid); substitution of one aromatic amino acid (tryptophan, tyrosine, or phenylalanine) for another.

A polypeptide has "substantially the same biological activity as the human Kir5.1 subunit protein" if that polypeptide is able to either form a functional potassium channel by itself, i.e., as a homomultimer, having properties similar to that of Kir5.1 channels, or combine with at least one other potassium channel subunit so as to form a complex that constitutes a functional potassium channel where the polypeptide confers upon the complex (as compared with the other subunit alone) altered electrophysiological or pharmacological properties that are similar to the electrophysiological or pharmacological properties that the native Kir5.1 protein (SEQ.ID.NO.:2) confers on the other subunit and where the polypeptide has an amino acid sequence that is at least about 50% identical to SEQ.ID.NO.:2 when measured by such standard programs as BLAST or FASTA. See, e.g.,Gish & States, 1993, Nature Genetics 3:266-272 and Altschul et al., 1990, J. Mol. Biol. 215:403-410. Examples of other potassium channel subunits with which the polypeptide may combine are: rat Kir4.1 (Bond et al., 1994, Recept. Channels 2:183-191 and GenBank accession no. X83585); human Kir4.1 (Schuck et al., 1997, J. Biol. Chem. 272: 586-593 and GenBank accession no, U52155); mouse Kir4.2 (Pearson et al., 1999, J. Physiol. 514: 639-653 and GenBank accession no. AF085696); or human Kir4.2 (Schuck et al., 1997, J. Biol. Chem. 272: 586-593 and GenBank accession no, Y10745). The present invention relates to the identification and cloning of DNA encoding the human Kir5.1 protein. Although cDNA encoding rat Kir5.1 has been isolated (Bond et al., 1994, Recept. Channels 2:183-191 and GenBank accession no. X83581), DNA encoding human Kir5.1 has not previously been reported and the rat Kir5.1 sequence contained an apparent sequencing artifact. The original rat sequence (GenBank accession no. X83581) contains an apparent sequencing insertion error of a single nucleotide at position 1080 which causes the C-terminal amino acid sequence to be out of frame and terminate prematurely. When corrected, this sequence was incomplete. The present inventors have cloned this region of the rat Kir5.1 cDNA to confirm the sequencing error in X83581, and have also identified the correct downstream sequence from another rat EST (GenBank accession no. AA892312). The presumptive correct rat Kir5.1 amino acid sequence is shown in Figure 2 as SEQ.ID.NO.:4. Mouse Kir5.1 DNA sequence (Mouri et al., 1998, Genomics 54: 181- 182 and GenBank accession no. AB016197) was used in a search of human EST databases to identify a homologous human sequence (GenBank accession no. AI636030). The DNA sequence of the entire open reading frame of human Kir5.1 was derived from a search of a human genomic DNA database, using mouse Kir5.1 (GenBank accession no. AB016197) as the query and the human sequence so obtained was confirmed by cloning human Kir5.1 cDNAs from human kidney RNA. Reverse transcriptase/polymerase chain reaction (RT-PCR) analysis showed that human Kir5.1 mRNA is expressed in human kidney, pancreas, brain, placenta, prostate, testis, lung, liver, spleen, ovary, thyroid, and small intestine. Because RT-PCR is highly sensitive and does not accurately reflect the relative abundance of mRNA, Northern blot analyses were performed as a more quantitative assessment of the expression pattern of human Kir5.1. These Northern analyses demonstrated strong expression in kidney, pancreas, and thyroid with lower levels of expression in brain, lung, liver, testes and prostate.

More detailed expression studies using in situ hybridization (see Figure 3) revealed Kir5.1 expression in the proximal and distal convoluted tubules of the human kidney. Glomeruli as well as collecting ducts were devoid of signal. In situ studies on human and monkey pancreas demonstrated that Kir5.1 expression is predominantly in acinar cells, with weaker signals, indicative of lower expression levels, noted in the cells surrounding the pancreatic ducts. Abundant expression of Kir5.1 mRNA in the convoluted tubules of the kidney and pancreatic acini, both sites of fluid regulation and maintenance of homeostatic pH balances, suggest that the Kir5.1 potassium channel subunit may have therapeutic relevance for the treatment of disorders of electrolyte balance, hypertension, renal failure, and pancreatic disease. The present invention provides DNAs encoding the human Kir5.1 subunit that are substantially free from other nucleic acids. The present invention also provides isolated and or recombinant DNA molecules encoding the human Kir5.1 subunit. The present invention provides DNA molecules substantially free from other nucleic acids comprising the nucleotide sequence shown in SEQ.ID.NO.:l.

The present invention includes isolated DNA molecules as well as DNA molecules that are substantially free from other nucleic acids comprising the coding region of SEQ.ID.NO.:l. Accordingly, the present invention includes isolated DNA molecules and DNA molecules substantially free from other nucleic acids having a sequence comprising positions 1 to 1254 of SEQJD.NO.:l.

Also included are recombinant DNA molecules having a nucleotide sequence comprising positions 1 to 1254 of SEQ.ID.NO.:l. The novel DNA sequences of the present invention encoding the human Kir5.1 subunit, in whole or in part, can be linked with other DNA sequences, i.e., DNA sequences to which the human Kir5.1 subunit is not naturally linked, to form "recombinant DNA molecules" encoding the human Kir5.1 subunit. Such other sequences can include DNA sequences that control transcription or translation such as, e.g., translation initiation sequences, internal ribosome entry sites, promoters for RNA polymerase π, transcription or translation termination sequences, enhancer sequences, sequences that control replication in microorganisms, sequences that confer antibiotic resistance, or sequences that encode a polypeptide "tag" such as, e.g., a polyhistidine tract, the FLAG epitope, or the myc epitope. The novel DNA sequences of the present invention can be inserted into vectors such as plasmids, cosmids, viral vectors, PI artificial chromosomes, or yeast artificial chromosomes.

Included in the present invention are DNA sequences that hybridize to SEQ.ID.NO: 1 under conditions of high stringency. By way of example, and not limitation, a procedure using conditions of high stringency is as follows: Prehybridization of filters containing DNA is carried out for 2 hr. to overnight at 65°C in buffer composed of 6X SSC, 5X Denhardt's solution, and 100 μg/ml denatured salmon sperm DNA. Filters are hybridized for 12 to 48 hrs at 65°C in prehybridization mixture containing 100 μg/ml denatured salmon sperm DNA and 5- 20 X lθ6 cpm of 32p_ι_abeled probe. Washing of filters is done at 37°C for 1 hr in a solution containing 2X SSC, 0.1% SDS. This is followed by a wash in 0.1X SSC, 0.1% SDS at 50°C for 45 min. before autoradiography.

Other procedures using conditions of high stringency would include either a hybridization carried out in 5XSSC, 5X Denhardt's solution, 50% formamide at 42°C for 12 to 48 hours or a washing step carried out in 0.2X SSPE, 0.2% SDS at 65°C for 30 to 60 minutes.

Reagents mentioned in the foregoing procedures for carrying out high stringency hybridization are well known in the art. Details of the composition of these reagents can be found in, e.g., Sambrook, Fritsch, and Maniatis, 1989, Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press. In addition to the foregoing, other conditions of high stringency which may be used are well known in the art.

The degeneracy of the genetic code is such that, for all but two amino acids, more than a single codon encodes a particular amino acid. This allows for the construction of synthetic DNA that encodes the human Kir5.1 subunit protein where the nucleotide sequence of the synthetic DNA differs significantly from the nucleotide sequences of SEQ.ID.NO:!, but still encodes the same human Kir5.1 subunit protein as SEQ.ID.NO: 1. Such synthetic DNAs are intended to be within the scope of the present invention.

Mutated forms of SEQ.ID.NO: 1 are intended to be within the scope of the present invention. In particular, mutated forms of SEQ.ID.NO: 1 encoding a protein that, when combined with other potassium channel subunits, gives rise to potassium channels having altered voltage sensitivity, current carrying properties, or other properties as compared to potassium channels formed by combination of wild- type Kir5.1 protein (SEQ.ID.NO:2) with the other potassium channel subunit, are within the scope of the present invention. Such mutant forms can differ from SEQ.ID.NO: 1 by having nucleotide deletions, substitutions, or additions.

Also intended to be within the scope of the present invention are RNA molecules having sequences corresponding to SEQ.ID.NO: 1. Antisense nucleotides, DNA or RNA, that are the reverse complements of SEQ.ID.NO: 1, or portions thereof, are also within the scope of the present invention. In addition, polynucleotides based on SEQ.ID.NO: 1 in which a small number of positions are substituted with non- natural or modified nucleotides such as inosine, methyl-cytosine, or deaza-guanosine are intended to be within the scope of the present invention. Polynucleotides of the present invention can also include sequences based on SEQ.ID.NO: 1 but in which non-natural linkages between the nucleotides are present. Such non-natural linkages can be, e.g., methylphosphonates, phosphorothioates, phosphorodithionates, phosphoroamidites, and phosphate esters. Polynucleotides of the present invention can also include sequences based on SEQ.ID.NO: 1 but having de-phospho linkages as bridges between nucleotides, e.g., siloxane, carbonate, carboxymethyl ester, acetamidate, carbamate, and thioether bridges. Other intemucletide linkages that can be present include N-vinyl, methacryloxyethyl, methacrylamide, or ethyleneimine linkages. Peptide nucleic acids based upon SEQ.ID.NO: 1 are also included in the present invention.

Another aspect of the present invention includes host cells that have been engineered to contain and/or express DNA sequences encoding the human Kir5.1 subunit protein. Such recombinant host cells can be cultured under suitable conditions to produce human Kir5.1 subunit protein. An expression vector containing DNA encoding the human Kir5.1 subunit protein can be used for the expression of the human Kir5.1 subunit protein in a recombinant host cell. Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to, bacteria such as E. coli, fungal cells such as yeast, mammalian cells including, but not limited to, cell lines of human, bovine, porcine, monkey and rodent origin, amphibian cells such as Xenopus oocytes, and insect cells including but not limited to Drosophila and silkworm derived cell lines. Cells and cell lines which are suitable for recombinant expression of the human Kir5.1 subunit protein and which are widely available, include but are not limited to, L cells L-M(TK^") (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171), CPAE (ATCC CCL 209), Saos-2 (ATCC HTB-85), ARPE-19 human retinal pigment epithelium (ATCC CRL-2302), Xenopus melanophores, and Xenopus oocytes.

A variety of mammalian expression vectors can be used to express recombinant human Kir5.1 subunit protein in mammalian cells. Commercially available mammalian expression vectors which are suitable include, but are not limited to, pMClneo (Stratagene), pSG5 (Stratagene), pcDNAI and pcDNAIamp, pcDNA3, pcDNA3.1, pCR3.1 (Invitrogen), EBO-pSV2-neo (ATCC 37593), pBPV- 1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pLZD35 (ATCC 37565), and pSV2-dhfr (ATCC 37146). Another suitable vector is the PT7TS oocyte expression vector.

Following expression in recombinant cells, human Kir5.1 subunit protein can be purified by conventional techniques to a level that is substantially free from other proteins. Techniques that can be used include ammonium sulfate precipitation, hydrophobic or hydrophilic interaction chromatography, ion exchange chromatography, affinity chromatography, phosphocellulose chromatography, size exclusion chromatography, preparative gel electrophoresis, and alcohol precipitation. In some cases, it may be advantageous to employ protein denaturing and/or refolding steps in addition to such techniques.

Certain potassium channel subunits have been found to require the expression of other potassium channel subunits in order to be properly expressed at high levels and inserted in membranes. For example, co-expression of KCNQ3 appears to enhance the expression of KCNQ2 in Xenopus oocytes (Wang et al., 1998, Science 282:1890-1893). Also, some voltage-gated potassium channel Kvα subunits require other related α subunits or Kvβ subunits (Shi et al., 1995, Neuron 16:843- 852). Accordingly, the recombinant expression of the human Kir5.1 subunit proteins may under certain circumstances benefit from the co-expression of other potassium channel proteins and such co-expression is intended to be within the scope of the present invention. A particularly preferred form of co-expression is the co-expression of a human Kir5.1 subunit protein with a human Kir4.1 subunit protein or with a human Kir4.2 subunit protein. Such co-expression can be effected by transfecting an expression vector encoding a human Kir5.1 subunit protein into a cell that naturally expresses a human Kir4.1 subunit protein or a human Kir4.2 subunit protein. Alternatively, an expression vector encoding a human Kir5.1 subunit protein can be transfected into a cell in which an expression vector encoding a human Kir4.1 subunit protein or a human Kir4.2 subunit protein has also been transfected. Preferably, such a cell does not naturally express human Kir4.1 subunit protein or human Kir4.2 subunit protein.

The present invention includes human Kir5.1 subunit proteins substantially free from other proteins. The deduced amino acid sequence of the full- length human Kir5.1 subunit protein is shown in SEQ.ID.NO. :2. Thus, the present invention includes human Kir5.1 subunit protein substantially free from other proteins having the amino acid sequence SEQ.ID.NO. :2. The present invention also includes isolated human Kir5.1 subunit protein having the amino acid sequence SEQ.ID.NO.:2.

Mutated forms of human Kir5.1 subunit proteins are intended to be within the scope of the present invention. In particular, mutated forms of SEQ.ID.NO:2 that give rise to potassium channels having altered electrophysiological or pharmacological properties when combined with other potassium channel subunits are within the scope of the present invention.

As with many proteins, it may be possible to modify many of the amino acids of the human Kir5.1 subunit protein and still retain substantially the same biological activity as for the original protein. Thus, the present invention includes modified human Kir5.1 subunit proteins which have amino acid deletions, additions, or substitutions but that still retain substantially the same biological activity as naturally occurring human Kir5.1 subunit protein. It is generally accepted that single amino acid substitutions do not usually alter the biological activity of a protein (see, e.g., Molecular Biology of the Gene, Watson et al., 1987, Fourth Ed., The Benjamin/Cummings Publishing Co., Inc., page 226; and Cunningham & Wells, 1989, Science 244:1081-1085). Accordingly, the present invention includes polypeptides where one amino acid substitution has been made in SEQ.ID.NO:2 wherein the polypeptides still retain substantially the same biological activity as naturally occurring human Kir5.1 subunit protein. The present invention also includes polypeptides where two or more amino acid substitutions have been made in SEQ.ID.NO:2 wherein the polypeptides still retain substantially the same biological activity as naturally occurring human Kir5.1 subunit protein. In particular, the present invention includes embodiments where the above-described substitutions are conservative substitutions. In particular, the present invention includes embodiments where the above-described substitutions do not occur in conserved positions.

Conserved positions are those positions in which the human Kir5.1, mouse Kir5.1, rat Kir5.1, and eel Kir5.0 proteins all have the same amino acid (see Figure 2).

The human Kir5.1 subunit proteins of the present invention may contain post-translational modifications, e.g., covalently linked carbohydrate, phosphorylation, myristoylation, palmytoylation, etc..

The present invention also includes chimeric human Kir5.1 subunit proteins. Chimeric human Kir5.1 subunit proteins consist of a contiguous polypeptide sequence of at least a portion of a human Kir5.1 subunit protein fused to a polypeptide sequence that is not from a human Kir5.1 subunit protein. Preferred chimeric human Kir5.1 subunit proteins are those in which a human Kir5.1 subunit protein is fused to a human, mouse, or rat Kir4.1 protein or is fused to a human, mouse, or rat Kir4.2 protein where the human Kir5.1 protein is in the C terminal position of the fusion protein.

The present invention also includes isolated human Kir5.1 subunit protein and DNA encoding the isolated subunit. Use of the term "isolated" indicates that the human Kir5.1 subunit protein or DNA has been removed from its normal cellular environment. Thus, an isolated human Kir5.1 subunit protein may be in a cell-free solution or placed in a different cellular environment from that in which it occurs naturally. The term isolated does not necessarily imply that an isolated human Kir5.1 subunit protein is the only protein present (although that is one of the meanings of isolated), but instead means that the isolated human Kir5.1 subunit protein is at least 95% free of non-amino acid material (e.g., nucleic acids, lipids, carbohydrates) naturally associated with the human Kir5.1 subunit protein. Thus, a human Kir5.1 subunit protein that is expressed in bacteria or even in eukaryotic cells which do not naturally (i.e., without human intervention) express it through recombinant means is an "isolated human Kir5.1 subunit protein."

It is known that certain potassium channel subunits can interact to form heteromeric complexes resulting in functional potassium channels. For example, KCNQ2 and KCNQ3 can assemble to form a heteromeric functional potassium channel (Wang et al., 1998, Science 282:1890-1893). Accordingly, it is believed likely that the human Kir5.1 subunit protein of the present invention will also be able to form heteromeric structures with other proteins where such heteromeric structures constitute functional potassium channels. Thus, the present invention includes such heteromers comprising human Kir5.1 subunit protein. Preferred heteromers are those in which the human Kir5.1 subunit proteins of the present invention forms heteromers with human Kir4.1 or Kir4.2.

DNA encoding the human Kir5.1 subunit protein can be obtained by methods well known in the art. For example, a cDNA fragment encoding full-length human Kir5.1 protein can be isolated from human kidney or pancreas cDNA by using the polymerase chain reaction (PCR) employing suitable primer pairs. Such primer pairs can be selected based upon the DNA sequence encoding the human Kir5.1 protein shown in Figure 1 as SEQ.ID.NO.: 1. Suitable primer pairs would be, e.g.:

5' ATG AGC TAT TAC GGC AGC AGC 3' (SEQ.ID.NO.:8)

5' CTA CAT TTG GGA TTC TAC AGA GAT T 3' (SEQ.ID.NO.:9)

The above primers are meant to be illustrative only; one skilled in the art would readily be able to design other suitable primers based upon SEQ.ID.NO.: 1. Such primers could be produced by methods of oligonucleotide synthesis that are well known in the art.

PCR reactions can be carried out with a variety of thermostable enzymes including but not limited to AmpliTaq, AmpliTaq Gold, or Vent polymerase. For AmpliTaq, reactions can be carried out in 10 mM Tris-Cl, pH 8.3, 2.0 mM MgCl2, 200 μM of each dNTP, 50 mM KCl, 0.2 μM of each primer, 10 ng of DNA template, 0.05 units/μl of AmpliTaq. The reactions are heated at 95°C for 3 minutes and then cycled 35 times using the cycling parameters of 95°C, 20 seconds, 62°C, 20 seconds, 72°C, 3 minutes. In addition to these conditions, a variety of suitable PCR protocols can be found in PCR Primer. A Laboratory Manual, edited by C.W. Dieffenbach and G.S. Dveksler, 1995, Cold Spring Harbor Laboratory Press; or PCR Protocols: A Guide to Methods and Applications, Michael et al., eds., 1990, Academic Press .

Since the Kir5.1 channel subunit of the present invention is highly homologous to other potassium channel subunits, it is desirable to sequence the clones obtained by the herein-described methods, in order to verify that the desired human Kir5.1 subunit has in fact been obtained.

By these methods, cDNA clones encoding the human Kir5.1 subunit protein can be obtained. These cDNA clones can be cloned into suitable cloning vectors or expression vectors, e.g., the mammalian expression vector pcDNA3.1

(Invitrogen, San Diego, CA). Human Kir5.1 subunit protein can then be produced by transferring expression vectors encoding the subunit or portions thereof into suitable host cells and growing the host cells under appropriate conditions. Human Kir5.1 subunit protein can then be isolated by methods well known in the art. As an alternative to the above-described PCR methods, cDNA clones encoding the human Kir5.1 subunit protein can be isolated from cDNA libraries using, as a probe, oligonucleotides specific for the human Kir5.1 subunit and methods well known in the art for screening cDNA libraries with oligonucleotide probes. Such methods are described in, e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, New York; Glover, D.M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K., Vol. I, π. Oligonucleotides that are specific for human Kir5.1 subunit protein and that can be used to screen cDNA libraries can be readily designed based upon the DNA sequence shown in Figure 1 and can be synthesized by methods well- known in the art.

Genomic clones containing the human Kir5.1 subunit gene can be obtained from commercially available human PAC or BAC libraries available from Research Genetics, Huntsville, AL. Alternatively, one may prepare genomic libraries, e.g., in PI artificial chromosome vectors, from which genomic clones containing the human Kir5.1 subunit gene can be isolated, using probes based upon the human

Kir5.1 subunit DNA sequence disclosed herein. Methods of preparing such libraries are known in the art (see, e.g., Ioannou et al.,1994, Nature Genet. 6:84-89).

The novel DNA sequences of the present invention can be used in various diagnostic methods. The present invention provides diagnostic methods for determining whether a patient carries a mutation in the human Kir5.1 subunit gene. In broad terms, such methods comprise determining the DNA sequence of a region in or near the human Kir5.1 subunit gene from the patient and comparing that sequence to the sequence from the corresponding region of the human Kir5.1 subunit gene from a non-affected person, i.e., a person who does not have the condition which is being diagnosed, where a difference in sequence between the DNA sequence of the gene from the patient and the DNA sequence of the gene from the non-affected person indicates that the patient has a mutation in the human Kir5.1 subunit gene.

The present invention also provides oligonucleotide probes, based upon SEQ.ID.NO: 1 that can be used in diagnostic methods to identify patients having mutated forms of the human Kir5.1 subunit, to determine the level of expression of RNA encoding the human Kir5.1 subunit, or to isolate genes homologous to the human Kir5.1 subunit from other species. In particular, the present invention includes DNA oligonucleotides comprising at least about 10, 15, or 18 contiguous nucleotides of SEQ.ID.NO: 1 where the oligonucleotide probe comprises no stretch of contiguous nucleotides longer than 5 from:SEQ.ID.NO:l other than the said at least about 10, 15, or 18 contiguous nucleotides. The oligonucleotides can be substantially free from other nucleic acids. Also provided by the present invention are corresponding RNA oligonucleotides. The DNA or RNA oligonucleotides can be packaged in kits. The present invention makes possible the recombinant expression of human Kir5.1 subunit protein in various cell types. Such recombinant expression makes possible the study of this protein so that its biochemical activity and its role in various diseases such as renal failure, hypokalemia, hypertension, hypotension, thyroid disease, and pancreatitis can be elucidated. The present invention also makes possible the development of assays which measure the biological activity of potassium channels containing human Kir5.1 subunit protein. Assays using recombinantly expressed human Kir5.1 subunit protein are especially of interest. Such assays can be used to screen libraries of compounds or other sources of compounds to identify compounds that are activators or inhibitors of the activity of potassium channels containing human Kir5.1 subunit protein. Such identified compounds can serve as "leads" for the development of pharmaceuticals that can be used to treat patients having diseases in which it is beneficial to enhance or suppress potassium channel activity. In versions of the above-described assays, potassium channels containing mutant human Kir5.1 subunit proteins are used and inhibitors or activators of the activity of the mutant potassium channels are identified.

Preferred cell lines for recombinant expression of human Kir5.1 subunit protein are those which do not express endogenous potassium channels (e.g., CV-1, NIH-3T3, CHO-K1, COS-7). Such cell lines can be exposed to 86R_D, an ion which can pass through potassium channels. The influx of 86Rb into such cells can be assayed in the presence and absence of collections of substances (e.g., combinatorial libraries, natural products, analogues of lead compounds produced by medicinal chemistry), or members of such collections, and those substances that are able to alter 86Rb influx thereby identified. Such substances are likely to be activators or inhibitors of potassium channels containing human Kir5.1 subunit protein.

Activators and inhibitors of potassium channels containing human Kir5.1 subunit protein are likely to be substances that are capable of binding to potassium channels containing human Kir5.1 subunit protein. Thus, one type of assay determines whether one or more of a collection of substances is capable of such binding.

Accordingly, the present invention provides a method for identifying substances that bind to potassium channels containing human Kir5.1 subunit protein comprising:

(a) providing cells expressing a potassium channel containing human Kir5.1 subunit protein;

(b) exposing the cells to a substance that is not known to bind potassium channels containing human Kir5.1 subunit protein;

(c) determining the amount of binding of the substance to the cells;

(d) comparing the amount of binding in step (c) to the amount of binding of the substance to control cells where the control cells are substantially identical to the cells of step (a) except that the control cells do not express human Kir5.1 subunit protein; where if the amount of binding in step (c) is greater than the amount of binding of the substance to control cells, then the substance binds to potassium channels containing human Kir5.1 subunit protein. An example of control cells that are substantially identical to the cells of step (a) would be a parent cell line where the parent cell line is transfected with an expression vector encoding Kir5.1 protein in order to produce the cells expressing a potassium channel containing human Kir5.1 protein of step (a). Another version of this assay makes use of compounds that are known to bind to potassium channels containing human Kir5.1 subunit protein. Substances that are new binders are identified by virtue of their ability to augment or block the binding of these known compounds. This can be done if the known compound is used at a concentration that is far below saturation, in which case a substance that is a new binder is likely to be able to either augment or block the binding of the known compound. Substances that have this ability are likely themselves to be inhibitors or activators of potassium channels containing human Kir5.1 subunit protein.

Accordingly, the present invention includes a method of identifying substances that bind potassium channels containing human Kir5.1 subunit protein and thus are likely to be inhibitors or activators of potassium channels containing human Kir5.1 subunit protein comprising:

(a) providing cells expressing potassium channels containing human Kir5.1 subunit protein;

(b) exposing the cells to a compound that is known to bind to the potassium channels containing human Kir5.1 subunit protein in the presence and in the absence of a substance not known to bind to potassium channels containing human Kir5.1 subunit protein;

(c) determining the amount of binding of the compound to the cells in the presence and in the absence of the substance not known to bind to potassium channels containing human Kir5.1 subunit protein; where if the amount of binding of the compound in the presence of the substance differs from that in the absence of the substance, then the substance binds potassium channels containing human Kir5.1 subunit protein and is likely to be an inhibitor or activator of potassium channels containing human Kir5.1 subunit protein. Generally, the known compound is labeled (e.g., radioactively, enzymatically, fluorescently) in order to facilitate measuring its binding to the potassium channels. Once a substance has been identified by the above-described methods, it can be assayed in functional tests, such as those described herein, in order to determine whether it is an inhibitor or an activator.

In particular embodiments, the compound known to bind potassium channels containing human Kir5.1 subunit protein is selected from the group consisting of:

Lq2 (a scorpion toxin) (Renisio et al., 1999, Proteins 34: 417- 426); delta dendrotoxin (a snake toxin) (Imredy et al., 1998, Biochemistry 37:14867-14874);

Bainh ( a sea anemone toxin) (Salinas et al., 1997, Toxicon 35:1699- 1709);

Tertiapin (a toxin from bee venom) (Jin & Lu, 1998, Biochemistry 37:13291-13299); RP58866 (Escande et al., 1992, J. Cardiovascular Pharmacol. 20

Suppl. 2: S106-113); terikalent (RP62719) (Escande et al., 1992, J. Cardiovascular Pharmacol. 20 Suppl. 2: S 106-113); quinine; and verapamil.

The present invention includes a method of identifying activators or inhibitors of potassium channels containing human Kir5.1 subunit protein comprising:

(a) recombinantly expressing human Kir5.1 subunit protein or mutant human Kir5.1 subunit protein in a host cell so that the recombinantly expressed human Kir5.1 subunit protein forms potassium channels either by itself or by forming heteromers with other potassium channel subunit proteins;

(b) measuring the biological activity of the potassium channels formed in step (a) in the presence and in the absence of a substance suspected of being an activator or an inhibitor of potassium channels containing human Kir5.1 subunit protein; where a change in the biological activity of the potassium channels formed in step (a) in the presence as compared to the absence of the substance indicates that the substance is an activator or an inhibitor of potassium channels containing human Kir5.1 subunit protein.

In particular embodiments of the methods described herein, the biological activity is the production of an inwardly rectifying potassium current or the influx of 86Rb.

In particular embodiments, it may be advantageous to recombinantly express the other subunits of potassium channels such as, e.g., the human Kir4.1 or Kir4.2 subunit. Alternatively, it may be advantageous to use host cells that endogenously express such other subunits. In particular embodiments, a vector encoding human Kir5.1 subunit protein is transferred into Xenopus oocytes in order to cause the expression of human Kir5.1 subunit protein in the oocytes. Alternatively, RNA encoding human Kir5.1 subunit protein can be prepared in vitro and injected into the oocytes, also resulting in the expression of human Kir5.1 subunit protein in the oocytes. Following expression of the human Kir5.1 subunit protein in the oocytes, and following the formation of potassium channels containing these subunits and other potassium subunits (which other subunits may also be transferred into the oocytes), membrane currents are measured after the transmembrane voltage is changed in steps. A change in membrane current is observed when the potassium channels open or close, modulating potassium ion flow. Similar studies were reported for KCNQ2 and KCNQ3 potassium channels in Wang et al., 1998, Science 282:1890-1893 and for MinK channels by Goldstein & Miller, 1991, Neuron 7:403-408. These references and references cited therein can be consulted for guidance as to how to carry out such studies. In such studies it is especially preferred to co-express Kir4.1 or Kir4.2 potassium channel subunits in the oocytes.

Inhibitors or activators of potassium channels containing human Kir5.1 subunit protein can be identified by exposing the oocytes to individual substances or collections of substances and determining whether the substances can block/diminish or enhance the membrane currents observed in the absence of the substance. Accordingly, the present invention provides a method of identifying inhibitors or activators of potassium channels containing human Kir5.1 subunit protein comprising:

(a) expressing human Kir5.1 subunit protein in cells such that potassium channels containing the human Kir5.1 subunit protein are formed; (b) changing the transmembrane potential of the cells in the presence and the absence of a substance suspected of being an inhibitor or an activator of potassium channels containing human Kir5.1 subunit protein;

(c) measuring membrane potassium currents following step (b); where if the potassium membrane currents measured in step (c) are greater in the absence rather than in the presence of the substance, then the substance is an inhibitor of potassium channels containing human Kir5.1 subunit protein; where if the potassium membrane currents measured in step (c) are greater in the presence rather than in the absence of the substance, then the substance is an activator of potassium channels containing human Kir5.1 subunit protein.

The present invention also includes assays for the identification of activators and inhibitors of potassium channels containing human Kir5.1 subunit protein that are based upon fluorescence resonance energy transfer (FRET) between a first and a second fluorescent dye where the first dye is bound to one side of the plasma membrane of a cell expressing potassium channels containing human Kir5.1 subunit protein and the second dye is free to shuttle from one face of the membrane to the other face in response to changes in membrane potential. In certain embodiments, the first dye is impenetrable to the plasma membrane of the cells and is bound predominately to the extracellular surface of the plasma membrane. The second dye is trapped within the plasma membrane but is free to diffuse within the membrane. At normal (i.e., negative) resting potentials of the membrane, the second dye is bound predominately to the inner surface of the extracellular face of the plasma membrane, thus placing the second dye in close proximity to the first dye. This close proximity allows for the generation of a large amount of FRET between the two dyes. Following membrane depolarization, the second dye moves from the extracellular face of the membrane to the intracellular face, thus increasing the distance between the dyes. This increased distance results in a decrease in FRET, with a corresponding increase in fluorescent emission derived from the first dye and a corresponding decrease in the fluorescent emission from the second dye. In this way, the amount of FRET between the two dyes can be used to measure the polarization state of the membrane. For a description of this technique, see Gonzalez & Tsien, 1997, Chemistry & Biology 4:269-277. See also Gonzalez & Tsien, 1995, Biophys. J. 69:1272-1280 and U.S. Patent No. 5,661,035. In certain embodiments, the first dye is a fluorescent lectin or a fluorescent phospholipid that acts as the fluorescent donor. Examples of such a first dye are: a coumarin-labeled phosphatidylethanolamine (e.g., N-(6-chloro-7-hydroxy- 2-OXO-2H— l-benzopyran-3-carboxamidoacetyl)-dimyristoylphosphatidyl- ethanolamine) or N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)- dipalmitoylphosphatidylethanolamine); a fluorescently-labeled lectin (e.g., fluorescein-labeled wheat germ agglutinin). In certain embodiments, the second dye is an oxonol that acts as the fluorescent acceptor. Examples of such a second dye are: bis(l,3-dialkyl-2-thiobarbiturate)trimethineoxonols (e.g., bis(l,3-dihexyl-2- thiobarbiturate)trimethineoxonol) or pentamethineoxonol analogues (e.g., bis(l,3- dihexyl-2-thiobarbiturate)pentamethineoxonol; or bis(l,3-dibutyl-2- thiobarbiturate)pentamethineoxonol). See Gonzalez & Tsien, 1997, Chemistry & Biology 4:269-277 for methods of synthesizing various dyes suitable for use in the present invention. In certain embodiments, the assay may comprise a natural carotenoid, e.g., astaxanthin, in order to reduce photodynamic damage due to singlet oxygen.

The above described assays can be utilized to discover activators and inhibitors of potassium channels containing human Kir5.1 subunit protein. Such assays will generally utilize cells that express potassium channels containing human Kir5.1 subunit protein, e.g., by transfection with expression vectors encoding human Kir5.1 subunit protein and, optionally, other potassium channel subunits.

The cellular membrane potential is determined by the balance between inward (depolarizing) and outward (repolarizing) ionic fluxes through various ion pumps and channels. Functional Kirs, such as a hetero-multimeric potassium channel containing Kir5.1 subunits, are typically highly selective for K+ and, therefore, exhibit reversal potentials close to the potassium equilibrium potential (EK)- Kirs thus function to maintain the resting membrane potential of a cell near EK- The presence of an inhibitor of a potassium channel containing Kir5.1 will prevent, or diminish, the ability of this channel to maintain this polarized (i.e., negative) membrane potential and the cell will, therefore, depolarize. Thus, membrane potential will tend to become more positive in the presence of Kir inhibitors. Changes in membrane potential that are caused by inhibitors of potassium channels containing Kir5.1 protein can be monitored by the assays using FRET described above. Accordingly, the present invention provides a method of identifying inhibitors of potassium channels containing human Kir5.1 subunit protein comprising:

(a) providing cells comprising: (1) an expression vector that directs the expression of human Kir5.1 subunit protein in the cells so that potassium channels containing human Kir5.1 subunit protein are formed in the cells;

(2) a first fluorescent dye, where the first dye is bound to one side of the plasma membrane of the cells; and (3) a second fluorescent dye, where the second fluorescent dye is free to distribute from one face of the plasma membrane of the cells to the other face in response to changes in membrane potential;

(b) exposing the cells to a substance that is suspected of being an inhibitor of potassium channels containing human Kir 5.1 subunit protein; (c) measuring the amount of fluorescence resonance energy transfer (FRET) in the cells in the presence and in the absence of the substance;

(d) comparing the amount of FRET exhibited by the cells in the presence and in the absence of the substance; where if the amount of FRET exhibited by the cells in the presence of the substance is less than the amount of FRET exhibited by the cells in the absence of the substance then the substance is an inhibitor of potassium channels containing human Kir5.1 subunit protein.

The membrane potential of the cells can be made more positive than the potassium equilibrium potential in a variety of ways. For example, ionophores, inhibitors of Na+/K+ ATPase, toxins or inhibitors of other potassium channels, modulators of C1-, Na+, and Ca++ channel, and detergent like compounds (to increase the permeability of the cellular membrane) could be used.

The resting membrane potential of a cell depends on the balance of inward and outward currents that are active in that cell. Thus, if an inwardly rectifying K+ current, e.g., Kir5.1, is the only or predominant current, the resting membrane potential will be near EK- However, if counteracting depolarizing currents are also found in the cell, the resting membrane potential will be more depolarized and the exact value will depend on the relative magnitudes of the depolarizing and hyperpolarizing currents. One can therefore construct a cell line that expresses an inwardly rectifying potassium channel and exhibits a more positive resting membrane potential by co-expression of a depolarizing current in the same cell. Examples of such depolarizing currents could be those carried by Na+, Ca++, or Cl- ions, or combinations thereof. In these cells, an activator of the inwardly rectifying potassium channel would increase the relative contribution of the Kir current compared to the depolarizing current and, in doing so, make the membrane potential more negative (i.e., drive it closer to EK)- Changes in membrane potential that are caused by activators of potassium channels containing Kir5.1 protein in such cells can be monitored by the assays using FRET described above. Accordingly, the present invention provides a method of identifying activators of potassium channels containing human Kir5.1 subunit protein comprising:

(a) providing cells comprising:

(1) expression vectors that direct the expression of human Kir5.1 subunit protein and a depolarizing channel in the cells so that potassium channels containing human Kir5.1 subunit protein and a depolarizing channel are both expressed in the same cells;

(b) exposing the cells to a substance suspected of being an activator of potassium channels containing the human Kir5.1 subunit protein; (c) measuring the amount of fluorescence resonance energy transfer (FRET) in the cells in the presence and in the absence of the substance;

(d) comparing the amount of FRET exhibited by the cells in the presence and in the absence of the substance; wherein if the amount of FRET exhibited by the cells in the presence of the substance is greater than the amount of FRET exhibited by the cells in the absence of the substance then the substance may be an activator of potassium channels containing human Kir5.1 subunit protein.

The substances identified by the above-described method may either be activators of potassium channels containing human Kir5.1 subunit protein or the substances may be inhibitors of the depolarizing current or currents. These two possibilities can be distinguished by expressing the depolarizing channels alone, i.e., without the potassium channels containing human Kir5.1 subunit protein, in another cell line. The substances can then be tested against cells containing the depolarizing currents alone, and it can be determined if the substances are able to inhibit the depolarizing currents. Alternatively, these substances can be directly tested on the potassium channel containing human Kir5.1 subunits in voltage clamp experiments to determine if they are activators of that channel.

The above-described method can be used to identify inhibitors of potassium channels containing human Kir5.1 subunit protein as well. This is because the presence of an inhibitor will block or diminish current movement through the Kir5.1 channels, thus preventing or lessening the ability of the Kir5.1 channels to counteract the effect of the depolarizing channels. Thus, the present invention includes a method of identifying inhibitors of potassium channels containing human Kir5.1 subunit protein comprising:

(a) providing cells comprising:

(2) a first fluorescent dye, where the first dye is bound to one side of the plasma membrane of the cells; and

(3) a second fluorescent dye, where the second fluorescent dye is free to distribute from one face of the plasma membrane of the cells to the other face in response to changes in membrane potential;

(b) exposing the cells to a substance that is suspected of being an inhibitor of potassium channels containing human Kir5.1 subunit protein;

(c) measuring the amount of fluorescence resonance energy transfer (FRET) in the cells in the presence and in the absence of the substance; (d) comparing the amount of FRET exhibited by the cells in the presence and in the absence of the substance; wherein if the amount of FRET exhibited by the cells in the presence of the substance is less than the amount of FRET exhibited by the cells in the absence of the substance then the substance may be an inhibitor of potassium channels containing human Kir5.1 subunit protein.

The substances identified by the above-described method may either be inhibitors of potassium channels containing human Kir5.1 subunit protein or the substances may be activators of the depolarizing current or currents. These two possibilities can be distinguished by expressing the depolarizing channels alone, i.e., without the potassium channels containing human Kir5.1 subunit protein, in another cell line. The substances can then be tested against cells containing the depolarizing currents alone, and it can be determined if the substances are able to activate the depolarizing currents. Alternatively, these substances can be directly tested on the potassium channel containing human Kir5.1 subunits in voltage clamp experiments to determine if they are inhibitors of that channel.

In particular embodiments of the above-described methods, the depolarizing channel is a sodium, calcium, non-specific cation, or chloride channel. In order to be sure that the effect of the substance in the above- described assays is arising through its action at potassium channels containing human Kir5.1 subunit protein, control experiments can be run in which the cells are as above, except that they do not contain an expression vector that directs the expression of human Kir5.1 subunit protein. In particular embodiments of the above-described methods, the expression vector is transfected into the test cells.

In particular embodiments of the above-described methods, the human Kir5.1 subunit protein has the amino acid sequence shown in SEQ.ID.NO. :2. In particular embodiments of the above-described methods, the expression vector comprises positions 1 to 1254 of SEQ.ID.NO.: 1.

In particular embodiments of the above-described methods, the first fluorescent dye is selected from the group consisting of: a fluorescent lectin; a fluorescent phospholipid; a coumarin-labeled phosphatidylethanolamine; N-(6-chloro- 7-hydroxy-2-oxo-2H~l-benzopyran-3-carboxamidoacetyl)-dimyristoylphosphatidyl- ethanolamine); N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)- dipalmitoylphosphatidylethanolamine); and fluorescein-labeled wheat germ agglutinin.

In particular embodiments of the above-described methods, the second fluorescent dye is selected from the group consisting of: an oxonol that acts as the fluorescent acceptor; bis(l,3-dialkyl-2-thiobarbiturate)trimethineoxonols; bis(l,3- dihexyl-2-thiobarbiturate)trimethineoxonol; bis(l,3-dialkyl-2-thiobarbiturate) quatramethineoxonols; bis(l,3-dialkyl-2-thiobarbiturate)pentamethineoxonols; bis(l,3-dihexyl-2-thiobarbiturate)pentamethineoxonol; bis(l,3-dibutyl-2- thiobarbiturate)pentamethineoxonol); and bis(l,3-dialkyl-2- thiobarbiturate)hexamethineoxonols.

In a particular embodiment of the above-described methods, the cells are eukaryotic cells. In another embodiment, the cells are mammalian cells. In other embodiments, the cells are L cells L-M(TK") (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26), or MRC-5 (ATCC CCL 171).

In assays to identify activators or inhibitors of potassium channels containing human Kir5.1 subunit protein, it may be advantageous to co-express another potassium channel subunit besides the human Kir5.1 subunit. In particular, it may be advantageous to co-express another inwardly rectifying potassium channel subunit such as Kir4.1 or Kir4.2. Preferably, this is done by co-transfecting into the cells an expression vector encoding the other subunit. The present invention also includes assays for the identification of inhibitors of potassium channels containing human Kir5.1 subunit protein that are based upon modulation of the growth phenotype of trklΔtrk2Δ mutant yeast that also express inwardly rectifying K+ channels containing the human Kir5.1 subunit. The products of the yeast trkl and trk2 genes are high affinity potassium transporters and their expression in wild type yeast allows growth under conditions in which the concentration of K+ in the medium is very low (e.g., <50 μM). Deletion, or inactivation, of these two genes abolishes high affinity K+ uptake and results in impaired growth in potassium limited (e.g, < 7 mM) media. In addition, growth of trklΔtrk2Δ yeast is also impaired by low (< 3.0) pH even in the presence of otherwise permissive K+ concentrations (Nakamura & Gaber, 1999, Methods. Enz. 293:89- 104). Heterologous expression of an inwardly rectifying K+ channel in trklΔtrk2Δ yeast can rescue the mutant growth phenotype (i.e., expression of such a channel can restore wild type growth to these cells in limiting K+ or low pH; see Anderson et al., 1992, Proc. Natl. Acad. Sci. USA 89:3736-3740; Sentenac et al., 1992, Science 256:663; Goldstein et al., 1996, Proc. Natl. Acad. Sci. USA 93:13256-13260; Tang et al., 1995, Mol. Biol. Cell 6:1231). Thus, inhibitors of the inwardly rectifying channel will negate its effects in these mutant yeast and result in their reversion to the mutant growth phenotype (i.e., impaired growth in low K+ or low pH) . Thus, the present invention includes a method of identifying inhibitors of potassium channels containing human Kir5.1 subunit protein comprising:

(a) providing a yeast strain that has been engineered to

(1) have functionally inactivated trkl and trk2 genes and

(2) heterologously express an inwardly rectifying K+ channel containing human Kir5.1 protein subunit;

(b) exposing the yeast to a substance that is suspected of being an inhibitor of potassium channels containing human Kir5.1 subunit protein;

(c) measuring the growth rate of the yeast in the presence and in the absence of the substance; (d) comparing the growth rate exhibited by the yeast in the presence and in the absence of the substance under either limiting K+ concentration or low pH and permissive K+; wherein if the growth rate exhibited by the yeast in the presence of the substance is less than the growth rate in the absence of the substance then the substance is an inhibitor of potassium channels containing human Kir5.1 subunit protein.

In certain embodiments, the yeast trkl and trk2 genes have been inactivated by deletion or mutagenesis.

Growth of the yeast is measured in media containing either 1) limiting K+ (e.g. , <7 mM K+) or 2) permissive K+ and low pH (e.g. , 100 mM K+ and pH <3.0). Growth rate may simply be measured as turbidity of the culture (e.g., as absorbance at 700 nm) as a function of time, or may be measured by other methods know in the art.

While the above-described methods are explicitly directed to testing whether "a" substance is an activator or inhibitor of potassium channels containing human Kir5.1 subunit protein, it will be clear to one skilled in the art that such methods can be adapted to test collections of substances, e.g., combinatorial libraries, to determine whether any members of such collections are activators or inhibitors of potassium channels containing human Kir5.1 subunit protein. Accordingly, the use of collections of substances, or individual members of such collections, as the substance in the above-described methods is within the scope of the present invention. In particular, it is envisioned that libraries that have been designed to incorporate chemical structures that are known to be associated with potassium ion channel modulation, e.g., dihydrobenzopyran libraries for potassium channel activators

(International Patent Publication WO 95/30642) or biphenyl-derivative libraries for potassium channel inhibitors (International Patent Publication WO 95/04277), will be of especial interest.

The present invention includes pharmaceutical compositions comprising activators or inhibitors of potassium channels comprising human Kir5.1 subunit protein that have been identified by the herein-described methods. The activators or inhibitors are generally combined with pharmaceutically acceptable carriers to form pharmaceutical compositions. Examples of such carriers and methods of formulation of pharmaceutical compositions containing activators or inhibitors and carriers can be found in Gennaro, ed., Remington's Pharmaceutical Sciences, 18^tn Edition, 1990, Mack Publishing Co., Easton, PA. To form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain a therapeutically effective amount of the activators or inhibitors. Therapeutic or prophylactic compositions are administered to an individual in amounts sufficient to treat or prevent conditions where the activity of potassium channels containing human Kir5.1 subunit protein is abnormal. The effective amount can vary according to a variety of factors such as the individual's condition, weight, gender, and age. Other factors include the mode of administration. The appropriate amount can be determined by a skilled physician. Generally, an effective amount will be from about 0.01 to about 1,000, preferably from about 0.1 to about 250, and even more preferably from about 1 to about 50 mg per adult human per day.

Compositions can be used alone at appropriate dosages. Alternatively, co-administration or sequential administration of other agents can be desirable.

The compositions can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration. For example, the compositions can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection. Likewise, they can also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts. Compositions can be administered in a single daily dose, or the total daily dosage can be administered in divided doses of two, three, four or more times daily. Furthermore, compositions can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.

The dosage regimen utilizing the compositions is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal, hepatic and cardiovascular function of the patient; and the particular composition thereof employed. A physician of ordinary skill can readily determine and prescribe the effective amount of the composition required to prevent, counter or arrest the progress of the condition. Optimal precision in achieving concentrations of composition within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the composition's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a composition.

The inhibitors and activators of potassium channels containing human Kir5.1 subunit protein will be useful for treating a variety of diseases involving excessive or insufficient potassium channel activity.

Expression of Kir5.1 in the human kidney was seen by in situ hybridization in the proximal and distal convoluted tubules, but no expression was seen in glomeruli or collecting ducts. These segments of the nephron are actively involved in the regulation of salt, water, and pH balance, and are the location of many transporters and channels, many of which are targets for currently available diuretics (Puschett, 1994, Cardiology 84 Suppl 2:4-13). In situ hybridization with the Kir5.1 probe in the human pancreas demonstrated expression in the acinar and ductal cells. The pancreatic acini produce the digestive enzymes and large amounts of bicarbonate and therefore require active ion transport systems. This bicarbonate, secreted in the pancreatic fluid into the duodenum, buffers the acidic pH of the stomach contents (Freedman & Scheele, 1994, Ann. N.Y. Acad. Sci. 713:199-206). The expression pattern of human Kir5.1 suggests that the inhibitors and activators of potassium channels containing human Kir5.1 subunit protein of the present invention are likely to be useful for the treatment of hypo- and hypertension, renal failure, and pancreatic disease.

The observation of relatively high levels of expression of Kir5.1 in human thyroid suggests that the inhibitors and activators of potassium channels containing human Kir5.1 subunit protein of the present invention are likely to be useful for the treatment of thyroid disease.

Potassium channels contribute to the repolarization, and thus the de- excitation, of neurons. Thus, inhibitors of potassium channels are expected to act as agents that tend to keep neurons in a depolarized, excited state. Many diseases, such as depression and memory disorders are thought to result from the impairment of neurotransmitter release. As agents that contribute to neuronal excitability, the inhibitors of the present invention are expected to useful in the treatment of such diseases since they will contribute to neuronal excitation and thus stimulate the release of neurotransmitters.

The activators of the present invention should be useful in conditions where it is desirable to decrease neuronal activity. Such conditions include, e.g., excessive smooth muscle tone, angina, asthma, hypertension, incontinence, pre-term labor, migraine, cerebral ischemia, and irritable bowel syndrome.

The Kir5.1 subunit of the present invention is useful in conjunction with screens designed to identify activators and inhibitors of other ion channels. When screening compounds in order to identify potential pharmaceuticals that specifically interact with a target ion channel, it is necessary to ensure that the compounds identified are as specific as possible for the target ion channel. To do this, it is necessary to screen the compounds against as wide an array as possible of ion channels that are similar to the target ion channel. Thus, in order to find compounds that are potential pharmaceuticals that interact with ion channel A, it is not enough to ensure that the compounds interact with ion channel A (the "plus target") and produce the desired pharmacological effect through ion channel A. It is also necessary to determine that the compounds do not interact with ion channels B, C, D, etc .(the "minus targets"). In general, as part of a screening program, it is important to have as many minus targets as possible (see Hodgson, 1992, Bio/Technology 10:973-980, at 980). Human Kir5.1 subunit protein, DNA encoding human Kir5.1 subunit protein, and recombinant cells that have been engineered to express human Kir5.1 subunit protein have utility in that they can be used as "minus targets" in screens designed to identify compounds that specifically interact with other ion channels. For example, Wang et al., 1998, Science 282: 1890-1893 have shown that KCNQ2 and KCNQ3 form a heteromeric potassium ion channel know as the "M- channel." The M-channel is an important target for drug discovery since mutations in KCNQ2 and KCNQ3 are responsible for causing epilepsy (Biervert et al., 1998, Science 279:403-406; Singh et al., 1998, Nature Genet. 18:25-29; Schroeder et al., Nature 1998, 396:687-690). A screening program designed to identify activators or inhibitors of the M-channel would benefit greatly by the use of potassium channels comprising human Kir5.1 subunit protein as minus targets.

The present invention also includes antibodies to the human Kir5.1 subunit protein. Such antibodies may be polyclonal antibodies or monoclonal antibodies. The antibodies of the present invention can be raised against the entire human Kir5.1 subunit protein or against suitable antigenic fragments that are coupled to suitable carriers, e.g., serum albumin or keyhole limpet hemocyanin, by methods well known in the art. Methods of identifying suitable antigenic fragments of a protein are known in the art. See, e.g., Hopp & Woods, 1981, Proc. Natl. Acad. Sci. USA 78:3824-3828; and Jameson & Wolf, 1988, CABIOS (Computer Applications in the Biosciences) 4:181-186.

For the production of polyclonal antibodies, human Kir5.1 subunit protein or antigenic fragments, coupled to a suitable carrier, are injected on a periodic basis into an appropriate non-human host animal such as, e.g., rabbits, sheep, goats, rats, mice. The animals are bled periodically and sera obtained are tested for the presence of antibodies to the injected subunit or antigen fragment. The injections can be intramuscular, intraperitoneal, subcutaneous, and the like, and can be accompanied with adjuvant. For the production of monoclonal antibodies, human Kir5.1 subunit protein or antigenic fragments, coupled to a suitable carrier, are injected into an appropriate non-human host animal as above for the production of polyclonal antibodies. In the case of monoclonal antibodies, the animal is generally a mouse. The animal's spleen cells are then immortalized, often by fusion with a myeloma cell, as described in Kohler & Milstein, 1975, Nature 256:495-497. For a fuller description of the production of monoclonal antibodies, see Antibodies: A Laboratory Manual, Harlow & Lane, eds., Cold Spring Harbor Laboratory Press, 1988.

Gene therapy may be used to introduce human Kir5.1 subunit protein into the cells of target organs. Nucleotides encoding human Kir5.1 subunit protein can be ligated into viral vectors which mediate transfer of the nucleotides by infection of recipient cells. Suitable viral vectors include retrovirus, adenovirus, adeno- associated virus, herpes virus, vaccinia virus, lentivirus, and polio virus based vectors. Alternatively, nucleotides encoding human Kir5.1 subunit protein can be transferred into cells for gene therapy by non-viral techniques including receptor-mediated targeted transfer using ligand-nucleotide conjugates, lipofection, membrane fusion, or direct microinjection. These procedures and variations thereof are suitable for ex vivo as well as in vivo gene therapy. Gene therapy with wild type human Kir5.1 subunit protein will be particularly useful for the treatment of diseases where it is beneficial to elevate inwardly rectifying potassium channel activity. Gene therapy with a dominant negative mutant of human Kir5.1 subunit protein will be particularly useful for the treatment of diseases where it is beneficial to decrease inwardly rectifying potassium channel activity.

The present invention provides methods for cloning orthologues of human Kir5.1 subunit protein from non-human species. In general, such processes include preparing a PCR primer or a hybridization probe based upon SEQ.ID.NO.: 1 that can be used to amplify a fragment containing the non-human Kir5.1 subunit (in the case of PCR) from a suitable DNA preparation or to select a cDNA or genomic clone containing the non-human Kir5.1 subunit from a suitable library. A preferred embodiment of this process is a process for cloning the Kir5.1 subunit from mouse. By providing DNA encoding mouse Kir5.1 subunit, the present invention allows for the generation of an animal model of human diseases in which Kir5.1 subunit activity is abnormal. Such animal models can be generated by making transgenic "knockout" or "knockin" mice containing altered Kir5.1 subunit genes. Knockout mice can be generated in which portions of the mouse Kir5.1 subunit gene have been deleted. Knockin mice can be generated in which mutations that have been shown to lead to human disease are introduced into the mouse gene. Such knockout and knockin mice will be valuable tools in the study of the relationship between potassium channels and disease and will provide important model systems in which to test potential pharmaceuticals or treatments for human diseases involving potassium channels.

Accordingly, the present invention includes a method of producing a transgenic mouse comprising: (a) designing PCR primers or an oligonucleotide probe based upon

SEQ.ID.NO. :1 for use in cloning the mouse Kir5.1 subunit gene or cDNA;

(b) using the PCR primers or the oligonucleotide probe to clone at least a portion of the mouse Kir5.1 subunit gene or cDNA, the portion being large enough to use in making a transgenic mouse; (c) producing a transgenic mouse having at least one copy of the mouse Kir5.1 subunit gene altered from its native state.

Methods of producing knockout and knockin mice are well known in the art. One method involves the use of gene-targeted ES cells in the generation of gene-targeted transgenic knockout mice and is described in, e.g., Thomas et al., 1987, Cell 51:503-512, and is reviewed elsewhere (Frohman et al., 1989, Cell 56:145-147; Capecchi, 1989, Trends in Genet. 5:70-76; Baribault et al., 1989, Mol. Biol. Med. 6:481-492).

Techniques are available to inactivate or alter any genetic region to virtually any mutation desired by using targeted homologous recombination to insert specific changes into chromosomal genes. Generally, use is made of a "targeting vector," i.e., a plasmid containing part of the genetic region it is desired to mutate. By virtue of the homology between this part of the genetic region on the plasmid and the corresponding genetic region on the chromosome, homologous recombination can be used to insert the plasmid into the genetic region, thus disrupting the genetic region. Usually, the targeting vector contains a selectable marker gene as well.

In comparison with homologous extrachromosomal recombination, which occurs at frequencies approaching 100%, homologous plasmid-chromosome recombination was originally reported to only be detected at frequencies between 10-6 and 10-3 (Lin et al., 1985, Proc. Natl. Acad. Sci. USA 82:1391-1395; Smithies et al., 1985, Nature 317: 230-234; Thomas et al., 1986, Cell 44:419-428).

Nonhomologous plasmid-chromosome interactions are more frequent, occurring at levels 105-fold (Lin et al., 1985, Proc. Natl. Acad. Sci. USA 82:1391-1395) to 102- fold (Thomas et al., 1986, Cell 44:419-428) greater than comparable homologous insertion. To overcome this low proportion of targeted recombination in murine ES cells, various strategies have been developed to detect or select rare homologous recombinants. One approach for detecting homologous alteration events uses the polymerase chain reaction (PCR) to screen pools of transformant cells for homologous insertion, followed by screening individual clones (Kim et al., 1988, Nucleic Acids Res. 16:8887-8903; Kim et al., 1991, Gene 103:227-233). Alternatively, a positive genetic selection approach has been developed in which a marker gene is constructed which will only be active if homologous insertion occurs, allowing these recombinants to be selected directly (Sedivy et al., 1989, Proc. Natl. Acad. Sci. USA 86:227-231). One of the most powerful approaches developed for selecting homologous recombinants is the positive-negative selection (PNS) method developed for genes for which no direct selection of the alteration exists (Mansour et al., 1988, Nature 336:348-352; Capecchi, 1989, Science 244:1288-1292; Capecchi, 1989, Trends in Genet. 5:70-76). The PNS method is more efficient for targeting genes which are not expressed at high levels because the marker gene has its own promoter. Nonhomologous recombinants are selected against by using the Herpes Simplex virus thymidine kinase (HSV-TK) gene and selecting against its nonhomologous insertion with herpes drugs such as gancyclovir (GANC) or FIAU (1- (2-deoxy 2-fluoro-B-D-arabinofluranosyl)-5-iodouracil). By this counter-selection, the percentage of homologous recombinants in the surviving transformants can be increased.

Other methods of producing transgenic mice involve microinjecting the male pronuclei of fertilized eggs. Such methods are well known in the art.

The present invention includes a transgenic, non-human animal in which the animal's genome contains DNA encoding at least a portion of the human Kir5.1 subunit.

The present invention includes isolated polypeptides comprising the rat Kir5.1 potassium channel subunit having the amino acid sequence SEQ.ID.NO. :4. The present invention also includes polypeptides that are substantially free from other proteins and that comprise the amino acid sequence SEQ.ID.NO. :4. The present invention also includes isolated and recombinant DNA encoding the rat Kir5.1 protein as well as host cells expressing the rat Kir5.1 protein by recombinant means. An example of such DNA encoding the rat Kir5.1 protein is the nucleic acid sequence SEQ.ID.NO. :7. The rat Kir5.1 protein of the present invention can be substituted for the human Kir5.1 protein in the assays described herein in order to identify activators and inhibitors of potassium channels containing the rat Kir5.1 protein.

The following non-limiting examples are presented to better illustrate the invention.

EXAMPLE 1

Identification of the human Kir5.1 subunit and cDNA cloning

Database Searching: The PROWEIGHT (GenCore Version4.5beta Compugen) algorithm was used to generate a weight matrix from a multiple sequence alignment of full length inward rectifier channels. The TPROFRAMESEARCH algorithm (GenCore Version 4.5beta, Compugen) was used to search the dbEST. Resulting ESTs and their contigs were evaluated and putative novel K+ channels were selected using the BLASTX (nucleotide sequence searches against protein database) and BLASTN (nucleotide searches against nucleotide databases) algorithms. Based upon Northern analysis, which demonstrated expression of

Kir5.1 in human kidney, synthetic oligonucleotide primers (5' -TAC TAC AAA ACT CAC CTG GAT-3' (SEQ.ID.NO.: 10) and 5'-CCT CAT AAT TGC AAT TTA GGA- 3') (SEQ.ID.NO.: 11) were used to amplify a cDNA fragment from human kidney mRNA. First strand kidney cDNA was synthesized from 1.5 μg human kidney poly A+ mRNA (Clontech) using 19 μM random hexamer primers in 50 mM Tris pH 8.3, 8 mM MgCl2, 3 mM KCl, 1 mM DTT, 2 mM dNTPs, and 24 units AMV reverse transcriptase at 42°C for 90 min. PCR was then carried out using 5% (2 μL) of the synthesized cDNA as the template in 20 mM Tris pH 8.75, 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% TritonXlOO, 0.1 mg/ml BSA, 200 μM dNTPs, 1 μM oligonuclotide primers, 5 Units Taq Plus Long (Stratagene). Cycling parameters were 25 cycles of 94°C for lmin, 56°C for 2min and 72°C for 3min. The cDNA fragment amplified in this manner was cloned into a TA cloning vector and sequenced. The sequence of the human kidney cDNA was identical to that derived from the genomic DNA database. EXAMPLE 2

Analysis of expression of human Kir5.1 subunit

RT-PCR: Panels of cDNAs prepared from poly(A+) mRNAs isolated from different human tissues (heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testes, ovary, small intestine, colon, and peripheral blood leukocytes) were purchased from Clontech (Palo Alto, CA). PCR reactions were carried out in a total volume of 20 μl containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 200 μM dNTPs, 250 nM of each primer, 0.05 U

Amplitaq Gold (Perkin Elmer Corp, Foster City, CA) and 0.4 ng cDNA. Oligonucleotide primers, specific for Kir5.1, were 5' - GAG CTA TTA CGG CAG CAG CTA- 3' (SEQ.ID.NO.: 12) and 5' - CAA CCA CAT AGC TTC CCC AT -3' (SEQ.ID.NO.: 13). PCR amplification was performed using the following cycling parameters: 10 min at 94°C, 35 cycles of 94°C for 30 sec, 56°C for 30 sec, and 72°C for 90 sec, and a final 7 min extension period at 72°C. Amplification products were analyzed by agarose gel electrophoresis.

Northern blot analysis: Northern blots (Multiple Tissue Northern blots and Master RNA blots) of poly(A+)-mRNAs isolated from human heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testes, ovary, small intestine, colon, and peripheral blood leukocytes were purchased from Clontech (Palo Alto, CA). Human Kir5.1 distribution was determined using a random-hexamer primed 32p_ι_abeled probe (~4 x 106 cpm ng) corresponding to nucleotides 2-177. The membranes were hybridized overnight at 42°C in 50% formamide, 5XSSPE, 10X Denhardts, 2% SDS, 100 μg/ml sheared, denatured salmon sperm DNA, and ~2x 10& cpm probe. Blots were then washed twice for 20 minutes in 2X SSC, 0.05% SDS at 42°C followed by two 20 minute washes in IX SSC, 0.05% SDS at 50°C. Hybridization was detected by analysis using a phosphorimager. The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties.

Claims

WHAT IS CLAIMED IS:

1. An isolated DNA comprising nucleotides encoding the human Kir5.1 subunit protein.

2. The DNA of claim 1 comprising nucleotides encoding a polypeptide having the amino acid sequence shown in SEQ.ID.NO. :2.

3. The DNA of claim 1 comprising a nucleotide sequence selected from the group consisting of: SEQ.ID.NO.: 1 and positions 1 to 1254 of

SEQ.ID.NO.:l.

4. An isolated DNA that hybridizes under stringent conditions to the DNA of claim 3.

5. An expression vector comprising the DNA of claim 3.

6. A recombinant host cell comprising the DNA of claim 3.

7. An isolated human Kir5.1 subunit protein.

8. The protein of claim 7 having the amino acid sequence shown in SEQ.ID.NO.: 2.

9. The protein of claim 8 containing a single amino acid substitution.

10. The protein of claim 8 containing two or more amino acid substitutions where the amino acid substitutions do not occur in conserved positions.

11. An antibody that binds specifically to the human Kir5.1 subunit protein.

12. A DNA or RNA oligonucleotide probe comprising at least 10 contiguous nucleotides from SEQ.ID.NO.:l.

13. A method for identifying substances that bind to potassium channels containing human Kir5.1 subunit protein comprising:

(b) exposing the cells to a substance that is not known to bind potassium channels containing human Kir5.1 subunit protein; (c) determining the amount of binding of the substance to the cells;

(d) comparing the amount of binding in step (c) to the amount of binding of the substance to control cells where the control cells are substantially identical to the cells of step (a) except that the control cells do not express human

Kir5.1 subunit protein; where if the amount of binding in step (c) is greater than the amount of binding of the substance to control cells, then the substance binds to potassium channels containing human Kir5.1 subunit protein.

14. A method of identifying substances that bind potassium channels containing human Kir5.1 subunit protein and thus are likely to be inhibitors or activators of potassium channels containing human Kir5.1 subunit protein comprising:

(a) providing cells expressing potassium channels containing human Kir5.1 subunit protein; (b) exposing the cells to a compound that is known to bind to the potassium channels containing human Kir5.1 subunit protein;

(c) determining the amount of binding of the compound to the cells in the presence and in the absence of a substance not known to bind to potassium channels containing human Kir5.1 subunit protein; where if the amount of binding of the compound in the presence of the substance differs from that in the absence of the substance, then the substance binds potassium channels containing human Kir5.1 subunit proteins and is likely to be an inhibitor or activator of potassium channels containing human Kir5.1 subunit protein.

15. A method of identifying activators or inhibitors of potassium channels containing human Kir5.1 subunit protein comprising:

(a) recombinantly expressing human Kir5.1 subunit protein or mutant human Kir5.1 subunit protein in a host cell so that the recombinantly expressed human Kir5.1 subunit protein forms potassium channels by forming heteromers with other potassium subunit proteins;

16. An isolated DNA comprising nucleotides encoding the rat Kir5.1 subunit protein having SEQ.ID.NO.:4.

17. An isolated rat Kir5.1 subunit protein having SEQ.ID.NO. :4.