WO1997012974A2 - Kidney atp-dependent potassium channels - Google Patents

Abstract

The present invention comprises human DNA compositions, including cDNA clones, with full sequences, called, KIRK-2 and KIRT-3, encoding proteins that confer potassium channel activity to membranes or recipient cell lines. The DNA compositions include structural genes coding for the potassium channel proteins, expression and replication plasmids or vectors containing the structural genes and host cells expressing those genes. Methods of screening compounds for potassium channel modulating activity are also described.

Description

KIDNEY ATP-DEPENDENT POTASSIUM CHANNELS Field of the Invention

The present invention comprises human DNA compositions encoding proteins that confer potassium channel activity to membranes of recipient cell lines. The DNA compositions include structural genes coding for the potassium channel proteins, expression and replication plasmids or vectors containing the structural genes and host cells expressing those genes. Methods of screening compounds for potassium channel modulating activity are also described. Information Disclosure

Bond, C.T., Pessia, M., Xia, X-M., Lagrutta, A., Kavanaugh, M.P. and Adelman, J.P. "Cloning and expression of a family of inward rectifier potassium channels" Receptors and Channels 2: 183-191 (1994). Descriptions of a rat BIRK clone. Takumi, T., Ishii, T., Horio, Y., Morishige, K- , Takahashi, N., Yamada, M.,

Yamashita, T., Kiyama, H., Sohmiya, K, Nakanishi, S. and Kurachi, Y. "A novel ATP-dependent inward rectifier potassium channel expressed predominantly in glial cells" J. Biol. Chem. 270: 16339-16346 (1995). Descriptions of a rat BIRK clone. Bredt, D.S., Wang, T-L., Cohen, N.A., Guggino, W.B. and Snyder, S.H. "Cloning and expression of two brain-specific inwardly rectifying potassium channels" Proc. Natl. Acad. Sci. USA 92: 6753-6757 (1995). Descriptions of a rat BIRK clone.

Bienkowski and Groppi, WO 94/19464, PCT US94/01210, published 1 September 1994. "Human DNA sequence encoding a kidney ATP-dependent potassium channel."

Ho, , et. al. "Cloning and expression of an inwardly rectifying ATP- regulated potassium channel." Nature (4 March 1993) Vol. 362 pp. 31-38. Describes the gene that encodes an ATP-regulated potassium channel protein from the inner stripe of outer medulla of rat kidneys. Chandy, K., et. al., WO 9202634, PCT/US91 05168, published 20 February

1992. Describes the gene product known as MK3, a voltage dependent, type n potassium channel protein in T lymphocytes.

Chandy, K, et. al., "A Family of Three Mouse Potassium Channel Genes with Intronless Coding Regions." Science (23 February 1990) Vol. 247, pp. 973-975 Harpold, M., and Brust P., WO92/02639, PCT/US91/05625, published 20 February 1992. Transcription assays that identify compounds that modulate the activity of cell surface proteins. Cells that contains DNA that encode reporter genes, transcriptional control elements and heterologous cell surface proteins that may be potassium ion channels. Luzdunski, M., "Potassium Channels: Structure-Function Relationships,

Diversity, and Pharmacology," Cardiovascular Drugs and Therapy, (1992) Vol. 6, pp. 312-319. General description and information concerning potassium channels. References listed above are incorporated by reference.

Background Ionic channels of cell membranes are the basic sites where ionic fluxes take place. The modern era of the study of drug-channel interactions began when voltage clamp techniques were used to demonstrate the block of Sodium, (Na⁺), and potassium, (K⁺), channels of squid axons caused by procaine and cocaine. Narahashi, Ann Neurology (1984); 16(suppl): S39-S51. This invention concerns potassium channels. Pharmacological and biophysical studies have revealed multiple subtypes for membrane ion channels that form potassium selective pores in the plasma membrane of many mammalian cells. Comparison of the pharmacological and electrophysiological properties of these potassium channels has given rise to an operational definition for grouping the various subtypes based largely on their gating properties.

Voltage-gated potassium channels sense changes in membrane potential and pass potassium ions in response to this alteration in the cell membrane potential. Ligand-gated potassium channels are regulated by small molecular weight effectors which include calcium, sodium, ATP or fatty acids (particularly arachidonic acid). Lazdunski, Cardiovascular Drugs and Therapy (1992) Vol. 6 pp. 313-319. Although these channel proteins share the common property that they selectively move potassium ions, their distinct biophysical, biochemical and pharmacological properties suggests that they are different gene products encoded by distinct genes. The ATP-Sensitive, or ATP-gated, potassium channel is an important class of channels that links the bioenergetic situation of the cell to its electrical excitability. The channel is blocked by high intracellular ATP concentrations and it opens when ATP decreases. Lazdunski (1992). Although ATP-gated potassium channels were originally described in cardiac tissue; Noma, A. Nature (1983) Vol. 305 pp. 147-148, they have subsequently been described in pancreatic β-cells; Cook et. al., Nature (1984) Vol. 311 pp. 271-273, vascular smooth muscle; Nelson, M.T. et. al., Am. J. Physiol. (1990) Vol. 259 pp. C3-C18 and in the thick ascending limb of the kidney; Wang, W. et. al. Am. J. Physiol. (1990) Vol. 258, pp. F244-F-253.

The expression cloning of the ROMK ⁽¹⁾, IRK ⁽²^ and G-protein regulated KGA ' potassium channels has defined a new genetic class of potassium channels that can be regarded as simplified versions of the voltage-gated potassium channels. All of these potassium channel polypeptides share a homologous H-5 region that is believed to form a part of the K-selective pore. In contrast to the predicted structure of the voltage-gated K channels which contain six transmembrane domains and relatively small cytoplasmic domains, these three K channels are predicted to contain only two transmembrane domains which flank the H-5 segment and relatively large COOH-terminal cytoplasmic domains. The discovery of these cDNA sequences has facilitated the isolation and characterization other members of this family of K channels that currently includes 11 distinct members. Classification of these DNA sequences based on homology reveals 3 distinct subfamilies in which the ROMK, IRK-1 and KGA/GIRK channels represent the charter members. The ROMK subfamily contains two members in addition to ROMK The closest genetic relative to ROMK, originally referred to as BIRK 10 ⁽ , has been independently cloned from rat brain [BIRK1 or

A more distantly related K channel that may actually define a new sub-family distinct from ROMK, referred to as uK_ATp-l, has been cloned from rat pancreatic islets ⁽ . The IRK family contains 4 distinct members including IRK-1 ⁽²'⁸'⁹⁾, IRK-2

IRK-3 ⁽¹³-¹⁷⁾ and BIRK-9 ⁽⁵⁾. Finally, the GIRK family contains 4 members including GIRK-1 ^3,18), GIRK-2 (also referred to as K_ATp-2) <¹⁹-²²⁾, GIRK-3 ^{( 19)} and GIRK-4 [also referred to as cardiac

Although our knowledge regarding the relationships between these cloned channels and their native counterparts is incomplete, there appear to be common functional features that support this genetic classification. All of these channels form inward rectifier K channels when expressed in heterologous systems. The IRK family all form K channels that show strong Mg^⁺-dependent inward rectification while all of the GIRK family members are regulated by G- proteins ' and are likely to form heteromultimers that have electrophysiological signatures that are distinct from the individual polypeptides ^^24»25\ Finally, the members of the ROMK family are regulated by ATP ^'°''' suggesting that these polypeptides may form the core pore-forming subunit of the K_ATp channel.

We have previously reported the cloning and characterization of multiple isoforms of the human ROMK channel from kidney that are formed by alternative splicing of a common gene ⁽²⁶l To extend our understanding of the expression of other members of the ROMK family in the kidney, we have used the BIRK- 10 cDNA cloned from rat brain as a probe to clone the human homolog from a kidney cDNA library. References cited in the Background

1. Ho, K, Nichols, C.G., Lederer, W.J., Lytton, J., Vassilev, P.M., Kanazirska, M.V. and Hebert, S.C. "Cloning and expression of an inwardly rectifying ATP-regulated potassium channel" Nature 362: 31-38 (1993).

2. Kubo, Y., Baldwin, T.J., Jan, Y.N. and Jan, L.Y. "Primary structure and functional expression of a mouse inward rectifier potassium channel" Nature 362:

127-133 (1993).

3. Dascal, N., Schreibmayer, W., Lim, N.F., Wang, W., Chavkin, C, DiMagno, L., Labarca, C, Kieffer, B.L., Gaveriaux-Ruff, C, Trollinger, D., Lester, HA. and Davidson, N. "Atrial G-protein-activated K* channel: Expression cloning and molecular properties" Proc. Natl. Acad. Sci. USA 90: 10235-10239 (1993).

4. Bond, C.T., Pessia, M., Xia, X-M., Lagrutta, A., Kavanaugh, M.P. and Adelman, J.P. "Cloning and expression of a family of inward rectifier potassium channels" Receptors and Channels 2: 183-191 (1994).

5. Takumi, T., Ishii, T., Horio, Y., Morishige, K-L, Takahashi, N., Yamada, M., Yamaahita, T., Kiyama, H., Sohmiya, K, Nakanishi, S. and Kurachi, Y. "A novel

ATP-dependent inward rectifier potassium channel expressed predominantly in glial cells" J. Biol. Chem. 270: 16339-16346 (1995).

6. Bredt, D.S., Wang, T-L., Cohen, N.A., Guggino, W.B. and Snyder, S.H. "Cloning and expression of two brain-specific inwardly rectifying potassium channels" Proc. Natl. Acad. Sci. USA 92: 6753-6757 (1995).

7. Inagaki, N., Tsuura, Y., Namba, N., Masuda, K, Gonoi, T., Horie, M., Seino, Y., Mizuta, M. and Seino, S. "Cloning and functional characterization of a novel K_ATp-sensitive potassium channel ubiquitously expressed in rat tissues including pancreatic islets, pituitary, skeletal muscle and heart" J. Biol. Chem. 270: 5691-5694 (1995).

8. Morishige, K-L, Takahashi, N., Findlay, I., Koyama, H., Zanelli, J.S., Peterson, C, Jenkins, N.A., Copeland, N.G., Mori, N. and Kurachi, Y. "Molecular cloning, functional expression and localization of an inward rectifier potassium channel in the mouse brain" FEBS Let. 336: 375-380 (1993). 9. Ashen, M.D., O'Rourke, B., Kluge, KA., Johns, D.C, and Tomaselli, G.F. "Inward rectifier K* channel from human heart and brain: cloning and stable expression in a human cell line" Am. J. Physiol. 268: (Heart Circ. Physiol.) H506- H511 (1995).

10. Koya a, H., Morishge, K-L, Takahashi, N., Zanelli, J.S., Fass, D.N. and Kurachi, Y. "Molecular cloning, functional expression of a novel inward rectifier potassium channel in the rat brain" FEBS Let. 341: 303-307 (1994).

11. Wible, B.A., Biasi, M., Majumder, K, Taglialatela, M. and Brown, AM. "Cloning and functional expression of an inwardly rectifying K* channel from human atrium" Circ. Res. 76: 343-350 (1995). 12. Raab-Graham, KF., Radeke, CM. and Vanderberg, CA. "Molecular cloning and expression of a human heart inward rectifier potassium channel" Neuroreport. 5: 2501-2505 (1994).

13. Morishe, K-L, Takahashi, N., Jahangir, A., Yamada, M., Koyama, H., Zanelli, J.S. and Kurachi, Y. "Molecular cloning and functional expression of a novel brain-specific inward rectifier potassium channel" FEBS Let. 346: 251-256 (1994).

14. Perier, F., Radeke, CM. and Vanderberg, CA. "Primary strucutre and characterization of a small conductance inwardly rectifying potassium channel from human hippocampus" Proc. Natl. Acad. Sci. USA 91: 6240-6244 (1994).

15. Makhina, E.N., Kelly, AJ., Lopatin, AN., Mercer, R.W. and Nichols, CG. "Cloning and expression of a novel brain inward rectifier potassium channel" J.

Biol. Chem. 269: 20468-20474 (1994).

16. Tang, W. and Yang, X-C "Cloning of a novel human brain inward rectifier potassium channel and its functional expression in Xenopus oocytes" FEBS Let. 348: 239-243 (1994). 17. Falk, T., Meyerhof, W., Corrette, B.J., Schafer, J., Bauer, CK, Schwarz,

J. and Richter, D. "Cloning, functional expression and mRNA distribution of an inwardly rectifying potassium channel protein" FEBS Let. 367: 127-131 (1995).

18. Kubo, Y., Reuveny, E., Slesinger, P.A., Jan Y.N. and Jan, L.Y. "Primary structure and functional expression of a rat G-protein-coupled muscarinic potassium channel" Nature 364: 802-806 (1993).

19. Lesage, F., Duprat, F., Fink, M., Guillemare, E., Coppola, T., Lazdunski, M. and Hugnot, J-P. "Cloning provides evidence for a family of inward rectifier and G-protein coupled K^"1" channels in the brain" FEBS Let. 353: 37-42 (1994).

20. Bond, CT., Ammala, C, Ashfield, R., Blair, T.A, Gribble, F., Khan, R.N., Lee, K, Proks, P., Rowe, I.C.M., Sakura, H., Ashford, M.J., Adelman, J.P. and Ashcroft, F.M. "Cloning and functional expression of the cDNA encoding an inwardly rectifying potassium channel expressed in pancreatic β-cells and in the brain" FEBS Let. 367: 61-66 (1995).

21. Sakura, H., Bond, C, Warren-Perry, M., Horsley, S., Kearney, L., Tucker, S., Adelman, J.P., Turner, R. and Ashcroft, F.M. "Characterization and variation of a human inwardly rectifying K-channel gene (KCNJ6): a putative ATP-sensitive K- channel subunit" FEBS Let. 367: 193-197 (1995).

22. Tsaur, M-L., Menzel, S., Lai, F-P., Espinosa, R., Concannon, P., Spielman, R.S., Hanis, C.L., Cox, N.J., Le Beau, M.M., German, M.S., Jan, L.Y., Bell, G.I. and Stoffel, M. "Isolation of a cDNA clone encoding a K r p channel-like protein expressed in insulin-secreting cells, localization of the human gene to Chromosome band 21q22.1 and linkage studies with NIDDM" Diabetes 44: 592-596 (1995).

23. Ashford, M.J., Bond, C.T., Blair, TA and Adelman, J.P. "Cloning and functional expression of a rat heart K_ATp channel" Nature 370: 456-459 (1994).

24. Krapivinsky, G., Gordon, E.A, Wickman, K, Velimirovic, B., Krapivinsky, L. and Clapman, D.E. "The G-protein-gated atrial K* channel

*⁸ a heteromultimer of two inwardly rectifying K⁺ channel proteins" Nature 374: 135- 141 (1995). 25. Duprat, F., Lesage, F., Guillemare, E., Fink, M., Hugnot, J-P., Bigay, J.,

Lazdunski, M., Romey, G. and Barhanin, J. "Heterologous multimeric assembly is essential for K* channel activity of neuronal and cardiac G-protein activated inward rectifiers" Biochem. Biophys. Res. Comm. 212: 657-663 (1995).

26. Shuck, M.E, Bock, J.H., Benjamin, C.W., Tsai, T-D., Lee, KS., Slightom, J.L. and Bienkowski, M. J. "Cloning and characterization of multiple forms of the human kidney ROM-K potassium channel" J. Biol. Chem. 269: 24261-24270 (1994).

SUMMARY OF THE INVENTION Two unique channel polypeptides and the cDNA that codes for them, KIRK-2, and KIRK-3, have been identified and cloned. KIRK-2 is related to a rat potassium channel protein coding cDNA called BIRK- 10. KIRK-2 is on chromosome 1 while IQRK-3 has no known homologs and maps to chromosome 21. DNA and Amino Acid sequences are provided. These two human K channels were compared by determining their tissue-distribution of expression, their human chromosome assignment and their electrophysiological properties when expressed in Xenopus oocytes and mammalian cells. KIRK- 2 and K-RK-3 have their cDNA sequences provided in Sequence Listings 1 and 2 and their protein sequences provided in Sequence Listings 3 and 4. c DNA molecules being about at least 80, 85, or 90 percent homologous to any of the above described DNA molecules thereof are described. Various vectors and plasmids comprising the DNA molecules of Sequence Listings 1-2 and selected derivatives thereof are described. Vectors and plasmids adapted for expression in a bacterial cell, yeast cell, or a mammalian cell are described. Use of the bacterial, yeast, or mammalian cell containing the vector or plasmid of Sequence Listings 1-2 or selected derivatives thereof, to screen for compounds that modulate human kidney ATP-gated and related potassium channel activity are described.

Also described is a method of using a mammalian, bacterial or, yeast cell as described above, to screen for compounds that modulate human kidney potassium channel activity. The method may be comprised of the following steps: a) growing cells expressing a cloned K^1" channel to confluence, b) equilibrating the cells of a) with a balanced salt solution, c) making baseline measurements of the equilibrated cells, d) adding one test compound or a cocktail of test compounds to the cells and recording changes in membrane potential, e) testing compounds that depolarize cells expressing the K* on wild type or mock transfected controls to establish selectivity, f) selecting the compounds that are shown to selectively block KT^1". DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention relates to the cloning and isolation of human DNAs encoding potassium ATP-gated channel proteins. In one embodiment this invention comprises isolated functional cDNA clones encoding human potassium ATP-gated channel proteins, verified by using Xenopus laevis oocytes as an expression system for studying ion channels. Mammalian and a bacterial cell lines expressing functional human potassium ATP-gated channels at the cell surface are described, as determined by pharmacologic and physiologic methods, thus establishing the first well-defined cell lines with which to study this particular member of a family of ATP-gated channel proteins. In another embodiment the human potassium ATP- gated channels are described as a possible high volume screen for novel compounds. Definitions. This document uses abbreviations and terms that should be well known to those skilled in the art. Some terms are more fully described in the sections below. This document also uses the terms: KIRK 1, KIRK 2 and KIRK 3, these terms are analogous to the terms: K_jrl.l (or "the ROM-K" or "ROMK"), K_jj.1.2 and K_j-,1.3, respectively. ISOLATION AND IDENTIFICATION OF A HUMAN POTASSIUM CHANNEL DNA CLONE.

The cDNA encoding the entire open-reading frame of a BIRK 10 transcript was isolated by RT-PCR and this sequence used as a probe to determine the tissue distribution of expression of the BIRK 10 transcript in various rat organs by Northern blot analysis. Of the tissues examined, a 5.2 kb transcript was only detected in the brain and kidney. A cDNA library prepared from the latter tissue was used to isolate the human BIRK 10. A human kidney cDNA library was screened at reduced stringency with the coding sequence of rat BIRK 10. Clonal cDNAs prepared from these positives were grouped by restriction mapping and representative clones subjected to DNA sequence analysis. Comparative analysis of these cDNA sequences revealed that among the clones that exhibited high homology to rat BIRK 10, none of them appeared to contain the entire open-reading frame. In addition, a second set of related cDNAs, were also identified and the latter sequence appeared novel. Consistent with the emerging nomenclature in this field, we have named these two new human K channel cDNAs KIRK- 2 and KIRK-3 (Kidney inward rectifier K channel), where the ROMK channel is referred to as KIRK-1. DETAILS OF PROCEDURES, METHODS AND EMBODIMENTS Cloning of rat brain BIRK 10. An on-line BLAST search of the GenBank database available through the National Center for Biotechnology Information

(NCBI) using the human ROMK1 cDNA as the query sequence (GenBank accession # U12541) identified a related rat cDNA sequence (GenBank accession # X83858). This cDNA had been cloned from rat brain (4) RNA and was referred to as brain inward rectifier channel 10 (BIRK 10). Based on the cDNA sequence in GenBank, a sense oligonucleotide primer (C12-A 5' CGC-TTT-GAA-TTC-ATG-ACA-TCA-GTT- GCC-AAG-GTC-TAT-TA 3' ahgnment positions 1-26 in X83858) and an antisense oligonucleotide primer (C12-B 5' CGC-TTT-GAA-TTC-TCA-GAC-GTT-ACT-AAT- GCG-CAC-ACT-A, alignment positions 1116-1140 in X83858) were synthesized. Total RNA (10 μg) isolated from rat brain was reversed transcribed using and oligo dT primer and reverse transcriptase. A portion of the resulting cDNA was then amplified in the PCR employing the C12-A and C12-B primers. The 1.2 kb product was digested to completion with EcoRI and subcloned into the EcoRI site of the plasmid vector pBK-CMV to yield pBK-CMV/BIRK 10.

Cloning af human kidney BIRK 10. Preliminary Northern blot analysis using the rat BIRK 10 cDNA as a probe revealed that in addition to the brain, BIRK 10 was also expressed in kidney. A human kidney cDNA Ubrary in the bacteriophage λ⁸^ was plated on E. coli strain DPδOsupF (500,000 recombinants). The rat BIRK 10 cDNA was excised from the plasmid pBK-CMV/BIRK 10 by digestion with EcoRI followed by preparative gel analysis. A portion of this fragment was then random prime labeled to a specific activity of > 1.0 x 10 dpm ³²P/μg DNA using α-³²P-dATP and Klenow DNA polymerase. Replicate lifts of the bacteriophage library were screened with the P-labeled rat BIRK 10 EcoRI fragment (5 x 10 dpm/ml) overnight at 65 °C The filters were then washed with 0.3 M NaCl/0.1% SDS at 65 °C and the coordinates of radioactivity on the filters determined by autoradiography at -70 °C with intensifying screens. Replicate positive bacteriophage clones from the primary screen were isolated from the agar plates and cloned by limiting dilution followed by rescreening as described above. Fourteen distinct clonal bacteriophage stocks prepared from the hybridization positive clones were used to prepare λ-DNA by infection of E. coli strain DPδOsupF at a multiplicity of infection of 0.02. Bacteriophage DNA was prepared for the infected cultures using polyethylene glycol precipitation of the bacteriophage particles followed by destruction of the bacteriophage protein coat. Bacteriophage DNAs prepared in this manner were digested with EcoRI to produce the cDNA inserts. The size of the liberated fragments was determined by agarose gel electrophoresis. Based on the restriction mapping, 4 representative clones were selected for DNA sequence analysis. DNA sequencing of the cDNA inserts was performed by cycle sequencing dideoxy-chain termination method (AmpliTaq kit, Perkin-Εlmer-Cetus, Norwalk, Connecticut). In the case of K-RK-2, a genomic clone was also isolated from a human genomic library in the vector λ using one of the partial cDNA clones obtained from the human kidney cDNA Ubrary as described above.

Northern blot analysis and human chromosome assignment The tissue distribution of expression of both KIRK-2 and KIRK-3 was determined by Northern blot analysis using MTN blots purchased from Clonetech (Palo Alto, CA). For KIRK- 2, a 1.3 kb EcoRI fragment containing the 3' end of the KIRK-2 open-reading frame and approximately 0.6 kb of 3' untranslated sequence was used as a probe. For KIRK-3, a 1.4 kb EcόRUMuήl fragment containing the entire open reading frame of KIRK-3 was used as a probe. In either case, the fragments were random prime labeled using α- P-dATP as described above foUowed by hybridization to Nylon membranes containing poly A⁺ RNAs from various human tissues or human brain regions as previously described (26). Somatic ceU hybrid panel blots were purchased for Oncor (Gaitherburg, MD) and hybridized to either of the KIRK cDNA probes described above under the conditions recommended by the manufacture (50% formamide/6X SSC/10% Dextran Sulfate/1.0% SDS/50 μg ml calf thymus DNA at 50 °C overnight). In aU cases, the blots were washed at high stringency (0.1X SSC) and labeled bands visualized by autoradiography.

Heterologous expression ofKTKK-2 and KIRKS. The open-reading frame of KIRK-2 was engineered for expression in Xenopus oocytes using the PCR. Sense (5* CAG-AAG-TTA-AGT-CGA-CAT-GAC-GTC-AGT-TGC-CAA-GGT-GTA-TT 3') and antisense (5' CAG-AAG-TTA-AGC-GGC-CGC-(T)₂₈-CAG-ACA-TTG-CTG-ATG-CGC- ACA-CT 3') primers that flank the open-reading frame of KIRK-2 were used to PCR amplify this region of the genomic clone for KIRK-2 (20 cycles with 0.5 μg template/reaction). See Shuck, M.E, Bock, J.H., Benjamin, C.W., Tsai, T-D., Lee, KS., SUghtom, J.L. and Bienkowski, M.J. "Cloning and characterization of multiple forms of the human kidney ROM-K potassium channel." J. Biol. Chem. 269: 24261- 24270 (1994), incorporated by reference..

The product was digested to completion with Sail and Notl and cloned into the plasmid vector pSPORT-1 to yield pSPORT/KIRK-2. For KIRK-3, the original λ- clone was double digested with EcoRI and Muni and subcloned into the plasmid vector pBK-CMV to yield pBK-CMV/KIRK-3. This plasmid was then double-digested with EcoRI AccI and directionaUy cloned into pSPORT/rROMKl to introduce the poly A tail from the rat ROMK1 cDΝA (1). See, Ho, K, Nichols, C.G., Lederer, W.J., Lytton, J., VassUev, P.M., Kanazirska, M.V. and Hebert, S.C. "Cloning and expression of an inwardly rectifying ATP-regulated potassium channel." Nature 362: 31-38 (1993), incorporated by reference. In each case, cRNA was synthesized from Notl-Unearized template use T7 RNA polymerase as previously described (26), incorporated by reference.

Oocytes were coUected under tricaine (3-aminobenzoic acid ethyl ester, 0.17%; Sigma, St. Louis, MO) and cold-induced anesthesia from adult African clawed frogs, Xenopus laevis (Nasco, Ft. Atkinson, WI), and defolUculated by blunt dissection in Ca²⁺ free ND-96 foUowing 40 minutes incubation in 2 mg ml collagenase (Type II; Sigma, St. Louis, MO). ND-96 consisted of 96 mM NaCl, 2 mM KCl, 1 mM MgCL_j, 0.3 mM CaCl₂, 5 mM HΕPΕS, pH 7.6. The following day, a glass injection pipette manually broken to -15 μm tip diameter was used to inject oocytes with 46 nl of polyadenylated cRNA dissolved in water at 15, 30 or 100 ng/μl resulting in injection of 0.6, 1.2, or 4 ng of cRNA. Prior to recordings, oocytes were maintained 2-7 days at 20° C in ND-96 supplemented with gentamycin (50 mg/ml; Bio-Whittaker, Walkersville, MD) and sodium-pyruvate (2.5 mM; Sigma).

Electrophysiological recordings were conducted at room temperature. Oocyte resting membrane potential was measured in ND-96 using 30-80 MΩ glass microelectrodes filled with 3 M KCl. For two-microelectrode voltage-clamp recordings, the voltage-measuring pipette had resistances of 1.5 to 2.0 MΩ, while the current-injection pipette had resistances of 0.7-0.9 MΩ; both pipettes were fiUed with 3 M KCl. AU voltage-clamp recordings were conducted in bath solutions containing 1 mM MgCl₂, 0.3 mM CaCl₂, and 5 mM HEPES. For experiments conducted in 50 mM K^1", the solution contained 50 mM KCl and 50 mM NaCl. For lower potassium concentrations, this solution was mixed with an identical solution in which choline was substituted for potassium. Pairs of solutions at pH 5.4 and 8.4, containing 50 mM KCl and either 50 mM potassium-acetate or 50 mM potassium-hydrogen phthalate (biphthalate; Sigma, St. Louis, MO) were mixed to vary pH.

Two-microelectrode, voltage-clamp recordings were conducted using the GeneClamp 500 (Axon Instruments, La JoUa, CA) interfaced with a 486 Computer running the PCLAMP 6.0 (Axon Instruments, La JoUa, CA) suite of programs. Currents were filtered at 2 kHz and sampled every 100 μs. Current amplitude was measured during the last 4 ms of a 400 ms test pulse. Data were analyzed and plotted using PCLAMP 6.0 and SIGMA PLOT 5.0 (Jandel Scientific, San Rafael, CA). Statistical significance was determined by the Student's T-test.

Composition ofthe cDNA's. The DNA sequence and predicted amino acid sequence of hKIRK-2 and hKIRK-3 are shown in Sequence Listings 1-4. The human KIRK-2 (BIRK 10) sequence, derived from a composite of both cDNA and genomic sequence, contains a 1128 bp open-reading frame that encodes a 376 amino acid polypeptide that showed 91% shared identity with the rat BIRK 10 amino acid sequence. Consistent with the predicted structures of other members of this family of K channels, the sequence of human KIRK-2 contains two putative transmembrane domains that flank an H-5 region that forms an integral part of the K*-selective pore. More detailed analysis of the predicted amino acid sequence of hKIRK-2 using the MOTIF algorithm revealed 2 canonical Asn-Unked glycosylation acceptor sites, 6 consensus acceptor sites for casein kinase II, and 6 consensus PKC acceptor sites. Also, the predicted Walker Type A ATP binding motif (GXfitK&_jd/V), is conserved in the hKIRK-2 sequence. The hKIRK-3 cDNAs predicted an 1125 bp open-reading frame that encodes a 375 amino acid polypeptide that was 62% identical and 47% identical to the hKIRK- 2 and hK-RK-1 amino acid sequences, respectively. The two putative transmembrane domains flanking an H-5 region were also evident in the predicted amino acid sequence of hK-RK-3. Analysis of the predicted amino acid sequence of hKIRK-3 using the MOTIF algorithm revealed 2 canonical acceptor sites for Asn- glycosylation, 5 consensus sites for casein kinase II, 4 consensus PKC sites and 3 consensus tyrosine kinase acceptor sites near the C-terminus of the protein. In contrast to the hKIRK-1 and hKIRK-2 sequences, the consensus Walker Type A ATP-binding motif GX₄GKXη(W), is not conserved in the hKIRK-3 sequence.

Heterologous expr^sion of KIRK 2 and KIRK 3 in Xenopus oocytes. The K channels synthesized foUowing injection of either KIRK 2 or KIRK 3 polyadenylated cRNAs into Xenopus oocytes were studied using standard electrophysiological procedures. Forty-eight hours post injection of 4 ng of KIRK 2 cRNA, the oocyte resting membrane potential was hyperpolarized by 49 mV (-93 ± 3 mV (n=25)) when compared to water-injected oocytes (-44 ± 3 mV, n=16, P < 0.001)). The observed resting membrane potential of KIRK 2-injected oocytes was close to the calculated potassium equilibrium potential of -106 mV at 2 mM extraceUular K and assuming 130 mM intraceUular K⁺. Thus, the KIRK 2 -injected ceUs expressed a potassium-selective ion channel that was not detected in water-injected ceUs. In contrast, injection of oocytes with up to 4 ng of polyadenylated KIRK 3 cRNA did not apparently significantly alter the membrane potential compared to water injected oocytes (- 49 ± 4 mV (n=17)-ι>ers--s- -44 ± 3 mV (n=17), respectively). The reasons for this apparent anomaly remain unknown at this time. Figure 1 shows current voltage curves from a single batch of oocytes injected with either water -- v, KIRK 2 = K_j.. 1.2 = O; or KIRK 3, = K^ 1.3 = *. The X- axis shows voltage (mV) the Y-axis is current (uA). Oocytes expressing KIRK 2 channels displayed large inward currents in response to voltage steps negative of the potassium equilibrium potential (- 20 mV in 50 mM K⁺) and these currents were not detected in water-injected ceUs. For example, whole-ceU current measured during a test pulse to -140 mV from a holding potential of -20 mV in 50 mM K⁺ was -4.4±0.5 (n=4) and -22.4±4.9 uA (n=7) in oocytes injected with 1.2 or 4 ng of KIRK 2 cRNA, respectively, but was only -125±13 nA in water-injected ceUs (n=8). Consistent with the lack of membrane hyperpolarization in oocytes injected with KIRK 3 cRNA, the whole-ceU current was actuaUy smaller than the current measured in water-injected oocytes (-161±27 nA at -140 mV, n=4). KIRK 2 currents measured during voltage steps positive of the potassium equilibrium potential were distinctly smaller than those measured during steps negative of the K* equilibrium potential, creating a mild, inward rectification. Some KIRK channel pore-forming polypeptides fail to produce detectable currents when expressed alone in oocytes. Co-expression of KIRK channel subunits has produced both reconstitution of channel activity, Krapivinsky, G., Gordon, EA, Wickman, K, Velimirovic, B., Krapivinsky, L. and Clapman, D.E. "The G-protein- gated atrial K* channel

*^{s a} heteromultimer of two inwardly rectifying K* channel proteins." Nature 374: 135-141 (1995)and Duprat, F., Lesage, F.,

Guillemare, E., Fink, M., Hugnot, J-P., Bigay, J., Lazdunski, M., Romey, G. and Barhanin, J. "Heterologous multimeric assembly is essential for K⁺ channel activity of neuronal and cardiac G-protein activated inward rectifiers." Biochem. Biophys. Res. Comm. 212: 657-663 (1995), as weU as decreases in currents compared to those produced by homomeric co-expression. Tucker, S.J., Bond, C.T., Herson, P., Pessia, M., and Adelman, J.P. (1996) "Inhibitory interactions between two inward rectifier K* channel subunits mediated by the transmembrane domains." J. Biol. Chem. 271: 5866-5870. The effect of coexpression of KIRK 3 cRNA with suboptimal amounts of either KIRK 1 (Figure 2A) or KIRK 2 (Figure 2B) cRNA (0.6 ng) was determined and the results are shown in Figures 2A and 2B. In Figures 2A and 2B the axis' are the same as for Figure 1. In Figure 2A, KIRK 1 = p _. 1.1 = v and KIRK 1 plus KIRK 3 = 1.1 plus K_j-. 1.3 = » . In Figure 2B, KIRK 2 = K^ 1.2 - O and KIRK 2 plus KIRK 3 -- K_j.. 1.2 plus K^ 1.3 - • . Expression of KIRK 1 or KIRK 2, channels alone produced - 1.14 ± 0.15 (n=5) and - 1.14 ± 0.34 (n=5) uA of current, respectively, at a holding potential of - 160 mV. Co-injection of a 6.7-fold excess of KIRK 3 decreased KIRK 1 and KIRK 2 currents by 54% and 51% to - 0.52 ± 0.03 (p<0.005, n=5) μA and - 0.56 ± 0.09 (p<0.05, n=4) μA, respectively.

The KIRK 2 channel forms a K* -selective, Be?⁺ -sensitive channel The dependence of reversal potential on extracellular K* and Ba²⁺ was determined, see Figures 3. The dependence of reversal potential on extracellular K* is similar to that expected for a potassium-selective channel at room temperature (slope-per- decade change of 55 mV. The effect of varying extraceUular [K*] on the current- voltage relationship of KIRK 2 expressing oocytes was investigated and the membrane conductance of KIRK 2 was dependent on extracellular [K⁺], Figure 3A. The axis for Figure 3A is the same as for Figure 1. In Figure 3A, 5mM K⁺ = O, lOmM K* m • , 25mM K⁺ = v , 50mM K⁺ = .

The relationship g = C[K⁺]₀ ^Z relates conductance, g, obtained by fitting the linear portion of the current-voltage curves (dotted Unes), to external potassium concentration. C and Z were varied to produce the best fit of these data, Figure SB. In Figure SB the y-axis is g/gmax and the x-axis is [K⁺](mM). The Z value for KIRK 2 was 0.54, simUar to the values of 0.62, 0.47, and 0.38-0.49 for the cardiac IK1 (28,29), mouse macrophage IRKl (3), and human ROMK isoforms 1-3, respectively. The square root dependence of membrane conductance on extraceUular K⁺ concentration is typical of the multi-ion pore of inward rectifier K* channels. Apphcation of Ba (0.03 μM to 10 mM) produced a reversible, concentration- dependent inhibition of KIRK 2 current, Figure 3C, n=8). In Figure 3C, the axis are the same as in Figures 1 and 2, control = O , 30 μM Ba²⁺ = •, 300 μM Ba²⁺ = v, and wash = ». The inhibition by Ba was time- and voltage-dependent as expected for the open-channel block typical of the effects of Ba on native and cloned inward-rectifier K⁺ channels.

The concentration-dependence of KIRK 2 inhibition by Ba at three different holding potentials is shown in Figure 3D. In Figure 3D, n=8) the x-axis is the Relative Current and the y-axis the concentration of Ba²⁺(M); the three curved lines show -120mV = O, -80mV = •, and -40mV a- v. These data were fitted by a simple logistic equation with a Hill coefficient of 2 and the resulting K_j values were plotted versus membrane potential in Figure 3E. Fit of these data by the Woodhull equation suggests that the Ba²⁺ binding site senβes 46% of the transmembrane electric field, in reasonable agreement with previously published results in BIRK- 10 injected oocytes. Inhibition of KIRK 2 by Cs⁺ was also reversible and voltage-dependent, being apparent only at the most negative potentials tested, but was far less effective than block by Ba (n=6, data not shown). Thus KIRK 2 encodes an inwardly-rectifying, Ba - and Cs⁺-sensitive potassium channel.

Tissue Distribution af Expression and Human Chromosome Assignment. KIRK-2 is expressed at high levels in whole brain and kidney and few other tissues. KIRK-3 also showed a limited tissue distribution of expression (kidney > pancreas > > lung) and multiple transcript sizes were detected. In contrast to KIRK- 2 which was most abundant in the brain, no KIRK-3 transcripts were detected in brain.

The human Chromosome location of the KIRK- 2 and KIRK-3 was determined by Southern blot analysis of genomic DNA prepared from a panel of mouse/human or hamster/human somatic ceU hybrids. The KIRK-2 and K3RK-3 genes localized to human Chromosome 1 and human Chromosome 21, respectively.

PREPARATION OF A BIOASSAY USING THE EXPRESSION SYSTEMS BASED ON THE PROTEINS DESCRIBED HEREIN By making appropriate modifications to the procedures described in:

Bienkowski and Groppi, WO 94 19464, PCT US94/01210, pubUshed 1 September 1994, "Human DNA sequence encoding a kidney ATP-dependent potassium channel," incorporated by reference, and using those procedures plus other appropriate procedures and techniques disclosed in other references cited herein, plus references and knowledge generaUy known to one skiUed in the art, in combination with the information and sequences described herein, one skilled in the art should be able to prepare suitable vectors and or plasmids comprising a DNA molecule that is of the KIRK- 2 or KIRK-3 type, Sequence Listings 1 and 2, or derivatives thereof having at least about 80, 85, or preferably 90 percent homology to KIRK-2 or KIRK-3, that are adapted for expression in a bacterial ceU, a mammalian ceU or a yeast cell and by using these bacterial, mammalian or yeast ceUs having those expressing vectors or plasmids one skilled in the art would be able to develop a method of using those ceUs to screen for compounds that modulate human kidney potassium channel activity.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: BIENKOWSKI, MICHAEL J.

(ϋ) TITLE OF INVENTION: NOVEL KIDNEY ATP-DEPENDENT POTASSIUM CHANNELS

(iii) NUMBER OF SEQUENCES: 8

(iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Pharmacia & Upjohn Company

(B) STREET: 301 Henrietta street

(C) CITY: Kalamazoo

(D) STATE: MI

(E) COUNTRY: USA (F) ZIP: 49001

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(Vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE:

(C) CLASSIFICATION:

(Viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Wootton, Thomas A. (B) REGISTRATION NUMBER: 35,004

(C) REFERENCE/DOCKET NUMBER: 6001.N CP

(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 616-833-7914 (B) TELEFAX: 616-833-6897

(2) INFORMATION FOR SEQ ID Nθ:l: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2896 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: N-terminal

(Xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:l:

CTGATCTGAT CTTATCTTCT CTCTTCTTTT CTTTGAGTGT GAATTTTCCT GTTTCCCCCA 60 GGCTGGAGTG CAGTGGCGCG ATGTCGGCTC ACTGCAACCT CTGTCTCCCG GGTTCAAGCG 120

ATTCTCCTGC CTCAGCCTCC TGAGTAGCTG GGACTACAGG CGCATGCCAC CATGCCCAGC 180 TAATTTTTGT ATTTTTAGTA GAGACAGGGT TTTGCCTTGT TGGCCAGGCT GGTCTTGAAC 240

TCCTGACCTC AGGCGATCCA CCCGCCTCGG CCCCTGCACA GTGCCTGGCA CATAGCAAGT 300 GCTCAATAAA TATTTGGTAA GACAAGAACA CATAAGCGAC ATTCAAATGA ATGTCAATTC 360

CTCCCTCCCA TGGGGTGAGG GTTAGGAGTC AGCTGGATTT CTACGATAAC CTCCATTATG 420

CTGTCTTGCT CCCTCCAGAT GACGTCAGTT GCCAAGGTGT ATTACAGTCA GACCACTCAG 480

ACAGAAAGCC GGCCCCTAAT GGGCCCAGGG ATACGACGGC GGAGAGTCCT GACAAAAGAT 540

GGTCGCAGCA ACGTGAGAAT GGAGCACATT GCCGACAAGC GCTTCCTCTA CCTCAAGGAC 600 CTGTGGACAA CCTTCATTGA CATGCAGTGG CGCTACAAGC TTCTGCTCTT CTCTGCGACC 660

TTTGCAGGCA CATGGTTCCT CTTTGGCGTG GTGTGGTATC TGGTAGCTGT GGCACATGGG 720

GACCTGCTGG AGCTGGACCC CCCGGCCAAC CACACCCCCT GTGTGGTACA GGTGCACACA 780

CTCACTGGAG CCTTCCTCTT CTCCCTTGAA TCCCAAACCA CCATTGGCTA TGGCTTCCGC 840

TACATCAGTG AGGAATGTCC ACTGGCCATT GTGCTTCTTA TTGCCCAGCT GGTGCTCACC 900 ACCATCCTGG AAATCTTCAT CACAGGTACC TTCCTGGCGA AGATTGCCCG GCCCAAGAAG 960

CGGGCTGAGA CCATTCGTTT CAGCCAGCAT GCAGTTGTGG CCTCCCACAA TGGCAAGCCC 1020

TGCCTCATGA TCCGAGTTGC CAATATGCGC AAAAGCCTCC TCATTGGCTG CCAGGTGACA 1080

GGAAAACTGC TTCAGACCCA CCAAACCAAG GAAGGGGAGA ACATCCGGCT CAACCAGGTC 1140

AATGTGACTT TCCAAGTAGA CACAGCCTCT GACAGCCCCT TCCTTATTCT ACCCCTTACC 1200 TTCTATCATG TGGTAGATGA GACCAGTCCC TTGAAAGATC TCCCTCTTCG CAGTGGTGAG 1260

GGTGACTTTG AGCTGGTGCT GATCCTAAGT GGGACAGTGG AGTCCACCAG TGCCACCTGT 1320

CAGGTGCGCA CTTCCTACCT GCCAGAGGAG ATCCTTTGGG GCTACGAGTT CACACCTGCC 1380

ATCTCACTGT CAGCCAGTGG TAAATACATA GCTGACTTTA GCCTTTTTGA CCAAGTTGTG 1440

AAAGTGGCCT CTCCTAGTGG CCTCCGTGAC AGCACTGTAC GCTACGGAGA CCCTGAAAAG 1500 CTCAAGTTGG AGGAGTCATT AAGGGAGCAA GCTGAGAAGG AGGGCAGTGC CCTTAGTGTG 1560

CGCATCAGCA ATGTCTGATG ACCTGTTCCC ACTCCCCCAT TCCTCTGGTC TCTTTTCCTC 1620

TCTTCCAATG CCCTGGTAAG GAATACTACC CGGGTTTACT GGAGATCCCC CGAAGCACCC 1680

ATCCTCCACT CCCTCTTCTT TAACCCAGTG GCCTGTTGGT AGCTTAGGCC AACTGGAGTC 1740

CAGGTTCGCC TCCCACTGTC CCCTTTCCAC TTCCCCAGCT TCTGCCCCAA TACACATACC 1800 TCCCTTAAGC CAGGATGGGG GAAAGAGTGG GATTAGGCTG AAGTGGCTTA GAAGGCCTCA 1860

GCCATGCTTG GATACTCACA TTAGGAGGAC CATGTGGTTG GAAGGATAGA CTGCCCCCTA 1920

CCTCCCACCA CCACCATGAA GTTTGGTGAC TTGAGGCTGG AGCTCCCTCT GTTACCTTTC 1980

CATCTAGCAA GTTCCCAAAG GCAAGACTCT CTCTGATGGT CACTTTGTGG TCTGTGCTTT 2040

CAGAAATACA GGAATCTGAT ATCAACATAT CCTAGGGTTT CTACCAATCT CTGTTGAAAG 2100 AAGCCAGGGT TTGCCACTGT GAAGCTTGAT TTCTGCTGGT GACTTCTGAC CATAAGCTAG 2160

AACCATGGTC GCCACTGTTT TCCCTCTGTA GTTTCTCAAG TGAACACTCT CAGGATACCC 2220 AGTTCCCTCA TAGCCTCTGT TCTCAGAGAA TTGGAGTTGG CCCAAGAAAC ATAAACATAT 2280

AACCACCCAT ATCTATCCTG GATTCTGAAC TCTTCAATTT GGAGTGACTA ACACAAGTTG 2340 TTATCTAAAC CTTTAAACCT ATCTTCCAGG CAGCCCAGAG AAGATCTGTT TCCCTGTGTC 2400

CTGTGAATGG AAGGACCCAA GCCAATATGT TCCTTTGAAA AGAGTCCAGT ACCCAGGCCC 2460

CATGGAAAGG TCTGAAAATA ATATTCCAGA TTACACTGTA CCTGGCTTCT CTTCTTCCTT 2520

TCCTGCTCAG CCTAGATCCT TCTTCCTTAA CCCCAACTCT TTGGGAGAAG GGAGGGAAAA 2580

TGCAAGGGCC TTCCTCTCTT AACACGGATG CTCAAGTAAA ACTAGATTCA CAGGGCACAG 2640 ATTCCCCAGA AAGTTAACAC AATCCCACCA TGAGGGATGG GTAAATTCTC AGATTTCCAA 2700

ACTGCTGTAC AGAGCCTCTG AGAATTGGTG ATGCTTTGTT AAGGTTTGGG CAGGAGCAGA 2760

ACTCTGTGGC TGGCAGCCAC TATTCTCAGT TACACCTCCC AGTGCCCTTC TGAAAAGTGC 2820

CAGCTATTTC ATTAGGCAAT GCTGGAAGGA AATGAAATTA TACCTTCTGA TCAAATAACC 2880 ATGGCTTCCC TCAGCC 2896 (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1625 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA to mRNA (iϋ) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (V) FRAGMENT TYPE: N-terminal

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: GAATTCCGGG TTCTACCTGC CTTGAAGAAG ACACCTGACC TGCGGAGTGA GTGACCAGTG 60

TTTCCAGAGC CTGGCAATGG ATGCCATTCA CATCGGCATG TCCAGCACCC CCCTGGTGAA 120

GCACACTGCT GGGGCTGGGC TCAAGGCCAA CAGACCCCGC GTCATGTCCA AGAGTGGGCA 180

CAGCAACGTG AGAATTGACA AAGTGGATGG CATATACCTA CTCTACCTGC AAGACCTGTG 240

GACCACAGTT ATCGACATGA AGTGGAGATA CAAACTCACC CTGTTCGCTG CCACTTTTGT 300 GATGACCTGG TTCCTTTTTG GAGTCATCTA CTATGCCATC GCGTTTATTC ATGGGGACTT 360

AGAACCCGAT GAGCCCATTT CAAATCATAC CCCCTGCATC ATGAAAGTGG ACTCTCTCAC 420

TGGGGCGTTT CTCTTTTCCC TGGAATCCCA GACAACCATT GGCTATGGAG TCCGTTCCAT 480

CACAGAGGAA TGTCCTCATG CCATCTTCCT GTTGGTTGCT CAGTTGGTCA TCACGACCTT 540

GATTGAGATC TTCATCACCG GAACCTTCCT GGCCAAAATC GCCAGACCCA AAAAGCGGGC 600 TGAGACCATC AAGTTCAGCC ACTGTGCAGT CATCACCAAG CAGAATGGGA AGCTGTGCTT 660

GGTGATTCAG GTAGCCAATA TGAGGAAGAG CCTCTTGATT CAGTGCCAGC TCTCTGGCAA 720 GCTCCTGCAG ACCCACGTCA CCAAGGAGGG GGAGCGGATT CTCCTCAACC AAGCCACTGT 780

CAAATTCCAC GTGGACTCCT CCTCTGAGAG CCCCTTCCTC ATTCTGCCCA TGACATTCTA 8 0 CCATGTGCTG GATGAGACGA GCCCCCTGAG AGACCTCACA CCCCAAAACC TAAAGGAGAA 900

GGAGTTTGAG CTTGTGGTCC TCCTCAATGC CACTGTGGAA TCCACCAGCG CTGTCTGCCA 960

GAGCCGAACA TCTTATATCC CAGAGGAAAT CTACTGGGGT TTTGAGTTTG TGCCTGTGGT 1020

ATCTCTCTCC AAAAATGGAA AATATGTGGC TGATTTCAGT CAGTTTGAAC AGATTCGGAA 1080

AAGCCCAGAT TGCACATTTT ACTGTGCAGA TTCTGAGAAA CAGCAACTCG AGGAGAAGTA 1140 CAGGCAGGAG GATCAGAGGG AAAGAGAACT GAGGACACTT TTATTACAAC AGAGCAATGT 1200

CTGATCACAG GGGCGCCATC CAGGTTTAAC CCTGCAAGCT GTTTCCACAT CAGAACTCCC 1260

TTCAAACACA AAGATTGCTG TGAAAACGAA AATGTGTAGA CGCACTCTCA AAAACTGCAC 1320

GGACATACAA AATCAATCTT TTCCTTTGAT CTTGTGGCTA AACCAGCATT TCTGTGTTTG 1380

AGAGATTTCC TGTTAGGTGC TTCGTCTGAA AGTGAACTCT CATAATTCAA ATTGTATAAA 1440 ATAAAGCTAC ATTTCTAAGA GCTTGGTGTA GGGCAATTGG AATAATGTCC TGTTAGATAA 1500

ACAGACATTT AGCAATGCTG ACATTAAAAG GAAATGTATT TCTATACAAG ATTATTAGCT 1560

GTAATACAAG ATATTTATTT AACCAATGAC CTTATGGCTG AGAGTTGAAT TGTGGTTCAG 1620

TATTC 1625 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 380 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(v) FRAGMENT TYPE: N-terminal

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Met Thr Ser Val Ala Lys Val Tyr Tyr Ser Gin Thr Thr Gin Thr Glu 1 5 10 15

Ser Arg Pro Leu Met Gly Pro Gly Ile Arg Arg Arg Arg Val Leu Thr 20 25 30

Lys Asp Gly Arg Ser Asn Val Arg Met Glu His Ile Ala Asp Lys Arg 35 40 45

Phe Leu Tyr Leu Lys Asp Leu Trp Thr Thr Phe Ile Asp Met Gin Trp 50 55 60 Arg Tyr Lys Leu Leu Leu Phe Ser Ala Thr Phe Ala Gly Thr Trp Phe 65 70 75 80 Leu Phe Gly Val Val Trp Tyr Leu Val Ala Val Ala His Gly Asp Leu 85 90 95

Leu Glu Leu Asp Pro Pro Ala Asn His Thr Pro Cys Val Val Gin Val 100 105 110

His Thr Leu Thr Gly Ala Phe Leu Phe Ser Leu Glu Ser Gin Thr Thr 115 120 125 Ile Gly Tyr Gly Phe Arg Tyr Ile Ser Glu Glu Cys Pro Leu Ala Ile 130 135 140

Val Leu Leu Ile Ala Gin Leu Val Leu Thr Thr Ile Leu Glu Ile Phe

145 150 155 160

Ile Thr Gly Thr Phe Leu Ala Lys Ile Ala Arg Pro Lys Lys Arg Ala

165 170 175

Glu Thr Ile Arg Phe Ser Gin His Ala Val Val Ala Ser His Asn Gly 180 185 190

Lys Pro Cys Leu Met Ile Arg Val Ala Asn Met Arg Lys Ser Leu Leu

195 200 205 Ile Gly Cys Gin Val Thr Gly Lys Leu Leu Gin Thr His Gin Thr Lys

210 215 220

Glu Gly Glu Asn Ile Arg Leu Asn Gin Val Asn Val Thr Phe Gin Val

225 230 235 240

Asp Thr Ala Ser Asp Ser Pro Phe Leu lie Leu Pro Leu Thr Phe Tyr 245 250 255

His Val Val Asp Glu Thr Ser Pro Leu Lys Asp Leu Pro Leu Arg Ser 260 265 270

Gly Glu Gly Asp Phe Glu Leu Val Leu Ile Leu Ser Gly Thr Val Glu 275 280 285 Ser Thr Ser Ala Thr Cys Gin Val Arg Thr Ser Tyr Leu Pro Glu Glu

290 295 300

Ile Leu Trp Gly Tyr Glu Phe Thr Pro Ala Ile Ser Leu Ser Ala Ser 305 310 315 320

Gly Lys Tyr Ile Ala Asp Phe Ser Leu Phe Asp Gin Val Val Lys Val 325 330 335

Ala Ser Pro Ser Gly Leu Arg Asp Ser Thr Val Arg Tyr Gly Asp Pro 340 345 350

Glu Lys Leu Lys Leu Glu Glu Ser Leu Arg Glu Gin Ala Glu Lys Glu 355 360 365 Gly Ser Ala Leu Ser Val Arg Ile Ser Asn Val Xaa 370 375 380

(2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 376 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein ( iii ) HYPOTHETICAL : NO

( iv ) ANTI - SENSE : NO ( v ) FRAGMENT TYPE : N-terminal

( i) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met Asp Ala Ile His Ile Gly Met Ser Ser Thr Pro Leu Val Lys His 1 5 10 15

Thr Ala Gly Ala Gly Leu Lys Ala Asn Arg Pro Arg Val Met Ser Lys 20 25 30

Ser Gly His Ser Asn Val Arg Ile Asp Lys Val Asp Gly Ile Tyr Leu

35 40 45 Leu Tyr Leu Gin Asp Leu Trp Thr Thr Val Ile Asp Met Lys Trp Arg 50 55 60

Tyr Lys Leu Thr Leu Phe Ala Ala Thr Phe Val Met Thr Trp Phe Leu 65 70 75 80

Phe Gly Val Ile Tyr Tyr Ala Ile Ala Phe Ile His Gly Asp Leu Glu 85 90 95

Pro Asp Glu Pro Ile Ser Asn His Thr Pro Cys Ile Met Lys Val Asp 100 105 110

Ser Leu Thr Gly Ala Phe Leu Phe Ser Leu Glu Ser Gin Thr Thr Ile

115 120 125 Gly Tyr Gly val Arg Ser Ile Thr Glu Glu Cys Pro His Ala Ile Phe

130 135 140

Leu Leu Val Ala Gin Leu Val Ile Thr Thr Leu Ile Glu Ile Phe Ile 145 150 155 160

Thr Gly Thr Phe Leu Ala Lys Ile Ala Arg Pro Lys Lys Arg Ala Glu 165 170 175

Thr Ile Lys Phe Ser His Cys Ala Val Ile Thr Lys Gin Asn Gly Lys 180 185 190

Leu Cys Leu Val lie Gin Val Ala Asn Met Arg Lys Ser Leu Leu Ile

195 200 205 Gin Cys Gin Leu Ser Gly Lys Leu Leu Gin Thr His Val Thr Lys Glu 210 215 220

Gly Glu Arg Ile Leu Leu Asn Gin Ala Thr Val Lys Phe His val Asp 225 230 235 240

Ser Ser Ser Glu Ser Pro Phe Leu Ile Leu Pro Met Thr Phe Tyr His 245 250 255

Val Leu Asp Glu Thr Ser Pro Leu Arg Asp Leu Thr Pro Gin Asn Leu 260 265 270

Lys Glu Lys Glu Phe Glu Leu Val Val Leu Leu Asn Ala Thr Val Glu 275 280 285 Ser Thr Ser Ala Val Cys Gin Ser Arg Thr Ser Tyr Ile Pro Glu Glu 290 295 300 Ile Tyr Trp Gly Phe Glu Phe Val Pro Val val Ser Leu Ser Lys Asn 305 310 315 320

Gly Lys Tyr Val Ala Asp Phe Ser Gin Phe Glu Gin Ile Arg Lys Ser 325 330 335

Pro Asp Cys Thr Phe Tyr Cys Ala Asp Ser Glu Lys Gin Gin Leu Glu 340 345 350 Glu Lys Tyr Arg Gin Glu Asp Gin Arg Glu Arg Glu Leu Arg Thr Leu 355 360 365

Leu Leu Gin Gin Ser Asn Val Xaa 370 375

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5 : CGCTTTGAAT TCATGACATC AGTTGCCAAG GTCTATTA 38

(2) INFORMATION FOR SEQ ID NO:6:

(1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: YES (v) FRAGMENT TYPE: N-terminal

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: CGCTTTGAAT TCTCAGACGT TACTAATGCG CACACTA 37

(2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 41 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO (v) FRAGMENT TYPE: N-terminal

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CAGAAGTTAA GTCGACATGA CGTCAGTTGC CAAGGTGTAT T 41

(2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 42 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES

(v) FRAGMENT TYPE: N-terminal

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: CAGAAGTTAA GCGGCCGCTC AGACATTGCT GATGCGCACA CT 42

Claims

CLAIMS What is claimed is:

1. An isolated DNA molecule encoding a human kidney potassium channel protein having the sequences shown in Sequence Listings 1 or 2, from the clones named KIRK 2 or KIRK 3, or derivatives selected from obvious variations thereof, or from a DNA molecule at least about 80, 85 or 90 percent homologous to Sequence Listings 1 or 2.

2. An isolated DNA molecule, of claim 1, encoding a human kidney potassium channel of claim 1, having the sequence shown in Sequence Listing 1, describing the clone KIRK 2, or an obvious variation from Sequence Listing 1, or from a DNA molecule at least about a 90 percent homologous to Sequence Listing 1.

3. An isolated DNA molecule, of claim 1, encoding a human kidney potassium channel of claim 1, having the sequence shown in Sequence Listing 2, describing the clone KIRK-3, or an obvious variation from Sequence Listing 2 or from a DNA molecule at least about a 90 percent homologous to Sequence Listing 2.

4. An essentially pure protein whose sequences are shown in Sequence Listings 3 or 4, or one that is coded for by the DNA shown in Sequence Listings 1 or 2, or from protein that is at least about 80, 85 or 90 percent homologous to the sequences in Sequence Listings 3 or 4 or from protein from a DNA molecule derived from DNA at least about 90 percent homologous to the DNA described in Sequence Listings 1 or 2.

5. An essentially pure protein or claim 4 whose sequences are shown in Sequence Listing 3, or one that is coded for by the DNA shown in Sequence Listing

1, or from protein that is at least about 90 percent homologous to the sequence in Sequence Listing 3 or from protein from a DNA molecule derived from DNA at least about 90 percent homologous to the DNA described in Sequence Listing 1.

6. An essentiaUy pure protein or claim 4 whose sequences are shown in Sequence Listing 4, or one that is coded for by the DNA shown in Sequence Listing

2, or from protein that is at least about 90 percent homologous to the sequence in Sequence Listing 4 or from protein from a DNA molecule derived from DNA at least about 90 percent homologous to the DNA described in Sequence Listing 2.

7. A vector comprising a DNA molecule described in claim 1.

8. A plasmid comprising a DNA molecule described in claim 1.

9. A vector of claim 7 adapted for expression in a bacterial ceU.

10. A vector of claim 7 adapted for expression in a mammaUan ceU.

11. A vector of claim 7 adapted for expression in a yeast ceU.

12. A plasmid of claim 8 adapted for expression in a bacterial ceU.

13. A plasmid of claim 8 adapted for expression in a mammalian ceU.

14. A plasmid of claim 8 adapted for expression in a yeast ceU.

15. A bacterial ceU comprising the plasmid of claim 12.

16. An isolated mammaUan ceU comprising the plasmid of claim 13.

17. A yeast ceU comprising the plasmid of claim 14.

18. The method of using the bacterial cell of claim 15 to screen for compounds that modulate human kidney potassium channel activity.

19. The method of using the isolated mammaUan ceU of claim 16 to screen for compounds that modulate human kidney potassium channel activity.

20. The method of claim 19, comprising the foUowing: a) growing ceUs expressing a cloned K* channel to confluence, b) equilibrating the ceUs of a) with a balanced salt solution, c) making baseUne measurements of the equilibrated ceUs, d) adding one test compoimd or a cocktaU of test compovmds to the ceUs and recording changes in membrane potential, e) testing compounds that depolarize ceUs expressing the K⁺ on wild type or mock transfected controls to estabUsh selectivity, f) selecting the compounds that are shown to selectively block K*.

21. The method of claim 20 comprising the foUowing: a) growing ceUs expressing the cloned KIRK 1 or 2 channel to confluence in a 96 weU microtest plate, b) equilibrating the ceUs of a) with a balanced salt solution, such as with 5 μM DiBAC₄(3) in Earle's balanced salt solution containing 20 mM HEPES, c) making baseline measurements of the equilibrated ceUs, d) adding one test compound or a cocktail of test compounds to the ceUs and recording changes in membrane potential, e) testing compounds that depolarize ceUs expressing the K* on wild type or mock transfected controls to estabUsh selectivity, f) selecting the compounds that are shown to selectively block ^1".

22. The method of using the yeast ceU of claim 19 to screen for compounds that modulate human kidney potassium channel activity.

23. An isolated protein functioning in a mammaUan ceU, as a human kidney potassium channel protein having the sequences shown in Sequence Listings 3 or 4 , or produced from the clones named KIRK 2 or KIRK 3,or derivatives selected from obvious variations thereof, or from derivative having at least about 90 percent homologous sequences thereof.

24. A isolated protein of claim 25, functioning in a mammalian ceU, as a human kidney potassium channel protein having the sequences shown in Sequence Listing 3, or from the clones named KIRK 2 or KIRK 3,or derivatives selected from obvious variations thereof, or from derivatives having at least about 90 percent homologous sequences thereof.

25. A isolated protein of claim 25, functioning in a mammaUan ceU, as a human kidney potassium channel protein having the sequences shown in Sequence Listing 4, or from the clone named KIRK 3, or derivatives selected from obvious variations thereof, or from derivatives having at least about 90 percent homologous sequences thereof.