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• W2150550641 abstract "We report the isolation of a novel mouse voltage-gated Shaker-related K+ channel gene,Kv1.7 (Kcna7/KCNA7). Unlike other known Kv1 family genes that have intronless coding regions, the protein-coding region of Kv1.7 is interrupted by a 1.9-kilobase pair intron. The Kv1.7 gene and the related Kv3.3(Kcnc3/KCNC3) gene map to mouse chromosome 7 and human chromosome 19q13.3, a region that has been suggested to contain a diabetic susceptibility locus. The mouse Kv1.7 channel is voltage-dependent and rapidly inactivating, exhibits cumulative inactivation, and has a single channel conductance of 21 pS. It is potently blocked by noxiustoxin and stichodactylatoxin, and is insensitive to tetraethylammonium, kaliotoxin, and charybdotoxin. Northern blot analysis reveals ∼3-kilobase pair Kv1.7transcripts in mouse heart and skeletal muscle. In situhybridization demonstrates the presence of Kv1.7 in mouse pancreatic islet cells. Kv1.7 was also isolated from mouse brain and hamster insulinoma cells by polymerase chain reaction. We report the isolation of a novel mouse voltage-gated Shaker-related K+ channel gene,Kv1.7 (Kcna7/KCNA7). Unlike other known Kv1 family genes that have intronless coding regions, the protein-coding region of Kv1.7 is interrupted by a 1.9-kilobase pair intron. The Kv1.7 gene and the related Kv3.3(Kcnc3/KCNC3) gene map to mouse chromosome 7 and human chromosome 19q13.3, a region that has been suggested to contain a diabetic susceptibility locus. The mouse Kv1.7 channel is voltage-dependent and rapidly inactivating, exhibits cumulative inactivation, and has a single channel conductance of 21 pS. It is potently blocked by noxiustoxin and stichodactylatoxin, and is insensitive to tetraethylammonium, kaliotoxin, and charybdotoxin. Northern blot analysis reveals ∼3-kilobase pair Kv1.7transcripts in mouse heart and skeletal muscle. In situhybridization demonstrates the presence of Kv1.7 in mouse pancreatic islet cells. Kv1.7 was also isolated from mouse brain and hamster insulinoma cells by polymerase chain reaction. Ion channels that exhibit a variety of gating patterns and ion selectivity are critical elements that transduce signals in diverse cell types (1Hille B. Ionic Channels of Excitable Membranes. 2nd Ed. Sinauer, Sunderland, MA1993Google Scholar). Voltage-gated potassium-selective (Kv) 1The abbreviations use are: Kv, voltage-gated potassium selective; PCR, polymerase chain reaction; RBL, rat basophilic leukemic; bp, base pair(s); mb, millibase pair(s); kb, kilobase pair(s). channels represent the largest family within this class of proteins (2Chandy K.G. Gutman G.A. North A. Handbook of Receptors and Channels: Ligand and Voltage-gated Ion Channels. CRC Press, Boca Raton, FL1995: 1-72Google Scholar), and perform many vital functions in both electrically excitable and nonexcitable cells. Following initiation of an action potential in nerve and muscle cells, Kv channels play the important role of repolarizing the cell membrane (1Hille B. Ionic Channels of Excitable Membranes. 2nd Ed. Sinauer, Sunderland, MA1993Google Scholar). Kv channels can also modulate hormone secretion, for example insulin release from pancreatic islet cells (3Smith P.A. Bokvist K. Arkhammar P. Berggren P.O. Rorsman P. J. Gen. Physiol. 1990; 95: 1041-1059Crossref PubMed Scopus (68) Google Scholar, 4Smith P.A. Ashcroft F.M. Rorsman P. FEBS Lett. 1990; 261: 187-190Crossref PubMed Scopus (155) Google Scholar, 5Philipson L.H. Rosenberg M. Kuznetsov A. Lancaster M.E. Worley III, J.F. Roe M.W. Dukes I.D. J. Biol. Chem. 1994; 269: 27787-27790Abstract Full Text PDF PubMed Google Scholar, 6Roe M.W. Worley 3rd, J.F. Mittal A.A. Kuznetsov A. DasGupta S. Mertz R.J. Witherspoon 3rd, S.M. Blair N. Lancaster M.E. McIntyre M.S. Shehee W.R. Dukes I.D. Philipson L.H. J. Biol. Chem. 1996; 271: 32241-32246Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), and regulate calcium signaling during mitogen-stimulated activation of lymphocytes (7Lewis R.S. Cahalan M.D. Annu. Rev. Immunol. 1995; 13: 623-653Crossref PubMed Scopus (447) Google Scholar). Kv channels in mammalian cells are encoded by an extended family of at least nineteen genes (2Chandy K.G. Gutman G.A. North A. Handbook of Receptors and Channels: Ligand and Voltage-gated Ion Channels. CRC Press, Boca Raton, FL1995: 1-72Google Scholar). The largest subfamily, Kv1, is related to the fly Shaker gene and contains six members,Kv1.1–Kv1.6 (2Chandy K.G. Gutman G.A. North A. Handbook of Receptors and Channels: Ligand and Voltage-gated Ion Channels. CRC Press, Boca Raton, FL1995: 1-72Google Scholar). The Shaker gene has 21 exons, which can be alternatively spliced to generate at least five functionally distinct transcripts (8Pongs O. Kecskemethy N. Muller R. Krah-Jentgens I. Baumann A. Kiltz H.H. Canal I. Llamazares S. Ferrsu A. EMBO J. 1988; 7: 1087-1096Crossref PubMed Scopus (316) Google Scholar, 9Schwarz T.L. Papazian D.M. Caretto R.C. Jan Y.N. Jan L.Y. Nature. 1988; 331: 137-142Crossref PubMed Scopus (370) Google Scholar). In contrast, the protein-coding regions of each of the six known mammalianKv1 genes and the three known Xenopus homologues are contained in a single exon (2Chandy K.G. Gutman G.A. North A. Handbook of Receptors and Channels: Ligand and Voltage-gated Ion Channels. CRC Press, Boca Raton, FL1995: 1-72Google Scholar, 10Chandy K.G. Williams C.B. Spencer R.H. Aguilar B.A. Ghanshani S. Tempel B.L. Gutman G.A. Science. 1990; 247: 973-975Crossref PubMed Scopus (130) Google Scholar), precluding alternative splicing as a means of generating functionally different proteins. The evolutionary significance of this pattern of organization remains a puzzle. Here we report the identification of a novel mammalian gene,Kv1.7 (Kcna7/KCNA7), that has a genomic organization distinct from the other members of the vertebrateKv1 subfamily. We have defined the chromosomal location of this gene in the mouse and human genome, determined its tissue distribution, and characterized the biophysical and pharmacological properties of the cloned channel. Three overlapping genomic clones (KC225, KC254, and KC256) were isolated from an AKR/J mouse genomic library screened with a mixture of mKv1.1 and rKv1.5 cDNA probes, as described previously (10Chandy K.G. Williams C.B. Spencer R.H. Aguilar B.A. Ghanshani S. Tempel B.L. Gutman G.A. Science. 1990; 247: 973-975Crossref PubMed Scopus (130) Google Scholar), and mapped by multiple and partial restriction endonuclease digests, and by dideoxy sequencing.Kv1.7 cDNAs were amplified by the polymerase chain reaction (PCR) from mouse brain and from the hamster insulinoma cell line, HIT-T1S, using intron-flanking primers (5′-TCTCCGTACTCGTCATCCTGG-3′ within S1 and 5′-AAATGGGTGTCCACCCGGTC-3′ on the 3′ side of S5). The resulting 588-bp PCR fragments were sequenced. Cosmid clones encoding hKv1.7 and hKv3.3 (11Ghanshani S. Pak M. McPherson J.D. Strong M. Dethlefs B. Wasmuth J.J. Salkoff L. Gutman G.A. Chandy K.G. Genomics. 1992; 12: 190-196Crossref PubMed Scopus (42) Google Scholar) were isolated from a human chromosome 19-enriched library, Library F (12deJong P.J. Yokabata K. Chen C. Lohman F. Pederson L. McNinch J. van Dilla M. Cytogenet. Cell Genet. 1990; 51: 985Google Scholar), screened with mKv1.7 and mKv3.3 probes. A 1.9-kb cDNA fragment of the Shaw family gene,hKv3.4, was isolated from a human pancreatic library (13Permutt M.A. Koranyi L. Keller K. Lacy P.E. Scharp D.W. Mueckler M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8688-8692Crossref PubMed Scopus (72) Google Scholar) screened with a mixture of hKv3.1 (0.9-kbXbaI/HindIII), hKv3.3 (1.4-kbPstI/EcoRI), and mKv3.4 (0.9-kbHindII/EcoRI) probes at a final stringency of 0.5 × SSC and 0.1% SDS at 55 °C (8 × 106phage screened). The isolated clone spans the region from S1 through the 3′ end of the coding region (0.6 kb), and 1.3 kb of the 3′-noncoding region. Pancreatic tissue sections were obtained from 9–16-week-old diabetic-prone (db/db) and healthy (db/+) C57BL/KsJ mice. Mice homozygous for the autosomal recessive diabetic susceptibility gene db, a mutated form of the leptin receptor (14Chen H. Charlat O. Tartaglia L.A. Woolf E.A. Weng X. Ellis S.J. Lakey N.D. Culpepper J. Moore K.J. Breitbart R.E. Duyk G.M. Tepper R. Morgenstern J.P. Cell. 1996; 84: 491-495Abstract Full Text Full Text PDF PubMed Scopus (1937) Google Scholar, 15Lee G.H. Proenca R. Montez J.M. Carroll K.M. Darvishzadeh J.G. Lee J.I. Friedman J.M. Nature. 1996; 379: 632-635Crossref PubMed Scopus (2111) Google Scholar) on chromosome 4, develop diabetes beginning at about 6 weeks of age (16Shafrir E. Diabetes Metab. Rev. 1992; 8: 179-208Crossref PubMed Scopus (158) Google Scholar). Interspecific backcross progeny were generated by mating (C57BL/6J × Mus spretus)F1 females and (C57BL/6J) males, and a total of 205 N2 mice were used to map the two mouse genes, mKv1.7/Kcna7 and mKv3.3/Kcnc3, as described previously (11Ghanshani S. Pak M. McPherson J.D. Strong M. Dethlefs B. Wasmuth J.J. Salkoff L. Gutman G.A. Chandy K.G. Genomics. 1992; 12: 190-196Crossref PubMed Scopus (42) Google Scholar, 17Green E.L. Genetics and Probability in Animal Breeding Experiments. Oxford University Press, New York1981: 77-113Crossref Google Scholar, 18Jenkins N.A. Copeland N.G. Taylor B.A. Lee B.K. J. Virol. 1982; 43: 26-36Crossref PubMed Google Scholar, 19Saunders A.M. Seldin M.F. Genomics. 1990; 8: 525-535Crossref PubMed Scopus (98) Google Scholar, 20Copeland N.G. Jenkins N.A. Trends Genet. 1991; 7: 113-118Abstract Full Text PDF PubMed Scopus (477) Google Scholar). The probe for mKv1.7 was the entire 6.4-kb EcoRI fragment shown in Fig. 1, and that for mouse Kv3.3 was a 4-kb genomic HindIII fragment containing the entire 3′-exon (11Ghanshani S. Pak M. McPherson J.D. Strong M. Dethlefs B. Wasmuth J.J. Salkoff L. Gutman G.A. Chandy K.G. Genomics. 1992; 12: 190-196Crossref PubMed Scopus (42) Google Scholar). Although 155 mice were analyzed for all markers and are shown in this segregation analysis, up to 188 mice were typed for some pairs of markers. Recombination frequencies were calculated as described (11Ghanshani S. Pak M. McPherson J.D. Strong M. Dethlefs B. Wasmuth J.J. Salkoff L. Gutman G.A. Chandy K.G. Genomics. 1992; 12: 190-196Crossref PubMed Scopus (42) Google Scholar, 17Green E.L. Genetics and Probability in Animal Breeding Experiments. Oxford University Press, New York1981: 77-113Crossref Google Scholar, 18Jenkins N.A. Copeland N.G. Taylor B.A. Lee B.K. J. Virol. 1982; 43: 26-36Crossref PubMed Google Scholar, 19Saunders A.M. Seldin M.F. Genomics. 1990; 8: 525-535Crossref PubMed Scopus (98) Google Scholar, 20Copeland N.G. Jenkins N.A. Trends Genet. 1991; 7: 113-118Abstract Full Text PDF PubMed Scopus (477) Google Scholar) using the computer program SPRETUS MADNESS. Gene order was determined by minimizing the number of recombination events required to account for the allele distribution patterns. Fluorescentin situ hybridization of cosmids to metaphase human chromosomes was carried out as described previously (21Brandriff B.F. Gordon L.A. Tynan K.T. Olsen A.S. Mohrenweiser H.W. Fertitta A. Carrano A.V. Trask B.J. Genomics. 1992; 12: 773-779Crossref PubMed Scopus (39) Google Scholar, 22Trask B. Fertitta A. Christensen M. Youngblom J. Bergmann A. Copeland A. de Jong P. Mohrenweiser H. Olsen A. Carrano A. Tynan K. Genomics. 1993; 15: 133-145Crossref PubMed Scopus (93) Google Scholar). A Northern blot of poly(A)+ RNA from mouse heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis (CLONTECH Inc., Palo Alto, CA) was probed with the mouse Kv1.7-specific 3′-noncoding region sequence. The PstI/SacIKv1.7 3′-noncoding region was labeled by the random primer method (Boehringer Mannheim Random Primed DNA labeling kit). The RNA blot was hybridized at 60 °C for 18 h, washed at a final stringency of 0.2 × SSC and 0.1% SDS at 60 °C for 1 h, and exposed to x-ray film at −80 °C with an intensifying screen for 3 days. cRNA probes labeled with α-35S-labeled UTP (1300 Ci/mmol) were alkaline-denatured to an average size of 100 nucleotides and used for in situhybridization on pancreatic frozen sections (6–10 μm thick) from db/db mice. Briefly, sections were hybridized overnight at 42 °C, RNase treated, washed five times in 0.5 × SSC at 65 °C, coated with Ilford K5 photographic emulsion, and exposed at 4 °C for varying times. After development, the sections were counterstained with hematoxylin and eosin Y and examined with a Leitz Aristoplan microscope equipped with reflected polarized light to visualize silver grains in dark field. The probes used for hybridization were as follows: insulin, 1.6-kb human insulin gene including the 5′- and 3′-flanking sequences (ATCC no. 57399);hKv3.4, 1.9-kb cDNA fragment spanning S1 through the 3′ end of the coding region (0.6 kb), and 1.3 kb of the 3′-noncoding region; mKv1.7, 540-bp PstI/SacI fragment containing 29 bp of C-terminal coding sequence and 511 bp of 3′-noncoding sequence. To make a mKv1.7expression construct we amplified a 588-bp fragment from mouse brain cDNA spanning the region encoded by the two Kv1.7 exons using reverse transcriptase PCR (5′-primer, 5′-TCTCCGTACTCGTCATCCTGG-3′; 3′-primer, 5′-AAATGGGTGTCCACCCGGTC-3′). Exon 1 (850-bpBspHI/BinI fragment), a 283-bpBinI/BglII fragment of our 588-bp PCR product, and exon 2 (747-bp BglII/HindIII), were ligated together at BinI and BglII sites to generate “fragment I” (1880 bp). Fragment I was blunt-ended at the 5′ end and cloned into the pBluescript vector atSmaI/HindIII sites. To remove the 5′-NCR from fragment I, and for the purpose of cloning this fragment into the pBSTA expression vector, we introduced a unique BamHI site just upstream of the initiator methionine using PCR: 5′-primer, 5′-ACAAAAGCTTCATATGACTACAAGGAAAGCT-3′; and 3′-primer: 5′-AAGCGCAACCCGGCCACG-3′. The resulting PCR product (corresponding to the first 233 nucleotides of the coding region) was spliced to fragment I at the NcoI site, and the 1870-nucleotide fragment was ligated to the pBSTA vector. mKv1.7 cRNA was transcribed in vitro (Ambion Kit, Austin, TX) and diluted in a 0.1–0.5% fluorescein-dextran (Mr 10,000, Molecular Probes, Eugene, OR) in 100 mm KCl. Rat basophilic leukemic (RBL) cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Hyclone, Logan, UT) and glutamine, and were plated onto glass coverslips one day prior to use for electrophysiological experiments. RBL cells were injected with cRNA using pre-pulled injection capillaries (Femtotip) in combination with an Eppendorf microinjection system (micromanipulator 5171 and transjector 5242; Madison, WI) as described previously (23Ikeda S.R. Soler F. Zuhlke R.D. Lewis D.L. Pflueg. Arch. Eur. J. Physiol. 1992; 422: 201-203Crossref PubMed Scopus (30) Google Scholar). Four to six hours later, fluorescent cRNA-injected cells were evaluated electrophysiologically. All membrane currents were recorded at room temperature (22–26 °C) with a LIST EPC-7 amplifier (Heka Elektronik, Germany). Series resistance compensation was employed if the current exceeded 2 nA, and the command input was controlled by a PDP 11/73 computer via a digital-to-analog converter. Capacitative and leak currents were subtracted using a P/8 procedure and the holding potential in all experiments was −80 mV. When membrane currents exceeded 2 nA 80% series resistance compensation was used. A restriction map of a 6.4-kb EcoRI DNA fragment containing the entire mouse Kv1.7 coding region is shown in Fig. 1. The coding region is contained in two exons separated by a 1.9-kb intron. The upstream exon encodes the entire N terminus, S1, and part of the S1-S2 loop. The downstream exon contains the region extending from the S1-S2 loop to the C-terminal end of the protein. The intron-exon splice sites were determined by comparing the genomic sequence with cDNA sequences obtained from the hamster insulinoma cell line, HIT-T1S, and from mouse brain (Fig. 1). The complete coding sequence of the mKv1.7 is shown in Fig. 2. The mKv1.7 sequence is identical in the N terminus from bp 52 to 996 with the mouse EST sequence AA021711. Betsholtz et al. (24Betsholtz C. Baumann A. Kenna S. Ashcroft F.M. Ashcroft S.J. Berggren P.O. Grupe A. Pongs O. Rorsman P. Sandblom J. Welsh M. FEBS Lett. 1990; 263: 121-126Crossref PubMed Scopus (31) Google Scholar) amplified a short segment of Kv1.7 cDNA spanning the S5/S6 region from mouse (MK-6), rat (RK-6), and hamster (HaK-6) insulin-producing cells using PCR. Our sequence is identical to their MK-6 sequence, except for four nucleotide changes. The deduced mKv1.7 protein consists of 532 amino acids and contains six putative membrane-spanning domains, S1–S6 (Fig. 2). The hydrophobic core of this protein shares considerable sequence similarity with otherShaker family channels, while the intracellular N and C termini and the external loops between S1/S2 and S3/S4 show little conservation. The protein contains conserved sites for post-translational modifications as indicated in Fig. 2. As do all other Shaker-related channels, mKv1.7 has a potential tyrosine kinase phosphorylation site (RPSFDAVLY) in its N-terminal region (2Chandy K.G. Gutman G.A. North A. Handbook of Receptors and Channels: Ligand and Voltage-gated Ion Channels. CRC Press, Boca Raton, FL1995: 1-72Google Scholar); the proline-rich stretch within the N terminus (see residues 29–42) may be a binding site for SH3 domains of tyrosine kinases. Two protein kinase C consensus sites (Ser/Thr-X-Arg/Lys) are present in the cytoplasmic loop between S4 and S5 of mKv1.7; at least one of these sites is present in all members of the Kv1 family (2Chandy K.G. Gutman G.A. North A. Handbook of Receptors and Channels: Ligand and Voltage-gated Ion Channels. CRC Press, Boca Raton, FL1995: 1-72Google Scholar). mKv1.7, like Kv1.6, lacks anN-glycosylation site in the extracellular loop linking the S1 and S2 transmembrane segments; this consensus sequence is conserved in all other Kv1 family genes. Fig. 3 shows a phylogenetic tree of the entire Shaker family of genes based on parsimony analysis of a nucleotide sequence alignment (generated from the amino acid sequence alignment) using the program PAUP (25Swofford D.L. PAUP: Phylogenetic analysis using parsimony. Computer program distributed by the Illinois Natural History Survey, Champaign, IL1993Google Scholar). Our analysis placesmKv1.7 within the Shaker family of genes. The mKv1.7 gene does not cluster with any of the known genes, and its position within the tree is not firmly established. The mKv1.7/Kcna7 gene resides on mouse chromosome 7 (Fig. 4 A), ∼0.5 centimorgan telomeric to the Shaw-related K+ channel gene,mKv3.3/Kcnc3, and ∼2.4 centimorgans centromeric of MyoD1 (myoblast differentiation factor). The most centromeric marker in this study was Gpi1 (glucose phosphate isomerase 1), which mapped ∼6.1 centimorgans centromeric tomKv3.3/Kcnc3. The interval on mouse chromosome 7 containing mKv1.7/Kcna7 and mKv3.3/Kcnc3 shares a region of homology with human chromosomes 19q13 and 11p15, and the human homologues of these K+ channel genes may therefore be expected to reside on one of these chromosomes. Confirming this prediction, we mapped both genes to human 19q13.3–13.4 using fluorescent in situhybridization. The idiogram of human chromosome 19 shown in Fig. 4 B, and a more detailed map shown in Fig. 4 C, indicate that hKv1.7/KCNA7 is located ∼1.3 mb centromeric of hKv3.3/KCNC3. The genes for both muscle glycogen synthase (GYS1) and the histidine-rich calcium protein (HRC) also map to this region; Kv1.7/KCNA7 lies ∼25 kb telomeric to GYS1 and ∼200 kb centromeric to HRC (Fig. 4 C). Interestingly, a putative diabetes susceptibility gene has been suggested to be present at 19q13.3 (26Groop L.C. Kankuri M. Schalin-Jantti C. Ekstrand A. Nikula-Ijas P. Widen E. Kuismanen E. Eriksson J. Franssila-Kallunki A. Saloranta C. Koskimies S. N. Engl. J. Med. 1993; 328: 10-14Crossref PubMed Scopus (184) Google Scholar, 27Elbein S.C. Hoffman M. Ridinger D. Otterud B. Leppert M. Diabetes. 1994; 43: 1061-1065Crossref PubMed Scopus (24) Google Scholar), especially in Finnish families with associated hypertension and difficulties in insulin-stimulated glucose storage. This region has also been suggested to contain a modifier locus for cystic fibrosis (28Estivill X. Nat. Genet. 1996; 12: 348-350Crossref PubMed Scopus (85) Google Scholar). We carried out a detailed characterization of mKv1.7 channels expressed in RBL cells which express no native Kv currents (29McCloskey M. Cahalan M.D. J. Gen. Physiol. 1990; 95: 208-222Crossref Scopus (67) Google Scholar, 30Nguyen QA Kath J. Hanson D.C. Biggers M.S. Canniff P.C. Donovan C.B. Mather R.J. Bruns M.J. Rauer H. Aiyar J. Lepple-Wienhues A. Gutman G.A. Grissmer S. Cahalan M.D. Chandy K.G. Mol. Pharmacol. 1996; 50: 1672-1679PubMed Google Scholar). The mKv1.7 gene expressed in Xenopus oocytes produced currents (data not shown) similar to those obtained in RBL cells (Fig. 5). Patch clamp studies were performed in the whole-cell mode. Fig. 5 A shows a family of outward currents elicited by a 200 ms depolarizing pulse from a holding potential of −80 mV in RBL cells injected with mKv1.7 cRNA; no outward currents were detected in control cells (data not shown). The currents activate rapidly, and from the conductance-voltage curve shown in Fig. 5 B we determined that the 1/2 activation potential (V1/2) is −20 mV. Inactivation of mKv1.7 channels was rapid following a 200 ms depolarizing pulse from −80 to 40 mV (Fig. 5 A). The rate of inactivation, measured by fitting the data to a single exponential function, yielded a time constant (τh) of 14 ± 2.1 ms (S.E., n = 7). As shown in Fig. 5 C, the current became progressively smaller following repeated depolarizing pulses at 1-s intervals. This phenomenon, termed “cumulative inactivation,” is due to the accumulation of channels in the inactivated state which are then unavailable for opening. The related channels, Kv1.3 (7Lewis R.S. Cahalan M.D. Annu. Rev. Immunol. 1995; 13: 623-653Crossref PubMed Scopus (447) Google Scholar) and Kv1.4 (31Wymore R. Korenberg J.R. Coyne C. Chen X-N Hustad C. Copeland N.G. Gutman G.A. Jenkins N.A. Chandy K.G. Genomics. 1994; 20: 191-202Crossref PubMed Scopus (32) Google Scholar), also display this behavior. The kinetics of channel closing (deactivation) was determined by first opening the channels with a 15 ms conditioning depolarizing pulse, and then forcing the channels to close by repolarizing to different potentials (Fig. 5 D). The time constant, τtail, of the resulting “tail” current was 5.1 and 5.3 ms at −60 mV in two cells. We measured single-channel currents in an outside-out patch by applying 450-ms voltage ramps from −90 to 80 mV every second (Fig. 5 E). Single channel events were seen at potentials more positive than ∼−15 mV. The single-channel conductance of 21 pS was estimated from the slope of the current recorded during an opening (Fig. 5 E). We determined the pharmacological sensitivity of the mKv1.7 channel using methods described previously (30Nguyen QA Kath J. Hanson D.C. Biggers M.S. Canniff P.C. Donovan C.B. Mather R.J. Bruns M.J. Rauer H. Aiyar J. Lepple-Wienhues A. Gutman G.A. Grissmer S. Cahalan M.D. Chandy K.G. Mol. Pharmacol. 1996; 50: 1672-1679PubMed Google Scholar, 32Grissmer S. Nguyen A.N. Aiyar J. Hanson D.C. Mather R.J. Gutman G.A. Karmilowicz M.J. Auperin D.D. Chandy K.G. Mol. Pharmacol. 1994; 45: 1227-1234PubMed Google Scholar), IC50 values in each case being determined when block reached steady-state. The channel was blocked by several non-peptide small molecule antagonists, 4-aminopyridine (IC50 = 245 μm), capsaicin (25 μm), cromakalim (450 μm), tedisamil (18 μm), nifedipine (13 μm), diltiazem (65 μm), and resiniferatoxin (20 μm). Surprisingly, the dihydroquinoline compound, CP-339,818, that blocks rapidly inactivating Kv1 channels in the nanomolar range (30Nguyen QA Kath J. Hanson D.C. Biggers M.S. Canniff P.C. Donovan C.B. Mather R.J. Bruns M.J. Rauer H. Aiyar J. Lepple-Wienhues A. Gutman G.A. Grissmer S. Cahalan M.D. Chandy K.G. Mol. Pharmacol. 1996; 50: 1672-1679PubMed Google Scholar), had little effect on mKv1.7 (IC50 = 10 μm). The channel was insensitive to externally applied tetraethylammonium (C50 = 86 mm), probably because the residue at the tetraethylammonium-binding site, Ala-441 (Fig. 2), is hydrophobic. The mKv1.7 channel is also potently blocked by a peptide (ShK toxin) obtained from sea anemone Stichodactyla helianthus(IC50 = 13 nm), and by the scorpion toxins, noxiustoxin (IC50 = 18 nm) and margatoxin (IC50 = 116 nm). The channel was resistant to charybdotoxin (IC50 >1000 nm) and kaliotoxin (IC50 >1000 nm). Northern blot assays using a mKv1.7-specific probe revealed strongly hybridizing 3-kb bands in heart and skeletal muscle; faint bands of similar size were visible in liver and lung (together with larger 7–8-kb bands), but none were seen in spleen, kidney, testis, or brain (Fig. 6) We were able to isolatemKv1.7 transcripts from mouse brain by PCR (see Fig. 1). mKv1.7 is also expressed in placenta, since the mouse EST AA021711 was derived from this tissue. PCR analysis demonstrated the presence of haKv1.7 mRNAs in hamster insulinoma cells (Fig. 1). We verified the presence of mKv1.7 mRNA in pancreatic islet cells obtained from 9–16-week-old diabetic db/db mice by in situhybridization (Fig. 7 C) using a mKv1.7-specific antisense probe (12deJong P.J. Yokabata K. Chen C. Lohman F. Pederson L. McNinch J. van Dilla M. Cytogenet. Cell Genet. 1990; 51: 985Google Scholar, 13Permutt M.A. Koranyi L. Keller K. Lacy P.E. Scharp D.W. Mueckler M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8688-8692Crossref PubMed Scopus (72) Google Scholar, 14Chen H. Charlat O. Tartaglia L.A. Woolf E.A. Weng X. Ellis S.J. Lakey N.D. Culpepper J. Moore K.J. Breitbart R.E. Duyk G.M. Tepper R. Morgenstern J.P. Cell. 1996; 84: 491-495Abstract Full Text Full Text PDF PubMed Scopus (1937) Google Scholar); mKv1.7 mRNA was also present in islets from normal db/+ mice (data not shown). Scattered acinar cells outside the islets also showed mKv1.7 hybridization (Fig. 7 C). In contrast, mKv3.4mRNA was found in acinar cells surrounding islets, but not in islets, of both db/db (Fig. 7B) and db/+ mice (data not shown). As a control, insulin mRNA was detected in both normal and diabetic islets, but not in acinar cells (Fig. 7 A). A Kv1.5-specific probe did not show appreciable hybridization to islets (data not shown), despite a report of Kv1.5 cDNA having been cloned from human insulinoma cells (33Philipson L.H. Hice R.E. Schaefer K. LaMendola J. Bell G.I. Nelson D.J. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 53-57Crossref PubMed Scopus (71) Google Scholar). Unlike all other known mammalian Shaker-related genes (Kv1.1–Kv1.6) that have intronless coding regions (2Chandy K.G. Gutman G.A. North A. Handbook of Receptors and Channels: Ligand and Voltage-gated Ion Channels. CRC Press, Boca Raton, FL1995: 1-72Google Scholar, 9Schwarz T.L. Papazian D.M. Caretto R.C. Jan Y.N. Jan L.Y. Nature. 1988; 331: 137-142Crossref PubMed Scopus (370) Google Scholar), the protein-coding region of mKv1.7 is interrupted by a single 1.9-kb intron. The fly Shaker gene also contains an intron in the S1-S2 loop, raising the possibility that the intron in Kv1.7 may be ancient, predating the divergence of flies and mammals. Both the mouse Kv.1.7 and the fly Shaker intron are placed between codons, i.e. they are “phase 0” introns. While this is consistent with their having a common origin it may also be fortuitous, since there are only three possible “phases.” Although we favor the idea that Kv introns were lost in the vertebrate lineage before their expansion by gene duplication (in which case the Kv1.7 intron would represent a more recent insertion), the evolutionary history of this complex gene family remains to be elucidated. Since Kv1.7 mRNA is expressed in the mouse heart, we searched the literature for native cardiac A-type Kv currents with properties resembling those of Kv1.7. The Kv1.7 homotetramer shares many properties with the rapidly inactivating transient outward (Ito) current in cardiac Purkinje fibers, but not the Ito current in atrial and ventricular myocytes. Kv1.7 and the Purkinje Ito currents activate at negative potentials (∼−30 to −20 mV), inactivate rapidly (τh < 25 ms), exhibit cumulative inactivation, are blocked by micromolar concentrations of 4-aminopyridine, and are resistant to >20 mm tetraethylammonium (34Reder R.F. Miura D.S. Danilo Jr., P. Rosen M.R. Circ. Res. 1981; 48: 658-668Crossref PubMed Scopus (30) Google Scholar, 35Gintant G.A. Cohen I.S. Datyner N.B. Kline R.P. Fozzard H. The Heart and Cardiovascular System. 2nd Ed. Raven Press, New York1992: 1122-1166Google Scholar, 36Dixon J.E. Shi W. Wang H.S. McDonald C. Yu H. Wymore R.S. Cohen I.S. McKinnon D. Circ. Res. 1996; 79: 659-668Crossref PubMed Scopus (395) Google Scholar) (this study). In contrast, the Ito current in atrial and ventricular muscle, a product of the Kv4.3 gene, does not exhibit cumulative inactivation (36Dixon J.E. Shi W. Wang H.S. McDonald C. Yu H. Wymore R.S. Cohen I.S. McKinnon D. Circ. Res. 1996; 79: 659-668Crossref PubMed Scopus (395) Google Scholar). These studies suggest that at least part of the Purkinje fiber Ito might be encoded by the Kv1.7 gene, although more extensive biophysical and pharmacological studies are required to confirm the link, and the presence of Kv1.7 mRNA and/or protein has yet to be demonstrated in these fibers. The abundant expression of Kv1.7 mRNA in mouse heart suggests that this channel is also likely to be present in ventricular and/or atrial muscle where it may co-assemble with other Kv1 family channels to form heterotetramers. Recent studies suggest an important role for Kv channels in regulating islet cell function, specifically in repolarizing the membrane potential following each action potential during the glucose-induced bursting phase associated with insulin secretion (3Smith P.A. Bokvist K. Arkhammar P. Berggren P.O. Rorsman P. J. Gen. Physiol. 1990; 95: 1041-1059Crossref PubMed Scopus (68) Google Scholar, 4Smith P.A. Ashcroft F.M. Rorsman P. FEBS Lett. 1990; 261: 187-190Crossref PubMed Scopus (155) Google Scholar, 5Philipson L.H. Rosenberg M. Kuznetsov A. Lancaster M.E. Worley III, J.F. Roe M.W. Dukes I.D. J. Biol. Chem. 1994; 269: 27787-27790Abstract Full Text PDF PubMed Google Scholar, 6Roe M.W. Worley 3rd, J.F. Mittal A.A. Kuznetsov A. DasGupta S. Mertz R.J. Witherspoon 3rd, S.M. Blair N. Lancaster M.E. McIntyre M.S. Shehee W.R. Dukes I.D. Philipson L.H. J. Biol. Chem. 1996; 271: 32241-32246Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Despite these interesting findings, the genes encoding Kv genes in β-cells have not been identified. Although the Kv1.5 gene was isolated from human insulinoma cells (33Philipson L.H. Hice R.E. Schaefer K. LaMendola J. Bell G.I. Nelson D.J. Steiner D.F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 53-57Crossref PubMed Scopus (71) Google Scholar), we did not detectKv1.5 mRNA in normal or diseased islets. We have, however, demonstrated the presence of Kv1.7 mRNA in these cells. Unlike the noninactivating Kv channels in pancreatic β-cells (3Smith P.A. Bokvist K. Arkhammar P. Berggren P.O. Rorsman P. J. Gen. Physiol. 1990; 95: 1041-1059Crossref PubMed Scopus (68) Google Scholar, 4Smith P.A. Ashcroft F.M. Rorsman P. FEBS Lett. 1990; 261: 187-190Crossref PubMed Scopus (155) Google Scholar), the Kv1.7 homotetramer exhibits rapid C-type inactivation. Since Kv1.7 mRNA is expressed in pancreatic islets, it is possible that heteromultimers composed of Kv1.7 and other Kv1 subunits constitute the native Kv channels in β-cells. Enhanced levels of such Kv channels would excessively hyperpolarize the membrane of β-cells and impair insulin secretion (5Philipson L.H. Rosenberg M. Kuznetsov A. Lancaster M.E. Worley III, J.F. Roe M.W. Dukes I.D. J. Biol. Chem. 1994; 269: 27787-27790Abstract Full Text PDF PubMed Google Scholar). The mapping of the Kv1.7 gene to human chromosome 19q13.3, a region thought to contain a diabetic susceptibility gene (26Groop L.C. Kankuri M. Schalin-Jantti C. Ekstrand A. Nikula-Ijas P. Widen E. Kuismanen E. Eriksson J. Franssila-Kallunki A. Saloranta C. Koskimies S. N. Engl. J. Med. 1993; 328: 10-14Crossref PubMed Scopus (184) Google Scholar, 27Elbein S.C. Hoffman M. Ridinger D. Otterud B. Leppert M. Diabetes. 1994; 43: 1061-1065Crossref PubMed Scopus (24) Google Scholar), also suggests that Kv1.7 might contribute to the pathogenesis of type II diabetes mellitus in some humans. In conclusion, we have described a novel Kv1 family gene with a genomic organization distinct from all the other members of the family. The Kv1.7 channel produces a typical A-type current, and transcripts are expressed in the heart, skeletal muscle, brain, placenta, and pancreatic β-cells. This channel is biophysically and pharmacologically similar to the Purkinje fiber Itocurrent, and the gene may contribute at least one subunit to heteromultimeric Kv channels in pancreatic β-cells. The assistance of F. Glaser, S. Plummer, B. Dethlefs, S. Hoffman, M. Christensen, T. Wymore, C. Chandy, and D. J. Gilbert is gratefully acknowledged. We are obliged to Dr. J. Aiyar for reading and improving our manuscript." @default.
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• W2150550641 title "Genomic Organization, Chromosomal Localization, Tissue Distribution, and Biophysical Characterization of a Novel MammalianShaker-related Voltage-gated Potassium Channel, Kv1.7" @default.
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