Pancreatic Islet Cells Express a Family of Inwardly Rectifying K+ Channel Subunits Which Interact to Form G-protein-activated Channels
1995; Elsevier BV; Volume: 270; Issue: 44 Linguagem: Inglês
10.1074/jbc.270.44.26086
ISSN1083-351X
AutoresJorge Ferrer, Colin G. Nichols, Elena Makhina, Lawrence Salkoff, Josh Bernstein, D.S. Gerhard, J. Wasson, Sasanka Ramanadham, Alan Permutt,
Tópico(s)Genetics and Neurodevelopmental Disorders
ResumoInsulin secretion is associated with changes in pancreatic β-cell K+ permeability. A degenerate polymerase chain reaction strategy based on the conserved features of known inwardly rectifying K+ (KIR) channel genes was used to identify members of this family expressed in human pancreatic islets and insulinoma. Three related human KIR transcript sequences were found: CIR (also known as cardiac KATP-1), GIRK1, and GIRK2 (KATP-2). The pancreatic islet CIR and GIRK2 full-length cDNAs were cloned, and their genes were localized to human chromosomes 11q23-ter and 21, respectively. Northern blot analysis detected CIR mRNA at similar levels in human islets and exocrine pancreas, while the abundance of GIRK2 mRNA in the two tissues was insufficient for detection by this method. Using competitive reverse-transcription polymerase chain reaction, CIR was found to be present at higher levels than GIRK2 mRNA in native purified β-cells. Xenopus oocytes injected with M2 muscarinic receptor (M2) plus either GIRK2 or CIR cRNA expressed only very small carbachol-induced currents, while co-injection of CIR plus GIRK2 along with M2 resulted in expression of carbachol-activated strong inwardly rectifying currents. Activators of KATP channels failed to elicit currents in the presence or absence of co-expressed sulfonylurea receptor. These results show that two components of islet cell KIR channels, CIR and GIRK2, may interact to form heteromeric G-protein-activated inwardly rectifying K+ channels that do not possess the typical properties of KATP channels. Insulin secretion is associated with changes in pancreatic β-cell K+ permeability. A degenerate polymerase chain reaction strategy based on the conserved features of known inwardly rectifying K+ (KIR) channel genes was used to identify members of this family expressed in human pancreatic islets and insulinoma. Three related human KIR transcript sequences were found: CIR (also known as cardiac KATP-1), GIRK1, and GIRK2 (KATP-2). The pancreatic islet CIR and GIRK2 full-length cDNAs were cloned, and their genes were localized to human chromosomes 11q23-ter and 21, respectively. Northern blot analysis detected CIR mRNA at similar levels in human islets and exocrine pancreas, while the abundance of GIRK2 mRNA in the two tissues was insufficient for detection by this method. Using competitive reverse-transcription polymerase chain reaction, CIR was found to be present at higher levels than GIRK2 mRNA in native purified β-cells. Xenopus oocytes injected with M2 muscarinic receptor (M2) plus either GIRK2 or CIR cRNA expressed only very small carbachol-induced currents, while co-injection of CIR plus GIRK2 along with M2 resulted in expression of carbachol-activated strong inwardly rectifying currents. Activators of KATP channels failed to elicit currents in the presence or absence of co-expressed sulfonylurea receptor. These results show that two components of islet cell KIR channels, CIR and GIRK2, may interact to form heteromeric G-protein-activated inwardly rectifying K+ channels that do not possess the typical properties of KATP channels. INTRODUCTIONThe permeability of K+ ions plays a crucial role in the control of pancreatic islet β-cell excitability and insulin secretion(1Misler S. Barnett D.W. Gillis K.D. Pressel D.M. Diabetes. 1992; 41: 1221-1228Crossref PubMed Google Scholar, 2Ashcroft F.M. Rorsman P. Prog. Biophys. Mol. Biol. 1991; 54: 87-143Crossref Scopus (945) Google Scholar). Electrophysiological studies have revealed at least four classes of functionally distinct K+ currents in β-cells: 1) ATP-sensitive K+ channels that close in response to increased intracellular ATP/ADP ratios generated by increased metabolic flux, 2) voltage-gated K+ channels activated by depolarization, 3) large and small conductance calcium-activated K+ channels, and 4) ligand-gated K+ channels that respond to physiological agonists acting through G-protein-coupled receptors(1Misler S. Barnett D.W. Gillis K.D. Pressel D.M. Diabetes. 1992; 41: 1221-1228Crossref PubMed Google Scholar, 3Cook D.L. Hales C.N. Nature. 1984; 311: 271-273Crossref PubMed Scopus (968) Google Scholar, 4Rorsman P. Bokvist K. Ammala C. Arkhammar P. Berggren P.O. Larsson O. Wahlander K. Nature. 1991; 349: 77-79Crossref PubMed Scopus (109) Google Scholar, 5Rorsman P. Trube G. J. Physiol. (Lond.). 1986; 374: 531-550Crossref Scopus (284) Google Scholar). Because pancreatic islet K+ channel genes are only beginning to be identified, the molecular basis for most of these currents remains unknown(6Inagaki N. Tsuura Y. Namba N. Masuda K. Gonoi T. Horie M. Seino Y. Mizuta M. Seino M. J. Biol. Chem. 1995; 270: 5691-5694Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar, 7Philipson L.H. Hice R.E. Schaefer K. LaMendola J. Bell G.I. Nelson D.J. Steiner D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 53-57Crossref PubMed Scopus (71) Google Scholar, 8Yano H. Philipson L.H. Kugler J.L. Tokuyama Y. David E.M. LeBeau M. Nelson D.J. Bell G.I. Takeda J. Mol. Pharmacol. 1994; 45: 854-860PubMed Google Scholar). Furthermore, their precise contribution to insulin secretory activity is largely not understood. The characterization of K+ channel proteins synthesized in islet cells is of great practical interest, as it will contribute to understanding β-cell electrophysiology and potentially enhance the development of more effective and specific drugs to manipulate insulin secretory function. Furthermore, because defective insulin release is central to the pathogenesis of non-insulin-dependent diabetes mellitus(9Porte Jr., D.J. Diabetes. 1991; 40: 166-180Crossref PubMed Scopus (0) Google Scholar), these molecules provide a valuable source of candidate genes to study the inherited basis of this disorder.A novel superfamily of genes encoding inward rectifying K+ (KIR) channels has been recently identified(10Ho K. Nichols C.G. Lederer W.J. Lytton J. Vassilev P.M. Kanazirska M.V. Hebert S.C. Nature. 1993; 362: 31-38Crossref PubMed Scopus (831) Google Scholar, 11Kubo Y. Baldwin T.J. Jan Y.N. Jan L.Y. Nature. 1993; 362: 127-133Crossref PubMed Scopus (936) Google Scholar, 12Kubo Y. Reuveny E. Slesinger P.A. Jan Y.N. Jan L.Y. Nature. 1993; 364: 802-806Crossref PubMed Scopus (542) Google Scholar, 13Stoffel M. Espinosa R. Powell K.L. Philipson L.H. Lebeau M.M. Bell G.I. Genomics. 1994; 21: 254-256Crossref PubMed Scopus (28) Google Scholar). Unlike voltage-activated K+ channels of the Shaker gene family which are opened by membrane depolarization(14Salkoff L. Baker K. Butler A. Covarrubias M. Pak M. Wei A. Trends Neurosci. 1992; 15: 161-166Abstract Full Text PDF PubMed Scopus (254) Google Scholar), KIR channels are open at hyperpolarized potentials. These channels share an underlying conserved structure, with two predicted membrane spanning domains, homologous to the fifth (S5) and sixth (S6) transmembrane domains of voltage-gated channels, encompassing a region homologous to the pore-forming portion of voltage-activated channels(14Salkoff L. Baker K. Butler A. Covarrubias M. Pak M. Wei A. Trends Neurosci. 1992; 15: 161-166Abstract Full Text PDF PubMed Scopus (254) Google Scholar). KIR channels, however, lack a portion homologous to the amino-terminal region of voltage-gated channels (S1-S4).Islet β-cells contain K+ channels which have gating properties similar to members of the KIR family of channels, including ATP-sensitive channels (KATP) and G-protein-activated K+ channels that do not possess features of KATP channels(2Ashcroft F.M. Rorsman P. Prog. Biophys. Mol. Biol. 1991; 54: 87-143Crossref Scopus (945) Google Scholar, 4Rorsman P. Bokvist K. Ammala C. Arkhammar P. Berggren P.O. Larsson O. Wahlander K. Nature. 1991; 349: 77-79Crossref PubMed Scopus (109) Google Scholar). These channels may be involved in the regulation of insulin secretion by glucose and/or neurotransmitters acting through G-protein-coupled receptors(1Misler S. Barnett D.W. Gillis K.D. Pressel D.M. Diabetes. 1992; 41: 1221-1228Crossref PubMed Google Scholar, 4Rorsman P. Bokvist K. Ammala C. Arkhammar P. Berggren P.O. Larsson O. Wahlander K. Nature. 1991; 349: 77-79Crossref PubMed Scopus (109) Google Scholar, 15Ashford M.L.J. Bond C.T. Blair T.A. Adelman J.P. Nature. 1994; 370: 456-459Crossref PubMed Scopus (168) Google Scholar, 16Dunne M.J. Bullett M.J. Li G. Wollheim C.B. Petersen O.H. EMBO J. 1989; 8: 413-420Crossref PubMed Scopus (130) Google Scholar). Because islet KIR proteins are likely to share homology to other KIR molecules, we have employed a degenerate polymerase chain reaction strategy based on the conserved features of known KIR genes to identify and clone members of this family expressed in pancreatic islets. We demonstrate here the presence of three related KIR transcript sequences, CIR, GIRK1, and GIRK2, in human pancreatic islet cells. In contrast to previous work based on studies with cultured tumor β-cell lines which disclosed the presence of GIRK2, but not CIR(15Ashford M.L.J. Bond C.T. Blair T.A. Adelman J.P. Nature. 1994; 370: 456-459Crossref PubMed Scopus (168) Google Scholar, 17Tsaur M.L. Menzel S. Lai F.P. Espinosa III, R. Concannon P. Spielman R.S. Hanis C.L. Cox N.J. Lebeau M.M. German M.S. Jan L.Y. Bell G.I. Stoffel M. Diabetes. 1995; 44: 592-596Crossref PubMed Scopus (40) Google Scholar), CIR was found to be more abundant than GIRK2 in native purified pancreatic β-cells. Cloned islet cell CIR and GIRK2 cDNAs are shown to express heteromultimeric G-protein-activated KIR channels that do not possess characteristic features of KATP channels in the presence or absence of co-expressed sulfonylurea receptor (18Aguilar-Bryan L. Nichols C.G. Wechsler S.W. Clement J.P.I. Boyd A.E.I. Gonzalez G. Herrera-Sosa H. Nguy K. Bryan J. Nelson D. Science. 1995; 268: 423-426Crossref PubMed Scopus (1279) Google Scholar).EXPERIMENTAL PROCEDURESTissue Procurement and Isolation of RNAPurified human pancreatic islets were obtained from the Human Islet Transplantation Center, Washington University School of Medicine (Dr. David Scharp), by a previously described method(19Ricordi C. Lacy P.E. Finke E.H. Olack B.J. Scharp D.W. Diabetes. 1988; 37: 413-420Crossref PubMed Google Scholar). A surgical specimen from a human insulinoma was kindly provided by Dr. W. Dilley, Department of Surgery, Washington University School of Medicine, St. Louis, MO. Rat pancreatic islets were obtained by collagenase digestion and Ficoll gradient purification, and single-cell preparations enriched in pancreatic islet β and non-β cells were obtained by fluorescence activated cell sorting, exactly as described previously(20Gross R.W. Ramanadham S. Kruszka K.K. Han X. Turk J. Biochemistry. 1993; 32: 327-336Crossref PubMed Scopus (112) Google Scholar). Total RNA was extracted by homogenization in guanidinium thiocyanate and cesium chloride gradient ultracentrifugation(21Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The poly(A)+-enriched fraction was purified by two successive passages through oligo(dT)-cellulose columns, using standard procedures(21Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar).Reverse Transcription PCR1 with Degenerate PrimersFour-hundred ng of total RNA derived from human pancreatic islets and from a human β-cell tumor specimen were primed with random hexamers to reverse-transcribed cDNA. Degenerate primers that contained flanking restriction sites were designed based on the existence of sequence conservation among known KIR genes(6Inagaki N. Tsuura Y. Namba N. Masuda K. Gonoi T. Horie M. Seino Y. Mizuta M. Seino M. J. Biol. Chem. 1995; 270: 5691-5694Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar, 10Ho K. Nichols C.G. Lederer W.J. Lytton J. Vassilev P.M. Kanazirska M.V. Hebert S.C. Nature. 1993; 362: 31-38Crossref PubMed Scopus (831) Google Scholar, 11Kubo Y. Baldwin T.J. Jan Y.N. Jan L.Y. Nature. 1993; 362: 127-133Crossref PubMed Scopus (936) Google Scholar, 12Kubo Y. Reuveny E. Slesinger P.A. Jan Y.N. Jan L.Y. Nature. 1993; 364: 802-806Crossref PubMed Scopus (542) Google Scholar, 15Ashford M.L.J. Bond C.T. Blair T.A. Adelman J.P. Nature. 1994; 370: 456-459Crossref PubMed Scopus (168) Google Scholar). The forward primer was 5′-TGAGAATTCTAGTGTTTCTGCTC(T/G)(T/C)TT(T/C)TTNGG-3′, corresponding to a portion of the pore domain, and the reverse primer was 5′-TTCTCCTTCTAGACTCAAGTTACNAT(A/C/T)GGNTA(C/T)GG-3′. PCR amplification was carried out for 30 cycles at 94, 56, and 72°C for 1 min each step. First round PCR products were used as templates for a second round of PCR with flanking primers 5′-ATGAGAATTCTAGTGTTTCTGCTC-3′ and 5′-TTCTCCTTCTAGACTCAAGTTAC-3′. PCR products within the expected region of 186 bp were purified and cloned into the XbaI-EcoRI site of M13mp18. Twenty-five colonies were picked, and single-stranded DNA was sequenced on an ABI 373A automated sequencer. Sequences were compared with the nonredundant nucleic acid and protein data bases using BLASTN and BLASTX algorithms.Construction of Human Insulinoma and Rat Islet cDNA LibrariesThree μg of poly(A)+-enriched RNA from purified Wistar rat pancreatic islets was used to synthesize an oligo(dT)-primed, directionally cloned cDNA library in lZAP Express (Stratagene, La Jolla, Ca). This library contained 1.5 × 102 primary pfu and had an average insert size of 1.5 kb. One μg of poly(A)+-enriched RNA was employed to construct random primed nondirectionally cloned library in lZAP II (Stratagene, La Jolla, CA), which contained 0.7 × 102 primary pfu and an average insert size of 1.1 kb. Three μg of poly(A)+-enriched RNA from human insulinoma was used to synthesize an oligo(dT)-primed, directionally cloned cDNA library in lZAP II, with 1.55 × 102 primary pfu and an average insert size of 1.7 kb. The libraries were amplified and stored as 2% MeSO stocks at −80°C.Screening of Human and Rat Islet cDNA LibrariesPCR products from islet KIR sequences were 32P-labeled by random priming to a specific activity greater than 1 × 109 cpm/μg and used as a hybridization probe to screen 5 × 105 pfu from the human insulinoma library, 3 × 105 pfu from the oligo(dT)-primed rat islet library, and 2 × 105 pfu from the random primed rat islet library. Hybridization conditions were 50% formamide, 2 × PIPES, 2% SDS, 100 μg/ml sonicated and denatured salmon sperm DNA, at 42°C for 16-20 h. Filters were washed in 0.5 × SSC, 0.1% SDS, first at room temperature and then at 50°C, and exposed to x-ray film with an intensifying screen for approximately 48 h. Plaques that showed hybridization signals on replica filters were purified by secondary screening and the pBluescript or pBK CMV sequence was excised for sequencing.Reverse Transcription-PCR and Northern Blot Analysis of Islet KIRGenesFor RT-PCR analysis of tissue distribution, RNAs from multiple human tissues were treated with RNase-free DNase (Life Technologies, Inc.), extracted with phenol-chloroform, ethanol-precipitated, and quantified by spectrophotometry. The integrity and accuracy of quantitation of RNA was ascertained by formaldehyde-agarose gel electrophoresis and ethidium bromide staining. cDNA was synthesized with oligo(dT). For each tissue, PCR with specific primers was carried out from cDNA corresponding to 80, 20, 5, and 1.25 ng of total RNA, using conditions described above except for modifications of annealing temperature and cycle number. A limited number of cycles, 25-28, were used to avoid the plateau phase of amplification. Reactions with H20 instead of cDNA and RNA lacking reverse transcriptase were used as controls. For human islet CIR, primers were 5′-GAAATGAAGAGGGAAGGCCG-3′ and 5′-GGCTCATCTTCTTCATTCTG-3′ (annealing temperature, 62°C), and primers for human islet GIRK2 were 5′- CCAATTCATTTCATCTACCA-3′ and 5′-CATGCTGGGTTTTATTACTA-3′ (annealing temperature, 58°C). Primers to co-amplify rat CIR and GIRK2 were 5′-(A/T)AGAGACAGAAAG(C/A)ACCATT-3′ and 5′-(T/C)TTC(C/T)CATCCCGCATGGAGA-3′ (annealing temperature, 58°C). PCR products were resolved on an ethidium bromide-stained 2% agarose gel and visualized under ultraviolet light.The 3′ rapid amplification of human islet KATP-1/CIR cDNA was performed as follows: reverse transcription of RNA was primed with T12(A/G/C)N, where N is A, G, C, and T. PCR amplification was performed for 35 cycles at 94, 48, and 72°C for 1 min each step with primer 5′-GGGCATTATACTCCTCTTGG-3′, derived from the insulinoma CIR degenerate PCR fragment sequence, and T12(A/G/C)N. A unique 2.4-kb band was purified and partially sequenced directly.Northern blots were prepared with selected human poly(A)+ RNA-enriched samples, hybridized with 32P-labeled probes, and washed at a final stringency of 0.1 × SSC, 0.1% SDS, 65°C before exposure to x-ray film for 48 h.Chromosomal Localization of Islet KIRGenesThe chromosomal localization of islet KIR genes was determined by PCR amplification of DNA from a panel of rodent/human somatic cell hybrids, each of which contained one of the 24 different human chromosomes(22Matsutani A. Janssen R. Donis Keller H. Permutt M.A. Genomics. 1992; 12: 319-325Crossref PubMed Scopus (53) Google Scholar). Primers and PCR conditions were identical to those used in tissue distribution studies. Sublocalization within chromosome 11 was achieved by typing a panel of well characterized human chromosome 11-Chinese hamster ovary cell hybrids containing only defined portions of human chromosome 11. A description of the chromosomal breakpoints is described in detail in (23Gerhard D.S. Lawrence E. Wu J. Chua H. Ma N. Bland S. Jones C. Genomics. 1992; 13: 1133-1142Crossref PubMed Scopus (34) Google Scholar, 24Glaser T. Housman D. Lewis W.H. Gerhard D. Jones C. Somatic Cell Mol. Genet. 1989; 15: 477-501Crossref PubMed Scopus (128) Google Scholar, 25van den Elsen P. Bruns G. Gerhard D.S. Pravtcheva D. Jones C. Housman D. Ruddle F.A. Orkin S. Terhorst C. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 2920-2924Crossref PubMed Scopus (38) Google Scholar).Oocyte Expression of KIRChannelsCapped cRNAs were transcribed in vitro from linearized cDNAs using T3 RNA polymerase (Promega, Madison, WI). Stage V-VI Xenopus oocytes were isolated by partial ovariectomy under tricaine anesthesia and then defolliculated by treatment with 1 mg/ml collagenase (Sigma Type 1A, Sigma) in zero Ca2+ ND96 for 1 h. Two to 24 h after defolliculation, oocytes were pressure-injected with ~50 nl of 1-100 ng/μl cRNA. Oocytes were kept in ND96 + 1.8 mM Ca2+ (below), supplemented with penicillin (100 units/ml) and streptomycin (100 μg/ml) for 1-7 days prior to experimentation.ElectrophysiologyOocytes were voltage-clamped using a commercial voltage-clamp amplifier (Warner Instruments, Inc.) in a small chamber (volume 200 μl) mounted on the stage of a SMZ-1 microscope (Nikon Instruments). The standard extracellular solution (KD98) contained (in mM): KCl, 98; MgCl2, 1; HEPES, 5; pH 7.5. Additions to this solution are described in the text. Electrodes were filled with 3 M KCl and had tip resistances of 1-5 M. Experiments were performed at room temperature. PClamp software and a Labmaster TL125 D/A converter were used to generate voltage pulses. Data were normally filtered at 1 kHz, signals were digitized at 22 kHz (Neurocorder, Neurodata, New York) and stored on video tape. Experiments could then be replayed onto a chart recorder or digitized into a microcomputer using Axotape software (Axon Instruments). Alternatively, signals were digitized on-line using Pclamp and stored on disk for off-line analysis.RESULTS AND DISCUSSIONCloning of Human Islet KIRmRNAs Using Degenerate PCRRT-PCR amplification of human islet and insulinoma RNA was carried out with degenerate primers corresponding to two conserved regions of KIR genes. PCR products corresponding to the expected 186-bp size were subcloned, and a total of 25 subclones were sequenced. The sequences were compared with nucleic acid and protein data bases, and 13 were found to be related to previously described KIR sequences. These could be grouped into three different islet KIR-related sequences (Fig. 1). The first sequence, labeled hi-CIR.pcr in Fig. 1, was derived from human β-cell tumor RNA. It was identical to a KIR described by different groups as the cardiac inward rectifying channel (CIR) (26Krapivinsky G. Gordon E.A. Wickman K. Velimirovic B. Krapivinski L. Clapham D.E. Nature. 1995; 394: 135-141Crossref Scopus (751) Google Scholar) or cardiac ATP-sensitive potassium channel (rat cardiac KATP-1)(15Ashford M.L.J. Bond C.T. Blair T.A. Adelman J.P. Nature. 1994; 370: 456-459Crossref PubMed Scopus (168) Google Scholar). Another islet KIR cDNA (hi-GIRK1.pcr) was represented by two clones derived from human islet RNA which had 97% nucleic acid identity with the rat G-protein-activated inward rectifying potassium channel cDNA (GIRK1)(12Kubo Y. Reuveny E. Slesinger P.A. Jan Y.N. Jan L.Y. Nature. 1993; 364: 802-806Crossref PubMed Scopus (542) Google Scholar). The translated open reading frame was identical to GIRK1(12Kubo Y. Reuveny E. Slesinger P.A. Jan Y.N. Jan L.Y. Nature. 1993; 364: 802-806Crossref PubMed Scopus (542) Google Scholar, 26Krapivinsky G. Gordon E.A. Wickman K. Velimirovic B. Krapivinski L. Clapham D.E. Nature. 1995; 394: 135-141Crossref Scopus (751) Google Scholar). While initial reports showed that GIRK1 is expressed in rat heart, muscle, and brain(12Kubo Y. Reuveny E. Slesinger P.A. Jan Y.N. Jan L.Y. Nature. 1993; 364: 802-806Crossref PubMed Scopus (542) Google Scholar), we now provide evidence for the expression of this mRNA in human islets. This is consistent with a recent publication that described cloning of a GIRK1 cDNA from a rat insulinoma cell line and subsequent mapping of the gene to chromosome 2q24(13Stoffel M. Espinosa R. Powell K.L. Philipson L.H. Lebeau M.M. Bell G.I. Genomics. 1994; 21: 254-256Crossref PubMed Scopus (28) Google Scholar). Finally, a third islet KIR cDNA fragment, hi-GIRK2.pcr, was also generated from non-tumoral human islets and had high identity (87%) with a mouse G-protein-activated brain inward rectifier mRNA which has been designated GIRK2(27Lesage F. Duprat F. Fink M. Guillemare E. Coppola T. Lazdunski M. Hugnot J.P. FEBS Lett. 1994; 353: 37-42Crossref PubMed Scopus (268) Google Scholar). A total of three distinct, though closely related, KIR sequences were thus identified in human pancreatic islet and insulinoma RNA.Isolation and Characterization of hi-GIRK2The partial hi-GIRK2 sequence derived from degenerate PCR was radiolabeled and used as a hybridization probe to screen for full-length cDNAs in pancreatic islet-cell libraries. Three clones were isolated from a human β-cell tumor library, which contained a novel 2.1-kb cDNA insert (hi-GIRK2). hi-GIRK2 was predicted to encode a 423-amino acid polypeptide, with a calculated Mr of 48,455. Two of the GIRK2 clones, however, contained a single adenosine nucleotide insertion at amino acid position 401 (nucleotide position 1416) which caused an open reading frameshift in the COOH terminus. This is presumed to have resulted from a cloning artifact given that human genomic and reverse-transcribed islet cDNA lacks this single nucleotide insertion (not shown). In addition, hi-GIRK2 cDNA contained 212 bp of 5′-untranslated region, and a 481-bp 3′-untranslated portion. A canonical polyadenylation site was present 23 bp upstream of the terminal poly(A) stretch. Overall, the nucleic acid sequence was 92% identical to that of mouse brain GIRK2. The translated product of hi-GIRK2 displayed 95% amino acid identity with the mouse brain GIRK2(27Lesage F. Duprat F. Fink M. Guillemare E. Coppola T. Lazdunski M. Hugnot J.P. FEBS Lett. 1994; 353: 37-42Crossref PubMed Scopus (268) Google Scholar), with most divergent residues present in the COOH and NH2 termini (Fig. 2). mb-GIRK2 was recently cloned from mouse brain, co-expressed in Xenopus oocytes along with opioid receptors and shown to express a G-protein-activated K+ channel(27Lesage F. Duprat F. Fink M. Guillemare E. Coppola T. Lazdunski M. Hugnot J.P. FEBS Lett. 1994; 353: 37-42Crossref PubMed Scopus (268) Google Scholar). Prior to submission of this manuscript, a hamster insulinoma clone homologous to mouse GIRK2 was reported, and designated KATP-2 based on its similarity to CIR/KATP-1(17Tsaur M.L. Menzel S. Lai F.P. Espinosa III, R. Concannon P. Spielman R.S. Hanis C.L. Cox N.J. Lebeau M.M. German M.S. Jan L.Y. Bell G.I. Stoffel M. Diabetes. 1995; 44: 592-596Crossref PubMed Scopus (40) Google Scholar), and a partial identical human cDNA sequence referred to as BIR1 was deduced from genomic DNA (28Sakura H. Bond C. Warren-Perry M. Horsley S. Kearney L. Tucker S. Adelman J. Turner R. Ashcroft F.M. FEBS Lett. 1995; 367: 193-197Crossref PubMed Scopus (46) Google Scholar). Like other KIR sequences, hi-GIRK2 had a primary structure compatible with a model that includes two hydrophobic membrane spanning domains, M1 and M2, which encompass a putative pore region, and a long COOH-terminal tail believed to be predominantly cytoplasmic (Fig. 2)(10Ho K. Nichols C.G. Lederer W.J. Lytton J. Vassilev P.M. Kanazirska M.V. Hebert S.C. Nature. 1993; 362: 31-38Crossref PubMed Scopus (831) Google Scholar, 26Krapivinsky G. Gordon E.A. Wickman K. Velimirovic B. Krapivinski L. Clapham D.E. Nature. 1995; 394: 135-141Crossref Scopus (751) Google Scholar). There are six potential protein kinase C phosphorylation sites (positions 49, 73, 212, 236, 236, 366, and 386), all of which are conserved between human and mouse. There are no putative N-glycosylation sites in regions commonly regarded as extracellular in KIR proteins. Interestingly, at residue 256 there is an N-glycosylation motif near several short stretches of hydrophobic segments of unknown topology, which is conserved among both CIR and GIRK2 in humans and rodents (Fig. 2). No consensus ATP-binding site was encountered. Other than mouse brain GIRK2, hi-GIRK2 resembled CIR more than any other KIR channel gene in nonredundant nucleic acid and protein data bases, with approximately 69% overall amino acid identity (Fig. 2). The homology between hi-GIRK2 and CIR was most pronounced in the central 360-amino acid segment, while a lower degree of conservation was apparent in the COOH- and NH2-terminal portions. Amino acid identity of hi-GIRK2 with GIRK1 was 57%.Figure 2Alignment of the deduced amino acid sequence of human islet GIRK2 (hi-GIRK2), mouse brain GIRK2 (mb-GIRK2), human cardiac KATP-1/CIR (hc-KATP-1/CIR), and rat islet CIR (ri CIR). Sequence alignments were created with Megalign (DNASTAR) and visual modification. Amino acid identities are indicated by background shading. Predicted M1 and M2 transmembrane domains and the P pore region are highlighted by a line above the sequence lineup. Conserved consensus serine/threonine phosphorylation sites are boxed. Two possible N-glycosylation sites are marked, one presumed extracellular site present only in the CIR sequences (∗), the other of uncertain topology but conserved in the four proteins (∗∗).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The expression of hi-GIRK2 in human tissues was evaluated by Northern blot and RT-PCR analysis. A distinct band of approximately 5.7 kb, and a more diffuse signal of approximately 2.4-2.8 kb, were observed by Northern analysis in poly(A)+-enriched RNA from human insulinoma (Fig. 3), while the abundance in purified human islets was insufficient to detect a similar signal. To further define the tissue distribution of hi-GIRK2 mRNA, RT-PCR analysis of a serial dilution of cDNAs was performed under reduced cycling conditions that allowed relative semiquantitation among tissues (Fig. 3). Using primers specific for hi-GIRK2, a unique PCR product of the expected size was observed to be most abundant in insulinoma and cerebellum RNA, while lower levels of expression were detected in all other tissues examined, including human islet and pancreatic exocrine tissue (Fig. 3). The fact that RT-PCR product signals were only slightly enhanced in islets relative to exocrine samples could reflect significant cross-contamination of the exocrine and islet preparations and/or the existence of GIRK2 mRNA at lower levels in exocrine tissue.Figure 3Distribution of GIRK2 and CIR mRNA in adult human tissues. A, Northern blot analysis. Poly(A)+ RNA from human islets (HI) (2 and 1.5 μg), insulinoma surgical specimen (INS) (2 μg), and pancreatic exocrine (EXO) (1.5 μg) was blotted and hybridized with 32P-labeled hi-GIRK2 cDNA first, then stripped and rehybridized with a 32P-labeled human islet CIR 0.7-kb PCR product. B, reverse transcription-PCR analysis. Total RNA from human tissues was treated with RNase-free DNase, reverse-transcribed, and cDNA corresponding to 80, 20, 5, and 1.25 ng of RNA was amplified for 25 or 28 cycles using primers specific for human islet GIRK2 and CIR, respectively. INS, insulinoma; HI, pancreatic islets; EXO, exocrine; LIV, liver; CER, cerebellum; MUS, muscle; VEN, left ventricle; and DUO, duodenum.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Segregation analysis of a panel of human-Chinese hamster ovary/mouse somatic cell hybrids with specific oligonucleotides that amplified a 130-bp fragment from the 3′-untranslated region of the hi-GIRK2 gene allowed unequivocal localization of this gene to chromosome 21 (data not shown). This confirms data reported by others during revision of this manuscript, w
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