NOVEL SEQUENCES OF p56 PROTEINS WHICH AFFECT K-ATP CHANNELS

Abstract: This invention describes the full length sequence of human p56 protein (p56-1), a related homolog (p56-2) and the nucleic acids that code for these proteins. The sequences are provided in Charts 1, 2, 3 and 4 and the sequence listings of the application.

Description: NOVEL SEQUENCES OF p56, PROTEINS WHICH AFFECT K-ATP CHANNELS FIELD OF THE INVENTION This invention relates to potassium channels and the p56 protein. INFORMATION DISCLOSURE The disclosures appearing in PCT/US95/14124, published 30 May 1996 as WO 96/16088, particularly page 1, is hereby incorporated by reference. Additional documents in the Detailed Description of the invention should be considered as part of the Information Disclosure. BACKGROUND The background appearing in PCT/US95/14124, published 30 May 1996 as WO 96/16088, particularly pages 1-5, are hereby incorporated by reference. The isolation and identification of p56, a protein useful for the identification of selective drugs that will selectively open or close K channels was described in PCT/US95/14124, published 30 May 1996 as WO 96/16088. Herein the full length amino acid sequence of p56 and the nucleic acid sequence that code for p56 are described. Also described are the amino acid sequences and coding DNA that code for p56-2, a different p56 protein.. SUMMARY OF THE INVENTION This invention describes the full length sequence of human p56 protein (p56- 1), a related homolog (p56-2) and the nucleic acids that code for these proteins. The sequences are provided in Charts 1, 2, 3, and 4 and the sequence listings of the application. There are two unique p56 DNA sequences and proteins disclosed in this document. For the first p56 sequence, p56-l, the protein's entire amino acid sequence of p56 is provided in Chart 1 and the DNA coding for p56 is provided in Chart 2. For the second sequence, p56-2, the DNA and amino acids are disclosed Charts 3 and 4. Equivalents and obvious homologues are disclosed. Also disclosed by reference to PCT/US95/14124, published 30 May 1996 as WO 96/16088, hereby incorporated by reference, are cloning and other useful vectors for the sequence. ADDITIONAL DESCRIPTION OF THE INVENTION AND DESCRIPTION OF THE PREFERRED EMBODIMENTS The entire document numbered PCT/US95/14124, published 30 May 1996 as WO 96/16088, particularly pages 6-11 are hereby incorporated by reference. UTILITY OF THE INVENTION The utility of this invention is disclosed and supported by the disclosure appearing in PCT/US95/14124, published 30 May 1996 as WO 96/16088, particularly pages 11-12 are hereby incorporated by reference. DETAILED DESCRIPTION OF THE INVENTION A detailed description of the invention may be found by reference to PCT/US95/14124, published 30 May 1996 as WO 96/16088, particularly page 12, lines 33-35 - page 20, hereby incorporated by reference. In addition to the PCT/WO publication the following additional remarks and materials disclose this invention. Type I ATP-sensitive K channels (IK-ATP-* were fi rst described in cardiac muscle (1) and have subsequently been characterized in skeletal muscle (2) vascular smooth muscle (3) and the β-cell of the pancreas (4-6). These type I channels are inhibited by micomolar concentrations of intracellular ATP and are insensitive to voltage and Ca (7). The β-cell IR-ATP channel is the most well understood in functional terms and it serves as a metabolic sensor that controls the release of insulin. Glucose stimulation of the β-cell results in an increase in the intracellular ATP/ADP ratio via glycolysis and the increase in ATP inhibits channel conductance. Because IR-ATP channels dominate the resting membrane potential of the β cell, inhibition of channel conductance results in a depolarization that activates voltage- n i , dependent Ca channels and the resultant increase in intracellular Ca triggers insulin release. Pharmacological agents like sulfonylureas [IK_ATP blocker] or diazoxide [IR-ATP opener] stimulate or inhibit insulin release, respectively (8). In cardiac and skeletal muscle, the IR.ATP channels serve a similar function, coupling cell metabolism and electrical activity. These channels have a low open probability under resting conditions and are activated by a decline in the intracellular ATP/ADP ratio in response to either ischemic injury and/or exercise (2,9). ^K-ATP channels are unique among potassium channels because their activity can be regulated by a wide variety of structurally diverse pharmacological agents (10-15). These include both channel blockers {eg. sulfonylureas, guanidines and cyanoguanidines) and channel openers (eg. diazoxide, pinacidil, chromakalim, and minoxidil sulfate). This rich pharmacology translates into many opportunities for therapeutic intervention such as antidiabetics (sulfonylureas blockers), diuretics (guanidine and cyanoguanidine blockers), antihypertensives (openers like pinacidil and minoxidil sulfate) and agents that promote hair growth (minoxidil sulfate). Given the structural diversity among the chemical classes of agents that modify the activity of IR-ATP channels in various cell types, it is likely that the structure of these channels are complex. This diversity, coupled to the key role that IR.A P channels play in linking cell excitability to metabolism, offers the hope that tissue selective modulators of IK_ATP channel activity can provide additional opportunities for the management of human disease. Elucidation of the minimal structural components necessary to form functional hannels was the result of two divergent efforts, namely the cloning and charaterization of either inward rectifier potassium channels or the high affinity sulfonylurea receptor (16,17). The first two members of the inward rectifier potassium channel gene family were isolated using expression cloning paradigms (18,19) and to date, homology cloning efforts have defined a total of 13 distinct genes in this class. Heterologous expression of the inward rectifier referred to as K^. 6.1 in human embryonic kidney cells led to the synthesis of a channel that displayed some of the properties of native channels (16). Subsequent cloning studies have defined a second member of the K^. 6 family, referred to as K^. 6.2 (19, 21-22). Molecular identification of the high affinity sulfonylurea receptor was facilitated by the use of sulfonylurea photoprobes to 'tag' the polypeptide responsible for high affinity sulfonylurea binding. Using either [ H]-glyburide (23-25), [ I]-glyburide analogs (26-28), or [ I]/azido analogs of glyburide (29,30), multiple investigators groups have identified a high affinity 140-150 kDa sulfonylurea binding protein in both β cells and in brain. Purification of photolabeled 140 kDa sulfonylurea receptor from β-cell lines followed by protein microsequence analysis and cDNA cloning has resulted in the molecular definition of the high affinity sulfonylurea receptor [SUR- 1] (17). Subsequent cloning efforts have resulted in the identification of a paralog of SUR-1 referred to as SUR-2 (31-33). The primary structure of SUR-1 and SUR-2 resembles ABC cassette transporter proteins and heterologous expression of either SUR-1 or SUR-2 does not led to the synthesis of functional channels. Alternatively, co-expression of K^. 6.1 SUR-2 or K^. 6.2/SUR-l combinations leads to the reconstitution of functional I jζ -ATP channels that display many of the electrophysiological and pharmacological signatures of native IR.ATP channels (31- 33). A parallel strategy for the identification of cyanoguanidine binding proteins was initiated with the synthesis and characterization of the azido photoprobe [ H]- probe 1. Because the value of this photoprobe was reduced by its relatively low specific activity, a second generation cyanoguanidine analog, [ I]-probe 2 was prepared and characterized. Photolabeling of intact rat aortic smooth muscle cells (A10) with 125 I-probe 2, identified both high affinity (p56) and a low affinity (p47) binding sites that were displaced by homologous competition. Purification of I- p56 from A10 cells and protein microsequence analysis identified a unique twelve amino acid residue sequence tag derived from the NH2 terminus of I-p56 and this identification was substantiated by the preparation and characterization of an antipeptide antibody that recognizes this sequence tag. This amino acid sequence tag was used to query expressed sequence tag (EST) databases to identify EST clones that potentially encode the human ortholog of the p56 sequence. Complete sequence analysis of multiple EST clones revealed a full-length cDNA that encoded the human ortholog of rat p56. Subsequent queries of an EST database with the full-length sequence of human p56 and sequence analysis revealed a second full- length human cDNA p56 paralog, referred to as p56-2. Human p56-l and p56-2 were characterized by determining their tissue distribution of expression, their glycosylation in vitro and their ability to reconstitute the I-probe 2 binding site following transient expression in COS cells. References by number above are reported here, all are incorporated by reference. 1. Noma, A. "ATP-regulated K + channels in cardiac muscle" Nature 305: 147-148 (1983). 2. Spruce, A.E., Standen, N.B. and Standfield, P.R. "Voltage-dependent ATP- sensitive potassium channels of skeletal muscle membrane" Nature 316: 736-738 (1985). 3. Standen, N.B., Quayle, J.M., Davies, N.W., Brayden, J.E., Huang, Y. and Nelson, M.T. "Hyperpolarizing vasodialators activate ATP-sensitive K + channels in arterial smooth muscle" Science 245: 177-180 (1989). 4. Cook, D.L. and Hales, C.N. "Intracellular ATP directly blocks K + channels in pancreatic beta cells" Nature 311: 271-273 (1984). 5. Sturgess, N.C., Ashford, M.L., Cook, D.L., and Hales, C.N. "The sulfonylurea receptor may be an ATP-sensitive potassium channel" Lancet 1:474-475 (1985). 6. Rorsman, P. and Trube, G. "Glucose-dependent K + channels in pancreatic beta cells are regulated by intracellular ATP" Pflugers Arch 405: 305-309 (1985). 7. Ashcroft, S. J.H. and Ashcroft F.M. "Properties and function of ATP-sensitive K + channels" Cell Signal 2:197-214 (1990). 8. Trube, G., Rorsman, P. and Ohno-Shosaku, T. "Opposite effects of tolbutamide and diazoxide on the ATP-dependent K + channel in mouse pancreatic β-cells" Pflugers Arch 407:493-499 (1986). 9. Escande, D. and Cavero, I. "K + channel openers and 'natural' cardioprotection" Trends Pharmacol. Sci. 13:269-271 (1992). 10. Gopalakrishnan, M., Janis, R.A. and Triggle, D.J. "ATP-sensitive K + channels: Pharmacologic properties, regulation and therapeutic potential" Drug Dev. Res. 28: 95-127 (1993). 11. Atwal, K.S. "Advances in the structure-activity relationships, mechanisms of action, and therapeutic utilities of ATP-sensitive potassium channel openers" Drug Dev. Res. 33:250-262 (1994). 12. Edwards, G. and Weston, A.H. "The pharmacology of ATP-sensitive potassium channels" Ann.Reυ. Pharmacol. Toxicol. 33: 597-637 (1993). 13. de Weille, J.R. "Modulation of ATP sensitive potassium channels" Cardiovascular Res. 26: 1017-1020 (1992). 14. Atwal, K.S. "Modulation of potassium channels by organic molecules" Medicinal Research Rev.12:569-591 (1992). 15. Robertson, D.W. and Steinberg, M.I. "Potassium channel modulators: Scientific applications and therapeutic promise" J. Med. Chem. 33: 1530-1541 (1990). 16. 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 ATP- sensitive potassium channel ubiquitously expressed in rat tissues, including pancreatic islets, pituitary, .skeletal muscle and heart" J. Biol. Chem. 270: 5691- 5694 (1995). 17. Aguilar-Bryan, L., Nichols, C.G., Wechsler, S.W., Clement, J.P., Boyd, A.E., Gonzalez, G.,Herrera-Sosa, H., Nguy, K, Bryan, J. and Nelson, D.A. "Cloning of the β-cell high affinity sulfonylurea receptor; a regulator of insulin secretion" Science 268:423-426 (1995). 18. 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). 19. Dascal, N., Schreibmayer, W., Li , N.F., Wang, W., Chavkin, C, DiMagno, L., Labarca, C, Kieffer, B.L., Gaveriaux-Ruff, C, Trollinger, D., Lester, H.A., and Davidson, N. "Atrial G-protein activated K + channel: Expression cloning and molecular properties" Proc. Natl. Acad. Sci. USA 90: 10235-10239 (1993). 21. Inagaki, N., Gonoi, T., Clement, J.P., Namba, N., Inazawa, J., Gonzalez, G., Aguilar- Bryan, L., Seino, S. and Bryan, J. "Reconstitution of an inward rectifier subunit plus the sulfonylurea receptor" Science 270: 1166-170 (1995). 22. Tokuyama, Y., Fan, Z., Furuta, H., Makielski, J.C., Polonsky, K.S., Bell, G.I. and Yano, H. "Rat inwardly rectifying potassium channel Kir 6.2: Cloning, electrophysiological characterization, and decreased expression in pancreatic islets of male Zucker diabetic fatty rats" Biochem. Biophys. Res. Comm. 220: 532-538 (1996). 23. Kramer, W., Oekonomopulos, R., Punter, J., and Summ, H.D. "Direct photolabeling of the putative sulfonylurea receptor in rat β-cell tumor membranes by [ 3 H]- glybenclamide" FEBS Let 229:355-359 (1988). 24. Bernardi, H., Fosset, M., and Lazdunski, M. "Characterization, purification and affinity labeling of the brain [ H]-glybenclamide-binding protein, a putative neuronal ATP-regulated K + channel" Proc. Natl. Acad. Sci. USA 85:9816-9820 (1988). 25. Benardi, H., Fosset, M., and Lazdunski, M. "ATP/ADP Binding sites are present in the sulfonylurea binding protein associated with brain ATP-sensitive K + channels" Biochemistry 31:6328-6332 (1992). 26. Aguilar-Bryan, L., Nelson, D.A., Vu, Q., Humphrey, M.B., and Boyd III, A.E. "Photoaffinity labeling and partial purification of the β-cell sulfonylurea receptor using a novel, biologically active glyburide analog" J. Biol. Chem. 265:8218-8224 (1990). 27. Nelson, D.A., Aguilar-Bryan, L., and Bryan, J. "Specficity of photolabeling of β- cell membrane proteins with an I-labeled glyburide analog" J. Biol. Chem. 267:14928-14933 (1992). 28. Bernardi, H., De Weille, J.R, Epelbaum, J., Mourre, C, Amoroso, S., Slama, A., Fosset, M., and Lazdunski, M. "ATP-modulated K + channels sensitive to antidiabetic sulfonylureas are present in adenohypophysis and are involved in growth hormone release" Proc. Natl. Acad. Sci. USA 90:1340-1344 (1993). 29. Schwanstecher, M., Loser, S., Chudziak, F., and Panten, U. "Identification of a 38 kDa high affinity sulfonylurea-binding peptide in insulin-secreting cells and cerebral cortex" J. Biol. Chem. 269:17768-17771 (1994). 30. Schwanstecher, M., Loser, S., Chudziak, F., Bachmann, C, and Panten, U. "Photoaffinity labeling of cerebral sulfonylurea receptor using a novel radioiodinated azidoglibenclamide analogue" J. Neurochem. 63:698-708 (1994). 31. Inagaki, N., Gonoi, T., Clement, J.P., Wang, C.-Z., Aguilar-Bryan, L., Bryan, J. and Seino, S. "A family of sulfonylurea receptors determines the pharmacological properties of ATP-sensitive K + channels" Neuron 16: 1011-1017 (1996). 32. Chutkow, W.A., Simon, M.C., Le Beau, M.M. and Burant, C.F. "Cloning, tissue expression and chromosomal localization of SUR2, the putative drug-binding subunit of cardiac, skeletal muscle and vascular K^p channels" Diabetes 45: 1439-1445 (1996). 33. Isomoto, S., Kondo, C, Ya ada, M., Matsumoto, S., Higashiguchi, O., Horio, Y., Matsuzawa, Y. and Kurachi, Y. "A novel sulfonylurea receptor forms with BIR (K^. 6.2) a smooth muscle type ATP-sensitive K + channel" J. Biol. Chem. 271:24321- 24324 (1996). The NH2 - terminal amino acid sequence was established, by the method described above in PCT/US95/14124, then various data bases and DNA libraries were repeatedly searched with no positive result. Finally, after numerous searches, the p56-l NH2-terminal amino acid sequence (EPRAPPEKIAIGAG), disclosed in PCT/US95/14124, published 30 May 1996 as WO 96/16088, led to the discovery of a human expressed sequence tag (EST) that displayed a significant match to the sequence disclosed in that PCT publication. A clone containing this sequence, identified as number 56-1, was obtained by searching various data bases and DNA libraries and the complete DNA sequence was determined. Clone 56-1 contained a 1515 bp open-reading frame that encoded a 505 amino acid polypeptide with a predicted Mr for the mature protein of 52 kDa, close to the expected p56. The predicted sequence contained a signal peptide followed by a mature NH2-terminus that showed 11/14 exact matches with the rat p56 NH2-terminal sequence disclosed in PCT/US95/14124, published 30 May 1996 as WO 96/16088. Consistent with biochemical data for rat p56, which demonstrated that p56 is a glycoprotein, the predicted polypeptide for clone 56-1 also contained 3 canonical acceptor sites for Asn-linked glycosylation. The predicted amino acid sequence has no significant homology to known sequences. The predicted amino acid from clone 56-1 was analyzed using a variety of secondary structure prediction algorithms. A Rossman fold was detected near the NH2-terminus, indicating that this protein was likely to bind nucleotides like ATP. In contrast to the biochemical data on rat p56, no predicted transmembrane segments were detected but an endoplasmic reticulum retention signal (KTEL-versus-the canonical KDEL) near the COOH-terminus was scored. A β-turn-β membrane association motif, similar to the P (pore)-region of voltage-gated-K + -channels, was also detected. It appears that this polypeptide may associate with other K-channel pore-forming polypeptides (e.g. Kir 6.1 or 6.2) and regulate K channel activity. See, 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 ATP-sensitive potassium channel ubiquitously expressed in rat tissues, including pancreatic islets, pituitary, skeletal muscle and heart" J. Biol. Chem. 270: 5691-5694 (1995). AND. Inagaki, N., Gonoi, T., Clement, J.P., Namba, N., Inazawa, J., Gonzalez, G., Aguilar- Bryan, L., Seino, S. and Bryan, J. "Reconstitution of Iκ-ATP : an mwa rd rectifier subunit plus the sulfonylurea receptor" Science 270: 1166-170 (1995). Both articles incorporated by reference, and in particular aspects describing functional channel systems. Using the sequence information disclosed herein one ordinarily skilled in the art should be able to use known PCT techniques to create, fashion, or produce clones having and or expressing the desired sequences described herein. The sequences can also be used in screens and assays for the detection of biologically active compounds. Additional descriptions of, procedures for and examples of these types of vectors, plasmids, cells, screens and assays can be found in case 6001. NCP, serial number 08/709,923 filed 9 September 1996, hereby incorporated by reference. In particular page 16 relating to procedures for preparing and using clones in assays especially relevant and incorporated by reference. In a similar manner additional descriptions of, procedures for and examples of these types of vectors, plasmids, cells, screens and assays can be found in WO 94/19464, PCT/US94/01210, published 1 September 1994, hereby incorporated by reference. In particular, the pages relating to procedures for preparing and using clones in assays is especially relevant and incorporated by reference. The second p56 sequence was discovered according to the following procedures. Various data bases were queried with the amino acid sequence of human p56 using the FASTA search tool. In addition to identifying the known EST matches to p56, three additional ESTs that shared approximately 38-48% identity with p56 were also scored [447210, 2607571 and 2663551]. Alignment of the 5' EST sequence reads for these 3 clones with the p56 sequence showed that it was unlikely that the EST sequences overlapped and that clone 2607571 was most likely to be full-length. These clones were obtained and clone 2607571 was completely sequenced. This clone contained a 2.6 kb insert complete with a 1482 bp open reading frame that showed 41% shared identity with human p56. Motifs that were common to both predicted amino acid sequences include (1) a signal sequence, (2) Rossman fold, (3) canonical acceptor sites for Asn-linked glycosylation [3 in p56-l and 6 in p56-2] and (4) an ER retention signal at the COOH-terminus [KTEL]. Comparison of the biochemical properties ofp56-l — Biochemical characterization of I-p56-l from A10 cells revealed a Triton X-100 soluble 56 kDa glycoprotein that could be de-glycosylated with N-glycanase to yield a 52 kDa form. These data are consistent with the M^ of human p56-l predicted from the cDNA sequence and the presence of canonical acceptor sites for Asn-linked glycosylation in the sequence. To establish these biochemical parameters experimentally for p56-l and to investigate the glycosylation pattern of p56-2, both polypeptides were prepared by in vitro translation. Each expression plasmid was linearized by digestion with Notl and capped cRNA synthesized using T7-RNA polymerase. These cRNAs were then used to direct the synthesis of S-methionine labeled protein using rabbit reticulocyte lysates ± canine pancreatic microsomes. The radiolabeled proteins were fractionated by SDS-PAGE and visualized by fluorography of the dried gel. P56-1 and p56-2 have unglycosylated M_ values of 52 kDa and 50 kDa, respectively. In the presence of canine pancreatic microsomes, multiple bands with higher M_ values, including 56kDa, were also detected, indicating the addition of Asn-linked oligosaccharides to p56-l. In contrast to p56-l, p56-2 did not appear to be glycosylated under these conditions. Tissue distribution of expression of human p56-l and p56-2.-Comparison of Northern analysis and transcript imaging — The expression pattern of p56-l and p56- 2 transcripts was determined using both classical Northern blot analysis and by BLAST searching of EST database. For Northern blot analysis, poly A + RNAs isolated from various peripheral tissues and various brain regions were fractionated by electrophoresis under denaturing conditions and displayed on a nylon membrane. t j The blots were then visualized by hybridization to coding sequence DNA probes prepared from either human p56-l or human p56-2. The p56-l probe visualized a 6.0 kb transcript that was expressed at the highest level in skeletal muscle and lower levels detected in heart and pancreas. Minor signal was also detected in brain, placenta and liver while no transcript was detected in either lung or kidney under these conditions. Alternatively, BLAST searching revealed 20 ESTs derived from 11 different tissues matching the query with the complete p56-l DNA sequence. These included the following ESTs; 5 from breast (4 normal/1 tumor), 3 from prostate (1 normal/2 tumor), 2 from brain, 2 from colon tumors, 2 from kidney and single ESTs from stomach, uterus, pituitary, nasal polyp, thyroid and mononuclear cells. The human p56-2 probe visualized a 2.8 kb transcript that was expressed at the highest level in heart, brain, pancreas and placenta. No signal for p56-2 was observed in lung, liver, skeletal muscle or kidney under these conditions. p56-2 transcripts were widely expressed in brain regions, with the highest levels in the amygdala and lower levels in the hippocampus, the caudate nucleus, the corpus callosum, the substantia nigra and the subthalamic nucleus. Twenty ESTs matched the p56-2 query sequence and these were derived from 13 different tissues including 3 from pancreas, 3 from lung tumors, 2 from synovial membrane, 2 from leukocytes, 2 from hippocampus and single EST matches from heart, kidney, bladder, small intestine, adrenal, breast, prostate and nasal polyp. Reconstitution of p56 photolabeling by transient expression of human p56-l and p56-2 — Photolabeling of p56-l with a cyanoguanidine opener photoprobe was reconstituted by transient heterologous expression of the p56-l cDNA in COS7 cells. The coding sequence of human p56-l was placed under the control of the SV40 immediate early promotor in the vector pSVL. This construct was introduced into COS7 cells using cationic liposomes and 48 hr post-infection, the transfected cells were photolabeled with I . Wild-type COS7 cells showed minimal photolabeling of a 52 kDa band and no p56. Alternatively, transient expression of human p56-l in these same cells led to photolabeling of a polypeptide that migrated with a M y of 56 kDa. CHARTS The following charts disclose the full length human p56-l and p56-2 proteins and the DNA that code for those proteins. Chart 1 provides the sequences for the full length of the p56-l protein. The sequences in Chart 1 include the signal or leader sequence. The sequences in Chart 1 are also in sequence listing no. 1. Chart 2 provides the cDNA residues that code for the p56-l protein and it includes untranslated sequences. All of the sequences in Chart 2 are provided for in sequence listing no. 2. The p56-l DNA sequence shown in Chart 2 contains one of many possible lengths of poly A tail, included as part of the cDNA sequence. This full-length cDNA contains: 1) a 5' untranslated sequence (alignment positions 1-39), 2) a coding sequence (alignment positions 40-1554), 3) a stop codon "TGA" (alignment position 1555-1557) and 4) a 3' untranslated sequence (alignment positions 1555-1724 with poly A). The coding sequence beginning with ATG at position 40 starts with a signal sequence that ends at position 123 while the mature sequence begins at position 124. These positions are noted on Charts 1 and 2 below. The signal sequence or leader sequence is a hydrophobic region, usually about 20-25 amino acids, here 28 aa, at the N-terminus that 'signals' attachment of the ribosome to the endoplasmic reticulum and aids in the extrusion of the nacent polypeptide chain into the lumen of the ER. This signal sequence is cleaved off in the lumen by the signal peptidase. The untranslated sequences may have important regulatory functions such as governing mRNA stability and the like. The poly A track is added after transcription, the length is variable, often from 10-200 A's, here we show a 27 track poly A. Charts 3 and 4 disclose the p56-2 proteins and the DNA that code for those proteins. Chart 3 provides the sequences for the full length of the p56-2 protein. The sequences in Chart 3 include the signal or leader sequence. The sequences in Chart 3 are also in sequence listing no. 3. Chart 4 provides the cDNA residues that code for the p56-2 protein and it includes untranslated sequences. All of the sequences in Chart 2 are provided for in sequence listing no. 4. The p56-2 DNA sequence shown in Chart 4 contains one of many possible lengths of poly A tail, included as part of the cDNA sequence. This full-length cDNA contains: 1) a 5' untranslated sequence (alignment positions 1-35), 2) a coding sequence (alignment positions 36-1517), 3) a stop codon "TGA" (alignment position 1518-1520) and 4) a 3' untranslated sequence (alignment positions 1518-2567 with poly A). The coding sequence beginning with ATG at position 36 starts with a signal sequence. These positions are noted on Charts 3 and 4 below. CHART 1 - Amino acid sequence of the p56-l protein 1 MGRWAELVS SLLG LLC SCGCPEGAEL RAPPDKIAII GAGIGGTSAA 28^29 51 YY RQKFGKD VKIDLFEREE VGGRLATMMV QGQEYEAGGS VIHP N HMK 101 RFVKDLGLSA VQASGGL GI YNGETLVFEE SN FIINVIK LVWRYGFQSL 151 RMHMWVEDVL DKFMRIYRYQ SHDYAFSSVE KL HALGGDD FLGM NRTLL 201 ETLQKAGFSE KF NEMIAPV MRVNYGQSTD INAFVGAVSL SCSDSGL AV 251 EGGNK VCSG L QASKSN I SGSVMYIEEK TKTKYTGNPT KMYEWYQIG 301 TETRSDFYDI V VATPLNRK MSNITFLNFD PPIEEFHQYY QHIVTTLVKG 351 ELNTSIFSSR PIDKFGLNTV LTTDNSD FI NSIGIVPSVR EKEDPEPSTD 401 GTYV KIFSQ ETLTKAQI K LFLSYDYAVK KP AYPHYK PPEKCPSIIL 451 HDR YYLNGI ECAASAMEMS AIAAHNAAL AYHRWNGHTD MIDQDGLYEK 501 LKTEL CHART 2 - Nucleotide sequence of the coding region for the p56-l protein . 1 CGCTCGGAAT TCGGCTCGAG TGCAGAGCTT GTGGAGGCCA TGGGGCGCGT 39^40 51 CGTCGCGGAG CTCGTCTCCT CGCTGCTGGG GTTGTGGCTG TTGCTGTGCA 101 GCTGCGGATG CCCCGAGGGC GCCGAGCTGC GTGCTCCGCC AGATAAAATC 123 T T 124 151 GCGATTATTG GAGCCGGAAT TGGTGGCACT TCAGCAGCCT ATTACCTGCG 201 GCAGAAATTT GGGAAAGATG TGAAGATAGA CCTGTTTGAA AGAGAAGAGG 251 TCGGGGGCCG CCTGGCTACC ATGATGGTGC AGGGGCAAGA ATACGAGGCA 301 GGAGGTTCTG TCATCCATCC TTTAAATCTG CACATGAAAC GTTTTGTCAA 351 AGACCTGGGT CTCTCTGCTG TTCAGGCCTC TGGTGGCCTA CTGGGGATAT 401 ATAATGGAGA GACTCTGGTA TTTGAGGAGA GCAACTGGTT CATAATTAAC 451 GTGATTAAAT TAGTTTGGCG CTATGGATTT CAATCCCTCC GTATGCACAT 501 GTGGGTAGAG GACGTGTTAG ACAAGTTCAT GAGGATCTAC CGCTACCAGT 551 CTCATGACTA TGCCTTCAGT AGTGTCGAAA AATTACTTCA TGCTCTAGGA 601 GGAGATGACT TCCTTGGAAT GCTTAATCGA ACACTTCTTG AAACCTTGCA 651 AAAGGCCGGC TTTTCTGAGA AGTTCCTCAA TGAAATGATT GCTCCTGTTA 701 TGAGGGTCAA TTATGGCCAA AGCACGGACA TCAATGCCTT TGTGGGGGCG 751 GTGTCACTGT CCTGTTCTGA TTCTGGCCTT TGGGCAGTAG AAGGTGGCAA 801 TAAACTTGTT TGCTCAGGGC TTCTGCAGGC ATCCAAAAGC AATCTTATAT 851 CTGGCTCAGT AATGTACATC GAGGAGAAAA CAAAGACCAA GTACACAGGA 901 AATCCAACAA AGATGTATGA AGTGGTCTAC CAAATTGGAA CTGAGACTCG 951 TTCAGACTTC TATGACATCG TCTTGGTGGC CACTCCGTTG AATCGAAAAA 1001 TGTCGAATAT TACTTTTCTC AACTTTGATC CTCCAATTGA GGAATTCCAT 1051 CAATATTATC AACATATAGT GACAACTTTA GTTAAGGGGG AATTGAATAC 1101 ATCTATCTTT AGCTCTAGAC CCATAGATAA ATTTGGCCTT AATACAGTTT 1151 TAACCACTGA TAATTCAGAT TTGTTCATTA ACAGTATTGG GATTGTGCCC 1201 TCTGTGAGAG AAAAGGAAGA TCCTGAGCCA TCAACAGATG GAACATATGT 1251 TTGGAAGATC TTTTCCCAAG AAACTCTTAC TAAAGCACAA ATTTTAAAGC 1301 TCTTTCTGTC CTATGATTAT GCTGTGAAGA AGCCATGGCT TGCATATCCT 1351 CACTATAAGC CCCCGGAGAA ATGCCCCTCT ATCATTCTCC ATGATCGACT 1401 TTATTACCTC AATGGCATAG AGTGTGCAGC AAGTGCCATG GAGATGAGTG 1451 CCATTGCAGC CCACAACGCT GCACTCCTTG CCTATCACCG CTGGAACGGG 1501 CACACAGACA TGATTGATCA GGATGGCTTA TATGAGAAAC TTAAAACTGA 1551 ACTATGAAGT GACACACTCC TTTTTCCCCT CCTAGTTCCA AATGACTATC 1554 T T f T T 1558 stop 1601 AGTGGCAAAA AAGAACAAAA TCTGAGCAGA GATGATTTTG AACCAGATAT 1651 TTTGCCATTA TCATTGTTTA ATAAAAGTAA TCCCTGCTGG TCATAGGAAA 1701 AAAAAAAAAA AAAAAAAAAA AAAA Chart 3. Amino acid sequence of the p56-2 protein. 1 MARAAP LAA LTALLAAAAA GGDAPPGKIA WGAGIGGSA VAHFLQQHFG 51 PRVQIDVYEK GTVGGRLATI SVNKQHYESG AASFHS SLH QDFVK LG 101 RHRREWGRS AIFGGEHFM EETD Y N FRL WHYGIS FLRLQ VEE 151 V EKFMRIYK YQAHGYAFSG VEELLYSLGE STFVNMTQHS VAESLLQVGV 201 TQRFIDDWS AVLRASYGQS AAMPAFAGAM SLAGAQGSLW SVEGGNKLVC 251 SG KLTKAN VIHATVTSVT HSTEGKALY QVAYENEVGN SSDFYDIWI 301 ATPLHLDNSS SNLTFAGFHP PIDDVQGSFQ PTWSLVHGY LNSSYFGFPD 351 PKLFPFANIL TTDFPSFFCT LDNICPVNIS ASFRRKQPQE AAVWRVQSPK 401 PLFRTQLKTL FRSYYSVQTA EWQAHPLYGS RPTLPRFALH DQLFYLNALE 451 WAASSVEV A VAAKNVALLA YNRWYQDLDK IDQKDLMHKV KTEL Chart 4. Nucleotide sequence of the coding region for the p56-2 protein . 1 CCTGAATCCG GCGTGCTGCC CGCTCGCCGC CCGCCATGGC CCGCGCAGCC 35 π 36 51 CCGCTGCTCG CCGCGTTGAC CGCGCTCCTC GCCGCCGCCG CTGCTGGCGG 101 AGATGCCCCG CCGGGCAAAA TCGCGGTGGT TGGGGCTGGG ATTGGGGGCT 151 CTGCTGTGGC CCATTTTCTC CAGCAGCACT TTGGACCTCG GGTGCAGATC 201 GACGTGTACG AGAAGGGAAC CGTGGGTGGC CGCTTGGCCA CCATCTCAGT 251 CAACAAGCAG CACTATGAGA GCGGGGCTGC CTCCTTCCAC TCCCTGAGCC 301 TGCACATGCA GGACTTCGTC AAGCTGCTGG GGCTGAGGCA CCGGCGCGAG 351 GTGGTGGGCA GGAGCGCCAT CTTCGGCGGG GAGCACTTCA TGCTGGAGGA 401 GACTGACTGG TACCTGCTGA ACCTCTTCCG CCTCTGGTGG CACTATGGCA 451 TCAGCTTCCT GAGGCTGCAG ATGTGGGTGG AGGAGGTCAT GGAGAAGTTC 501 ATGAGGATCT ATAAGTACCA GGCCCACGGC TATGCCTTCT CGGGTGTGGA 551 GGAGCTGCTC TACTCACTGG GGGAGTCCAC CTTTGTTAAC ATGACCCAGC 601 ACTCTGTGGC TGAGTCCCTG CTGCAGGTGG GCGTCACGCA GCGCTTTATT 651 GATGATGTCG TTTCTGCTGT CCTGCGGGCC AGCTATGGCC AGTCAGCAGC 701 GATGCCCGCC TTTGCAGGAG CCATGTCACT AGCCGGGGCC CAAGGCAGCC 751 TGTGGTCTGT GGAAGGAGGC AATAAGCTGG TTTGTTCCGG TTTGCTGAAG 801 CTCACCAAGG CCAATGTGAT CCATGCCACA GTGACCTCTG TGACCCTGCA 851 CAGCACAGAG GGGAAAGCCC TGTACCAGGT GGCGTATGAG AATGAGGTAG 901 GCAACAGCTC TGACTTCTAT GACATCGTGG TCATCGCCAC CCCCCTGCAC 951 CTGGACAACA GCAGCAGCAA CTTAACCTTT GCAGGCTTCC ACCCGCCCAT 1001 TGATGACGTG CAGGGCTCTT TCCAGCCCAC CGTCGTCTCC TTGGTCCACG 1051 GCTACCTCAA CTCGTCCTAC TTCGGTTTCC CAGACCCTAA GCTTTTCCCC 1101 TTTGCCAACA TCCTTACCAC AGATTTCCCC AGCTTCTTCT GCACTCTGGA 1151 CAACATCTGC CCTGTCAACA TCTCTGCCAG CTTCCGGCGA AAGCAGCCCC 1201 AGGAGGCAGC TGTTTGGCGA GTCCAGTCCC CCAAGCCCCT CTTTCGGACC 1251 CAGCTAAAGA CCCTGTTCCG TTCCTATTAC TCAGTGCAGA CAGCTGAGTG 1301 GCAGGCCCAT CCCCTCTATG GCTCCCGCCC CACGCTCCCG AGGTTTGCAC 1351 TCCATGACCA GCTCTTCTAC CTCAATGCCC TGGAGTGGGC GGCCAGCTCC 1401 GTGGAGGTGA TGGCCGTGGC TGCCAAGAAT GTGGCCTTGC TGGCTTACAA 1451 CCGCTGGTAC CAGGACCTAG ACAAGATTGA TCAAAAAGAT TTGATGCACA 1501 AGGTCAAGAC TGAACTGTGA GGGCTCTAGG GAGAGCCTGG GAACTTTCAT 1518*. Tt f 1521 Stop 1551 CCCCCACTGA AGATGGATCA TCCCACAGCA GCCCAGGACT GAATAAGCCA 1601 TGCTCGCCCA CCAGGCTTCT TTCTGACCCC TCATGTATCA AGCATCTCCA 1651 GGTGACCTAC TGTCTGCCTA TATTAAGGGT CCACACGGCG GCTGCTGCTT 1701 TTTTTTAAGG GGGAAAGTAA GAAAAGAGAA GGAAATCCAA GCCAGTATAT 1751 TTGTTTTATT TATTTTTTTT AAGAAGAAAA AAGTTCATCT TCACAAGGTG 1801 CTTCAGACTT GGTTTCTTAG CTAGAAACCA GAAGACTACG GGAGGGAATA 1851 TAAGGCAGAG AACTATGAGT CTTATTTTAT TACTGTTTTT CACTACCTAC 1901 TCCCACAATG GACAATCAAT TGAGGCAACC TACAAGAAAA CATTTACAAC 1951 CAGATGGTTA CAAATAAAGT AGAAGGGAAG ATCAGAAAAC CTAAGAAATG 2001 ATCATAGCTC CTGGTTACTG TGGACTTGAT GGATTTGAAG TACCTAGTTC 2051 AGAACTCCCT AGTCACCATC TCCAAGCCTG TCAACATCAC TGCATATTGG 2101 AGGAGATGAC TGTGGTAGGA CCCAAGGAAG AGATGTGTGC CTGAATAGTC 2151 GTCACCATAT CTCCAAGCTT CCTGGCAACC AGTGGGAAAA GAAACATGCG 2201 AGGCTGTAGG AAGAGGGAAG CTCTTCCTTG GCACCTAGAG GAATTAGCCA 2251 TTCTCTTCCT TATGCAAAGA TTGAGGAATG CAACAATATA AAGAAGAGAA 2301 GTCCCCAGAT GGTAGAGAGC AGTCATATCT TACCCCTAGA TGTTCATCCC 2351 AGCAGAAGAA AGAAGAAGGT GTTGGGGTAG GATTCTTCAG AGGTTAGCCT 2401 GGTACTTTCT CATCAGACAC TAGCTTGAAG TAAGAGGAGA ATTATGCTTT 2451 TCTTTGCTTT TTCTACAAAC CCTTAAAAAT CACTTGTTTT AAAAAGAAAG 2501 TAAAAGCCCT TTTCATTCAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 2551 AAAAAAAAAA AAAAAAA 2567 SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Pharmacia & Upjohn Company (ii) TITLE OF INVENTION: Novel Sequences of p56, Proteins Which Affect K-ATP Channels (iii) NUMBER OF SEQUENCES: 4 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Pharmacia and Upjohn, Co., Intel. Prop. Law (1920-32-LAW) (B) STREET: 301 Henrietta Street (C) CITY: Kalamazoo (D) STATE: Michigan (E) COUNTRY: U.S.A. (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: Patentln Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: ( iii) ATTORNEY/AGENT INFORMATION: (A) NAME: Wootto , Thomas A. (B) REGISTRATION NUMBER: 35,004 (C) REFERENCE/DOCKET NUMBER: 6092 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (616) 833-7914 (B) TELEFAX: (616) 833-8897 (2) INFORMATION FOR SEQ ID NO : 1 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 505 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 : 1 : Met Gly Arg Val Val Ala Glu Leu Val Ser Ser Leu Leu Gly Leu Trp 1 5 10 15 Leu Leu Leu Cys Ser Cys Gly Cys Pro Glu Gly Ala Glu Leu Arg Ala 20 25 30 Pro Pro Asp Lys lie Ala lie lie Gly Ala Gly lie Gly Gly Thr Ser 35 40 45 Ala Ala Tyr Tyr Leu Arg Gin Lys Phe Gly Lys Asp Val Lys lie Asp 50 55 60 Leu Phe Glu Arg Glu Glu Val Gly Gly Arg Leu Ala Thr Met Met Val 65 70 75 80 Gin Gly Gin Glu Tyr Glu Ala Gly Gly Ser Val lie His Pro Leu Asn 85 90 95 Leu His Met Lys Arg Phe Val Lys Asp Leu Gly Leu Ser Ala Val Gin 100 105 110 Ala Ser Gly Gly Leu Leu Gly lie Tyr Asn Gly Glu Thr Leu Val Phe 115 120 125 Glu Glu Ser Asn Trp Phe lie lie Asn Val lie Lys Leu Val Trp Arg 130 135 140 Tyr Gly Phe Gin Ser Leu Arg Met His Met Trp Val Glu Asp Val Leu 145 150 155 160 Asp Lys Phe Met Arg lie Tyr Arg Tyr Gin Ser His Asp Tyr Ala Phe 165 170 175 Ser Ser Val Glu Lys Leu Leu His Ala Leu Gly Gly Asp Asp Phe Leu 180 185 190 Gly Met Leu Asn Arg Thr Leu Leu Glu Thr Leu Gin Lys Ala Gly Phe 195 200 205 Ser Glu Lys Phe Leu Asn Glu Met lie Ala Pro Val Met Arg Val Asn 210 215 220 Tyr Gly Gin Ser Thr Asp lie Asn Ala Phe Val Gly Ala Val Ser Leu 225 230 235 240 Ser Cys Ser Asp Ser Gly Leu Trp Ala Val Glu Gly Gly Asn Lys Leu 245 250 255 Val Cys Ser Gly Leu Leu Gin Ala Ser Lys Ser Asn Leu lie Ser Gly 260 265 270 Ser Val Met Tyr lie Glu Glu Lys Thr Lys Thr Lys Tyr Thr Gly Asn 275 280 285 Pro Thr Lys Met Tyr Glu Val Val Tyr Gin lie Gly Thr Glu Thr Arg 290 295 300 Ser Asp Phe Tyr Asp lie Val Leu Val Ala Thr Pro Leu Asn Arg Lys 305 310 315 320 Met Ser Asn lie Thr Phe Leu Asn Phe Asp Pro Pro lie Glu Glu Phe 325 330 335 His Gin Tyr Tyr Gin His lie Val Thr Thr Leu Val Lys Gly Glu Leu 340 345 350 Asn Thr Ser lie Phe Ser Ser Arg Pro lie Asp Lys Phe Gly Leu Asn 355 360 365 Thr Val Leu Thr Thr Asp Asn Ser Asp Leu Phe lie Asn Ser lie Gly 370 375 380 lie Val Pro Ser Val Arg Glu Lys Glu Asp Pro Glu Pro Ser Thr Asp 385 390 395 400 Gly Thr Tyr Val Trp Lys lie Phe Ser Gin Glu Thr Leu Thr Lys Ala 405 410 415 Gin lie Leu Lys Leu Phe Leu Ser Tyr Asp Tyr Ala Val Lys Lys Pro 420 425 430 Trp Leu Ala Tyr Pro His Tyr Lys Pro Pro Glu Lys Cys Pro Ser lie 435 440 445 lie Leu His Asp Arg Leu Tyr Tyr Leu Asn Gly lie Glu Cys Ala Ala 450 455 460 Ser Ala Met Glu Met Ser Ala lie Ala Ala His Asn Ala Ala Leu Leu 465 470 475 480 Ala Tyr H s Arg Trp Asn Gly His Thr Asp Met lie Asp Gin Asp Gly 485 490 495 Leu Tyr Glu Lys Leu Lys Thr Glu Leu 500 505 (2) INFORMATION FOR SEQ ID NO : 2 : (1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1724 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (11) MOLECULE TYPE: cDNA (ill) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO (XI) SEQUENCE DESCRIPTION: SEQ ID NO : 2 : CGCTCGGAAT TCGGCTCGAG TGCAGAGCTT GTGGAGGCCA TGGGGCGCGT CGTCGCGGAG 60 CTCGTCTCCT CGCTGCTGGG GTTGTGGCTG TTGCTGTGCA GCTGCGGATG CCCCGAGGGC 120 GCCGAGCTGC GTGCTCCGCC AGATAAAATC GCGATTATTG GAGCCGGAAT TGGTGGCACT 180 TCAGCAGCCT ATTACCTGCG GCAGAAATTT GGGAAAGATG TGAAGATAGA CCTGTTTGAA 240 AGAGAAGAGG TCGGGGGCCG CCTGGCTACC ATGATGGTGC AGGGGCAAGA ATACGAGGCA 300 GGAGGTTCTG TCATCCATCC TTTAAATCTG CACATGAAAC GTTTTGTCAA AGACCTGGGT 360 CTCTCTGCTG TTCAGGCCTC TGGTGGCCTA CTGGGGATAT ATAATGGAGA GACTCTGGTA 420 TTTGAGGAGA GCAACTGGTT CATAATTAAC GTGATTAAAT TAGTTTGGCG CTATGGATTT 480 CAATCCCTCC GTATGCACAT GTGGGTAGAG GACGTGTTAG ACAAGTTCAT GAGGATCTAC 540 CGCTACCAGT CTCATGACTA TGCCTTCAGT AGTGTCGAAA AATTACTTCA TGCTCTAGGA 600 GGAGATGACT TCCTTGGAAT GCTTAATCGA ACACTTCTTG AAACCTTGCA AAAGGCCGGC 660 TTTTCTGAGA AGTTCCTCAA TGAAATGATT GCTCCTGTTA TGAGGGTCAA TTATGGCCAA 720 AGCACGGACA TCAATGCCTT TGTGGGGGCG GTGTCACTGT CCTGTTCTGA TTCTGGCCTT 780 TGGGCAGTAG AAGGTGGCAA TAAACTTGTT TGCTCAGGGC TTCTGCAGGC ATCCAAAAGC 840 AATCTTATAT CTGGCTCAGT AATGTACATC GAGGAGAAAA CAAAGACCAA GTACACAGGA 900 AATCCAACAA AGATGTATGA AGTGGTCTAC CAAATTGGAA CTGAGACTCG TTCAGACTTC 960 TATGACATCG TCTTGGTGGC CACTCCGTTG AATCGAAAAA TGTCGAATAT TACTTTTCTC 1020 AACTTTGATC CTCCAATTGA GGAATTCCAT CAATATTATC AACATATAGT GACAACTTTA 1080 GTTAAGGGGG AATTGAATAC ATCTATCTTT AGCTCTAGAC CCATAGATAA ATTTGGCCTT 1140 AATACAGTTT TAACCACTGA TAATTCAGAT TTGTTCATTA ACAGTATTGG GATTGTGCCC 1200 TCTGTGAGAG AAAAGGAAGA TCCTGAGCCA TCAACAGATG GAACATATGT TTGGAAGATC 1260 TTTTCCCAAG AAACTCTTAC TAAAGCACAA ATTTTAAAGC TCTTTCTGTC CTATGATTAT 1320 GCTGTGAAGA AGCCATGGCT TGCATATCCT CACTATAAGC CCCCGGAGAA ATGCCCCTCT 1380 ATCATTCTCC ATGATCGACT TTATTACCTC AATGGCATAG AGTGTGCAGC AAGTGCCATG 1440 GAGATGAGTG CCATTGCAGC CCACAACGCT GCACTCCTTG CCTATCACCG CTGGAACGGG 1500 CACACAGACA TGATTGATCA GGATGGCTTA TATGAGAAAC TTAAAACTGA ACTATGAAGT 1560 GACACACTCC TTTTTCCCCT CCTAGTTCCA AATGACTATC AGTGGCAAAA AAGAACAAAA 1620 TCTGAGCAGA GATGATTTTG AACCAGATAT TTTGCCATTA TCATTGTTTA ATAAAAGTAA 1680 TCCCTGCTGG TCATAGGAAA AAAAAAAAAA AAAAAAAAAA AAAA 1724 (2) INFORMATION FOR SEQ ID NO .3 (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 494 amino acids (B) TYPE: amino acid (C) STRANDEDNESS single (D) TOPOLOGY, linear ( i) MOLECULE TYPE: protein (111) HYPOTHETICAL. NO (IV) ANTI -SENSE NO (v) FRAGMENT TYPE N-termmal (X ) SEQUENCE DESCRIPTION SEQ ID NO : 3 Met Ala Arg Ala Ala Pro Leu Leu Ala Ala Leu Thr Ala Leu Leu Ala 1 5 10 15 Ala Ala Ala Ala Gly Gly Asp Ala Pro Pro Gly Lys lie Ala Val Val 20 25 30 Gly Ala Gly lie Gly Gly Ser Ala Val Ala His Phe Leu Gin Gin H s 35 40 45 Phe Gly Pro Arg Val Gin lie Asp Val Tyr Glu Lys Gly Thr Val Gly 50 55 60 Gly Arg Leu Ala Thr lie Ser Val Asn Lys Gin His Tyr Glu Ser Gly 65 70 75 80 Ala Ala Ser Phe His Ser Leu Ser Leu His Met Gin Asp Phe Val Lys 85 90 95 Leu Leu Gly Leu Arg His Arg Arg Glu Val Val Gly Arg Ser Ala lie 100 105 110 Phe Gly Gly Glu His Phe Met Leu Glu Glu Thr Asp Trp Tyr Leu Leu 115 120 125 Asn Leu Phe Arg Leu Trp Trp His Tyr Gly lie Ser Phe Leu Arg Leu 130 135 140 Gin Met Trp val Glu Glu Val Met Glu Lys Phe Met Arg lie Tyr Lys 145 150 155 160 Tyr Gin Ala His Gly Tyr Ala Phe Ser Gly Val Glu Glu Leu Leu Tyr 165 170 175 Ser Leu Gly Glu Ser Thr Phe Val Asn Met Thr Gin His Ser Val Ala 180 185 190 Glu Ser Leu Leu Gin Val Gly Val Thr Gin Arg Phe lie Asp Asp Val 195 200 205 Val Ser Ala Val Leu Arg Ala Ser Tyr Gly Gin Ser Ala Ala Met Pro 210 215 220 Ala Phe Ala Gly Ala Met Ser Leu Ala Gly Ala Gin Gly Ser Leu Trp 225 230 235 240 Ser Val Glu Gly Gly Asn Lys Leu Val Cys Ser Gly Leu Leu Lys Leu 245 250 255 Thr Lys Ala Asn Val lie His Ala Thr Val Thr Ser Val Thr Leu His 260 265 270 Ser Thr Glu Gly Lys Ala Leu Tyr Gin Val Ala Tyr Glu Asn Glu Val 275 280 285 Gly Asn Ser Ser Asp Phe Tyr Asp lie Val Val He Ala Thr Pro Leu 290 295 300 H s Leu Asp Asn Ser Ser Ser Asn Leu Thr Phe Ala Gly Phe His Pro 305 310 315 320 Pro He Asp Asp Val Gin Gly Ser Phe Gin Pro Thr Val Val Ser Leu 325 330 335 Val His Gly Tyr Leu Asn Ser Ser Tyr Phe Gly Phe Pro Asp Pro Lys 340 345 350 Leu Phe Pro Phe Ala Asn He Leu Thr Thr Asp Phe Pro Ser Phe Phe 355 360 365 Cys Thr Leu Asp Asn He Cys Pro Val Asn He Ser Ala Ser Phe Arg 370 375 380 Arg Lys Gin Pro Gin Glu Ala Ala Val Trp Arg Val Gin Ser Pro Lys 385 390 395 400 Pro Leu Phe Arg Thr Gin Leu Lys Thr Leu Phe Arg Ser Tyr Tyr Ser 405 410 415 Val Gin Thr Ala Glu Trp Gin Ala His Pro Leu Tyr Gly Ser Arg Pro 420 425 430 Thr Leu Pro Arg Phe Ala Leu His Asp Gin Leu Phe Tyr Leu Asn Ala 435 440 445 Leu Glu Trp Ala Ala Ser Ser Val Glu Val Met Ala Val Ala Ala Lys 450 455 460 Asn Val Ala Leu Leu Ala Tyr Asn Arg Trp Tyr Gin Asp Leu Asp Lys 465 470 475 480 He Asp Gin Lys Asp Leu Met His Lys Val Lys Thr Glu Leu 485 490 (2) INFORMATION FOR SEQ ID NO : 4 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2567 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 : CCTGAATCCG GCGTGCTGCC CGCTCGCCGC CCGCCATGGC CCGCGCAGCC CCGCTGCTCG 60 CCGCGTTGAC CGCGCTCCTC GCCGCCGCCG CTGCTGGCGG AGATGCCCCG CCGGGCAAAA 120 TCGCGGTGGT TGGGGCTGGG ATTGGGGGCT CTGCTGTGGC CCATTTTCTC CAGCAGCACT 180 TTGGACCTCG GGTGCAGATC GACGTGTACG AGAAGGGAAC CGTGGGTGGC CGCTTGGCCA 240 CCATCTCAGT CAACAAGCAG CACTATGAGA GCGGGGCTGC CTCCTTCCAC TCCCTGAGCC 300 TGCACATGCA GGACTTCGTC AAGCTGCTGG GGCTGAGGCA CCGGCGCGAG GTGGTGGGCA 360 GGAGCGCCAT CTTCGGCGGG GAGCACTTCA TGCTGGAGGA GACTGACTGG TACCTGCTGA 420 ACCTCTTCCG CCTCTGGTGG CACTATGGCA TCAGCTTCCT GAGGCTGCAG ATGTGGGTGG 480 AGGAGGTCAT GGAGAAGTTC ATGAGGATCT ATAAGTACCA GGCCCACGGC TATGCCTTCT 540 CGGGTGTGGA GGAGCTGCTC TACTCACTGG GGGAGTCCAC CTTTGTTAAC ATGACCCAGC 600 ACTCTGTGGC TGAGTCCCTG CTGCAGGTGG GCGTCACGCA GCGCTTTATT GATGATGTCG 660 TTTCTGCTGT CCTGCGGGCC AGCTATGGCC AGTCAGCAGC GATGCCCGCC TTTGCAGGAG 720 CCATGTCACT AGCCGGGGCC CAAGGCAGCC TGTGGTCTGT GGAAGGAGGC AATAAGCTGG 780 TTTGTTCCGG TTTGCTGAAG CTCACCAAGG CCAATGTGAT CCATGCCACA GTGACCTCTG 840 TGACCCTGCA CAGCACAGAG GGGAAAGCCC TGTACCAGGT GGCGTATGAG AATGAGGTAG 900 GCAACAGCTC TGACTTCTAT GACATCGTGG TCATCGCCAC CCCCCTGCAC CTGGACAACA 960 GCAGCAGCAA CTTAACCTTT GCAGGCTTCC ACCCGCCCAT TGATGACGTG CAGGGCTCTT 1020 TCCAGCCCAC CGTCGTCTCC TTGGTCCACG GCTACCTCAA CTCGTCCTAC TTCGGTTTCC 1080 CAGACCCTAA GCTTTTCCCC TTTGCCAACA TCCTTACCAC AGATTTCCCC AGCTTCTTCT 1140 GCACTCTGGA CAACATCTGC CCTGTCAACA TCTCTGCCAG CTTCCGGCGA AAGCAGCCCC 1200 AGGAGGCAGC TGTTTGGCGA GTCCAGTCCC CCAAGCCCCT CTTTCGGACC CAGCTAAAGA 1260 CCCTGTTCCG TTCCTATTAC TCAGTGCAGA CAGCTGAGTG GCAGGCCCAT CCCCTCTATG 1320 GCTCCCGCCC CACGCTCCCG AGGTTTGCAC TCCATGACCA GCTCTTCTAC CTCAATGCCC 1380 TGGAGTGGGC GGCCAGCTCC GTGGAGGTGA TGGCCGTGGC TGCCAAGAAT GTGGCCTTGC 1440 TGGCTTACAA CCGCTGGTAC CAGGACCTAG ACAAGATTGA TCAAAAAGAT TTGATGCACA 1500 AGGTCAAGAC TGAACTGTGA GGGCTCTAGG GAGAGCCTGG GAACTTTCAT CCCCCACTGA 1560 AGATGGATCA TCCCACAGCA GCCCAGGACT GAATAAGCCA TGCTCGCCCA CCAGGCTTCT 1620 TTCTGACCCC TCATGTATCA AGCATCTCCA GGTGACCTAC TGTCTGCCTA TATTAAGGGT 1680 CCACACGGCG GCTGCTGCTT TTTTTTAAGG GGGAAAGTAA GAAAAGAGAA GGAAATCCAA 1740 GCCAGTATAT TTGTTTTATT TATTTTTTTT AAGAAGAAAA AAGTTCATCT TCACAAGGTG 1800 CTTCAGACTT GGTTTCTTAG CTAGAAACCA GAAGACTACG GGAGGGAATA TAAGGCAGAG 1860 AACTATGAGT CTTATTTTAT TACTGTTTTT CACTACCTAC TCCCACAATG GACAATCAAT 1920 TGAGGCAACC TACAAGAAAA CATTTACAAC CAGATGGTTA CAAATAAAGT AGAAGGGAAG 1980 ATCAGAAAAC CTAAGAAATG ATCATAGCTC CTGGTTACTG TGGACTTGAT GGATTTGAAG 2040 TACCTAGTTC AGAACTCCCT AGTCACCATC TCCAAGCCTG TCAACATCAC TGCATATTGG 2100 AGGAGATGAC TGTGGTAGGA CCCAAGGAAG AGATGTGTGC CTGAATAGTC GTCACCATAT 2160 CTCCAAGCTT CCTGGCAACC AGTGGGAAAA GAAACATGCG AGGCTGTAGG AAGAGGGAAG 2220 CTCTTCCTTG GCACCTAGAG GAATTAGCCA TTCTCTTCCT TATGCAAAGA TTGAGGAATG 2280 CAACAATATA AAGAAGAGAA GTCCCCAGAT GGTAGAGAGC AGTCATATCT TACCCCTAGA 2340 TGTTCATCCC AGCAGAAGAA AGAAGAAGGT GTTGGGGTAG GATTCTTCAG AGGTTAGCCT 2400 GGTACTTTCT CATCAGACAC TAGCTTGAAG TAAGAGGAGA ATTATGCTTT TCTTTGCTTT 2460 TTCTACAAAC CCTTAAAAAT CACTTGTTTT AAAAAGAAAG TAAAAGCCCT TTTCATTCAA 2520 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAA 2567

Claims: Claims 1. A composition of nucleic acids, separated from the native genome, comprising the nucleic acids that can code for a glycoprotein of about 54,000 to 60,000 daltons and having a core protein mass (free of sugars) of about 51,000 daltons, capable of being isolated from rat A10 cells, that contains at least 60, 70, 80, 90, 95 or a substantial portion of the amino acids shown in Chart 2 or 4 or sequence listing 2, or 4, in sequential order, and capable of binding with N-(3-azido-5-iodophenyl)-N'-cyano-N"- (1,1-dimethylpropyl) -guanidine or substantially similar analogues. 2. A composition of claim 1 having 70, 80, 90, or 95 percent homology to the nucleic acids listed in Charts 2 or 4. 3. A composition of claim 2 that codes for human p56 protein. 4. A composition of claim 3 that codes for the amino acids disclosed in Chart 2 or 4 or sequence listing 2 or 4. 5. A composition of nucleic acids of claim 4 having 90 percent homology to the nucleic acids listed in Chart 2 or 4 or of sequence listing 2 or 4. 6. A composition of nucleic acids of claim 5 having 95 percent homology to the nucleic acids listed in Chart 2 or 4 or of sequence listing 2 or 4. 7. A composition of nucleic acids of claim 4 comprising the nucleic acids listed in Chart 2 or 4 or of sequence listing 2 or 4. 8. A composition of nucleic acids of claim 7 comprising the coding nucleic acids listed in Chart 2, or of sequence listing 2, where the sequences before position 124 may be any appropriate alignment sequences and the sequences after position 1558, which includes any stop codon, may be any appropriate poly A sequences, and substantially similar sequences or a sequence obtained by deleting, adding or replacing one to several amino acid residues of any of the sequence. 9. A composition of nucleic acids of claim 8 comprising the coding nucleic acids listed in Chart 2, starting at position 124 through and including position 1558, including any stop codon, and substantially similar sequences or a sequence obtained by deleting, adding or replacing one to several amino acid residues of that sequence. 10. A composition of nucleic acids of claim 7 comprising the coding nucleic acids listed in Chart 4, or of sequence listing 4, where the sequences before position 36 may be any appropriate alignment sequences and the sequences after position 1521, which includes any stop codon, may be any appropriate poly A sequences, and substantially similar sequences or a sequence obtained by deleting, adding or replacing one to several amino acid residues of any of the sequence. 11. A composition of nucleic acids of claim 10 comprising the coding nucleic acids listed in Chart 4, starting at position 36, through and including position 1520, including any stop codon, and substantially similar sequences or a sequence obtained by deleting, adding or replacing one to several amino acid residues of that sequence. 12. A composition of consecutive linked amino acids, a protein, in essentially purified form, or expressed in an isolated manner, that create a glycoprotein of about 54,000 to 60,000 daltons and having a core protein mass (free of sugars) of about 51,000 daltons, that is capable of being isolated from rat A10 cells and capable of binding with N-(3-azido-5-iodophenyl)-N'-cyano-N"- (1,1-dimethylpropyl), that contains at least 50, 60, 70, 80, 90 or a substantial portion of the amino acids shown in Chart 1 or 3 sequence listing 1 or 3, in the order shown. 13. A composition of claim 1 having 70, 80, 90, or 95 percent homology to the nucleic acids listed in Chart 1 or 3. 14. A composition of amino acids of claim 13 where the amino acid sequences are substantially similar sequences to those in Chart 1 or 3 or sequence listing 1 or 3 or a sequence obtained by deleting, adding or replacing one to several amino acid residues of that sequence. 15. A composition of amino acids of claim 14 where the amino acid sequences are substantially similar sequences to those in Chart 1 or sequence listing 1 or a sequence obtained by deleting, adding or replacing one to several amino acid residues of that sequence. 16. A composition of amino acids of claim 15 where the amino acid sequences are substantially similar sequences to those in Chart 3 or sequence listing 3 or a sequence obtained by deleting, adding or replacing one to several amino acid residues of that sequence. 17. The DNA disclosed in claiml, above, incorporated into a vector selected from a cloning vector, a shuttle vector or an expression vector. 18. A vector of Claim 17 where the vector is selected from cloning vectors, shuttle vectors or expression vectors and where these vectors are plasmids. 19. A cloning vector of Claim 18 where the cloning vector or plasmid is selected from any widely available or commercially available plasmids. 20. A plasmid of claim 18 adapted for expression in a bacterial cell. 21. A plasmid of claim 18 adapted for expression in a mammalian cell. 22. A plasmid of claim 18 adapted for expression in a yeast cell. 23. The method of using the bacterial cell of claim 20 to screen for compounds that modulate human potassium channel activity. 24. The method of using the isolated mammalian cell of claim 21 to screen for compounds that modulate human potassium channel activity. 25. The method of claim 21, comprising the following: a) growing cells expressing a cloned ^p channel, such channel comprising Kir type channel and a p56-l or p56-2 protein 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^p on wild type or mock transfected controls to establish selectivity, f) selecting the compounds that are shown to selectively modulate K^-pp channel activity. 26. The method of claim 25 where the K^. type channel is type K^. 6.1 channel and the p56 protein is p56-2, described by sequence listing 3, and related homologues. 27. The method of claim 25 where the K^. type channel is type K^. 6.2 channel and the p56 protein is p56-2, described by sequence listing 1, and related homologues. 28. The method of claim 21 comprising the following: a) growing cells expressing a cloned K + channel, that includes the p56-l or p56-2 protein, to confluence in a 96 well microtest plate, b) equilibrating the cells of a) with a balanced salt solution, such as with 5 μM DiBAC 4 (3) in Earle's balanced salt solution containing 20 mM HEPES, 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 on wild type or mock transfected controls to establish selectivity, f) selecting the compounds that are shown to selectively modulate channel activity. 29. The method of using either a yeast cell of claim 22 or a bacterial cell of claim 20, according to the procedures in claim 25, to screen for compounds that modulate human potassium channel activity. 30. A protein of claim 12, functioning in a mammalian cell, as a human cyanoguanidine binding protein having the sequences shown in Sequence Listings 1, or 2 or produced from the clones named p56-l or p56-2, (Seq. ID. No. 2 or 4) or homologues selected from obvious variations thereof, or from homologues having at least about 90 percent homologous sequences thereof . 31. A isolated protein of claim 30, having at least about 95 percent homologous sequences thereof. 32. A isolated protein of claim 31, having substantially the same sequences as those in Chart 2 or sequence listing 2. 33. A isolated protein of claim 32, having substantially the same sequences as those in Chart 4 or sequence listing 4.

Inventors: BIENKOWSKI MICHAEL J, BIENKOWSKI, MICHAEL, J.
Applicants: BIENKOWSKI MICHAEL J, UPJOHN CO, BIENKOWSKI, MICHAEL, J., PHARMACIA & UPJOHN COMPANY
Application year: 1997
Main classification: C12N15
IPC codes: C07K14, C12N15, C12Q1, G01N33