Cloning and Functional Expression of Two Families of β-Subunits of the Large Conductance Calcium-activated K+ Channel
2000; Elsevier BV; Volume: 275; Issue: 30 Linguagem: Inglês
10.1074/jbc.m910187199
ISSN1083-351X
AutoresVictor N. Uebele, Armando Lagrutta, Theresa Wade, David J. Figueroa, Yuan Liu, Edward J. McKenna, Christopher P. Austin, Paul B. Bennett, Richard Swanson,
Tópico(s)Nicotinic Acetylcholine Receptors Study
ResumoWe report here a characterization of two families of calcium-activated K+ channel β-subunits, β2 and β3, which are encoded by distinct genes that map to 3q26.2–27. A single β2 family member and four alternatively spliced variants of β3 were investigated. These subunits have predicted molecular masses of 27.1–31.6 kDa, share ∼30–44% amino acid identity with β1, and exhibit distinct but overlapping expression patterns. Coexpression of the β2 or β3a–c subunits with a BK α-subunit altered the functional properties of the current expressed by the α-subunit alone. The β2 subunit rapidly and completely inactivated the current and shifted the voltage dependence for activation to more polarized membrane potentials. In contrast, coexpression of the β3a–c subunits resulted in only partial inactivation of the current, and the β3b subunit conferred an apparent inward rectification. Furthermore, unlike the β1 and β2 subunits, none of the β3 subunits increased channel sensitivity to calcium or voltage. The tissue-specific expression of these β-subunits may allow for the assembly of a large number of distinct BK channels in vivo, contributing to the functional diversity of native BK currents. We report here a characterization of two families of calcium-activated K+ channel β-subunits, β2 and β3, which are encoded by distinct genes that map to 3q26.2–27. A single β2 family member and four alternatively spliced variants of β3 were investigated. These subunits have predicted molecular masses of 27.1–31.6 kDa, share ∼30–44% amino acid identity with β1, and exhibit distinct but overlapping expression patterns. Coexpression of the β2 or β3a–c subunits with a BK α-subunit altered the functional properties of the current expressed by the α-subunit alone. The β2 subunit rapidly and completely inactivated the current and shifted the voltage dependence for activation to more polarized membrane potentials. In contrast, coexpression of the β3a–c subunits resulted in only partial inactivation of the current, and the β3b subunit conferred an apparent inward rectification. Furthermore, unlike the β1 and β2 subunits, none of the β3 subunits increased channel sensitivity to calcium or voltage. The tissue-specific expression of these β-subunits may allow for the assembly of a large number of distinct BK channels in vivo, contributing to the functional diversity of native BK currents. calcium-activated K+ channel untranslated region reverse transcription polymerase chain reaction rapid amplification of cDNA ends expressed sequence tag base pair(s) fluorescence in situ hybridization group of overlapping clones bacterial artificial chromosome log of the ratio of odds large conductance calcium-activated potassium channel. Calcium-activated K+ channels (KCa)1 modulate cellular electrical excitability. These channels are gated by both cytoplasmic calcium and membrane potential and, therefore, provide feedback mechanisms to modulate Ca2+ influx. Activation of KCa channels hyperpolarizes cells, and the way in which this hyperpolarization regulates Ca2+ entry is dependent upon the nature of the influx pathway. For example, entry through voltage-gated calcium channels (e.g. in myocytes) may be decreased, due to voltage-dependent deactivation of the calcium channels (1Heppner T.J. Bonev A.D. Nelson M.T. Am. J. Physiol. 1997; 273: C110-C117Crossref PubMed Google Scholar, 2Marrion N.V. Tavalin S.J. Nature. 1998; 395: 900-905Crossref PubMed Scopus (464) Google Scholar, 3Vergara C. Latorre R. Marrion N.V. Adelman J.P. Curr. Opin. Neurobiol. 1998; 8: 321-329Crossref PubMed Scopus (638) Google Scholar). However, influx through voltage-independent channels (e.g. in endothelial cells) may be enhanced due to an increase in the driving force for Ca2+ (4Sharma N.R. Davis M.J. Am. J. Physiol. 1994; 266: H156-H164PubMed Google Scholar, 5Sullivan R. Koliwad S.K. Kunze D.L. Am. J. Physiol. 1998; 275: C1342-C1348Crossref PubMed Google Scholar). Thus, the regulatory roles of KCachannels are context-dependent and may vary with cell type. KCa currents have been recorded from a variety of tissues and have traditionally been classified into broad categories based on single channel conductance (e.g. large, intermediate, or small conductance). However, in addition to these differences in unitary current amplitude, distinct KCa currents may also vary in terms of their calcium- and voltage dependence, kinetics, or pharmacologic properties (3Vergara C. Latorre R. Marrion N.V. Adelman J.P. Curr. Opin. Neurobiol. 1998; 8: 321-329Crossref PubMed Scopus (638) Google Scholar, 6Lingle C.J. Solaro C.R. 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Neuron. 1994; 13: 1315-1330Abstract Full Text PDF PubMed Scopus (384) Google Scholar). Two distinct genes encoding BK β-subunits have been identified: KCNMB1, which encodes the β1 subunit originally isolated from airway smooth muscle (11Knaus H.G. Folander K. Garcia-Calvo M. Garcia M.L. Kaczorowski G.J. Smith M. Swanson R. J. Biol. Chem. 1994; 269: 17274-17278Abstract Full Text PDF PubMed Google Scholar) has been localized to human chromosome 5q34 (14Tseng-Crank J. Godinot N. Johansen T.E. Ahring P.K. Strobaek D. Mertz R. Foster C.D. Olesen S.P. Reinhart P.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9200-9205Crossref PubMed Scopus (142) Google Scholar), and a recently isolated homologue encoding the β2 subunit (15Wallner M. Meera P. Toro L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4137-4142Crossref PubMed Scopus (325) Google Scholar, 16Xia X.M. Ding J.P. Lingle C.J. J. Neurosci. 1999; 19: 5255-5264Crossref PubMed Google Scholar). Although functional BK channels can be expressed from α-subunits alone, coassembly with β-subunits can alter the biophysical and pharmacologic properties of the channel (15Wallner M. Meera P. Toro L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4137-4142Crossref PubMed Scopus (325) Google Scholar, 16Xia X.M. Ding J.P. Lingle C.J. J. Neurosci. 1999; 19: 5255-5264Crossref PubMed Google Scholar, 17Hanner M. Schmalhofer W.A. Munujos P. Knaus H.G. Kaczorowski G.J. Garcia M.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2853-2858Crossref PubMed Scopus (82) Google Scholar, 18McManus O.B. Helms L.M. Pallanck L. Ganetzky B. Swanson R. Leonard R.J. Neuron. 1995; 14: 645-650Abstract Full Text PDF PubMed Scopus (418) Google Scholar, 19Valverde M.A. Rojas P. Amigo J. Cosmelli D. Orio P. Bahamonde M.I. Mann G.E. Vergara C. Latorre R. Science. 1999; 285: 1929-1931Crossref PubMed Scopus (458) Google Scholar). However, the properties of some native BK currents are not well reproduced by combinations of currently known α- and β-subunits, suggesting the possibility that novel subunits of these channels may still exist. We identified two families of BK β-subunits. The first, which, to date, contains only a single member (β2), is identical to that recently identified in a lung carcinoid EST library (15Wallner M. Meera P. Toro L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4137-4142Crossref PubMed Scopus (325) Google Scholar, 16Xia X.M. Ding J.P. Lingle C.J. J. Neurosci. 1999; 19: 5255-5264Crossref PubMed Google Scholar). The second, the β3 family, comprises four distinct subunits (β3a–d) that arise by alternative splicing of a single gene. Coexpression of the β2 or β3a, -b, or- c subunits with a BK α-subunit alters the functional properties of the current from that of the α-subunit expressed alone. However, unlike the β2 subunit, which both inactivates the channel and increases its calcium and voltage sensitivity, the β3 subunits do not increase the calcium or voltage sensitivity of the current. The differential expression of these novel β-subunits may underlie part of the large functional diversity observed in native BK currents. Sequence encoding the β1 subunit (U61537) was used to search the GenBank™ data base for homologues using the BLASTN and TBLASTN algorithms of the GCG software package (Wisconsin Genetics Group). This search identified an EST (AA904191) that, when completely sequenced, was demonstrated to encode a full-length KCaβ-subunit, β2. A fragment of this cDNA containing the coding region and 105 bp of the 3′-UTR was amplified by PCR using gene-specific oligonucleotide primers, cloned into pMVpl+ (a modified version of pSP64T (20Krieg P.A. Melton D.A. Nucleic Acids Res. 1984; 12: 7057-7070Crossref PubMed Scopus (1081) Google Scholar) containing an expanded polylinker), and confirmed by complete sequencing of both strands. The open reading frame and deduced amino acid sequence of β2 were then used to query the GenBank™ data base again. Iterative searches identified several human ESTs (AA195381, AA236930, AA236968,AA279911, AA761761, and AA934876) encoding partial sequences of novel putative BK β-subunits (β3). Commercially available cDNAs encoding these ESTs (AA195381, AA279911, and AA761761) were purchased and sequenced, which demonstrated that none encoded a full-length protein. To isolate the entire coding regions, 5′-RACE (21Frohman M.A. Dush M.K. Martin G.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8998-9002Crossref PubMed Scopus (4341) Google Scholar) was performed using Marathon-Ready spleen cDNA (CLONTECH) and nested gene-specific antisense primers, both of which were derived from the 3′-UTR of the putative β3. Specifically amplified products were identified in two ways: 1) by cloning and sequencing the major PCR products identified by agarose gel electrophoresis and 2) by cloning the products of the entire unfractionated amplification reaction and probing resultant colonies with a β3-specific probe (nucleotides 84–449 of AA195381). The amplification reactions were performed several times on two different lots of spleen cDNA. RACE products obtained in this manner were sequenced on both strands and shown to encode four distinct members of a novel family of β-subunits, β3a–d. For functional expression, the coding regions of the β3 cDNAs were amplified by PCR using sense primers that removed the 5′-UTR and improved the translation initiation sequence. The amplified fragments were cloned into pMVpl+ and confirmed by sequencing both strands. Sequences encoding β3a–d were used to search the high through put genomic sequence data base, resulting in the identification of several β3 gene fragments (AA195511, AA279608, AC007823, AQ093921, AQ096353,AQ590923, and AQ673110). Sequence comparisons showed thatAC007823 contains the alternatively spliced exons and the beginning of the conserved domain of the β3 cDNAs. The other genomic sequences each contain different amounts of the conserved core. To obtain the complete coding sequence, an arrayed human genomic DNA library was screened by PCR (Genome Systems) for the β3 gene using two sets of primers, one pair annealing in the β3b 5′-UTR and the other in the conserved core domain. Four BACs, each encoding part of the β3 gene, were identified and analyzed by PCR for exons encoding the splice variant-specific and conserved domains. These amplifications also enabled approximation of intron/exon boundaries and sizes within the gene. Appropriate regions of each clone were then sequenced to confirm and refine the results. The chromosomal locations of the β2, β3, and GCF2 genes were mapped by radiation hybrid analysis, which was carried out using DNAs isolated from the Stanford G3 (22Stewart E.A. McKusick K.B. Aggarwal A. Bajorek E. Brady S. Chu A. Fang N. Hadley D. Harris M. Hussain S. Lee R. Maratukulam A. O'Connor K. Perkins S. Piercy M. Qin F. Reif T. Sanders C. She X. Sun W.L. Tabar P. Voyticky S. Cowles S. Fan J.B. Cox D.R. et al.Genome Res. 1997; 7: 422-433Crossref PubMed Scopus (280) Google Scholar) and GeneBridge4 (23Walter M.A. Spillett D.J. Thomas P. Weissenbach J. Goodfellow P.N. Nat. Genet. 1994; 7: 22-28Crossref PubMed Scopus (353) Google Scholar) radiation hybrid panels (Research Genetics). The presence or absence of the human gene in each of the DNA samples was determined by amplification of a fragment of that gene by PCR. Control experiments demonstrated no amplification of homologous hamster genes by any of the primer pairs used. To distinguish between amplification of the β3b and GCF2 genes, a BsaBI restriction fragment length polymorphism was utilized. This enzyme distinguishes β3b from GCF2 by virtue of a BsaBI site unique to the β3b gene. Thus, only fragments cleaved by BsaBI were scored as positive for the presence of the β3b exon. Similarly, a complementaryPstI restriction fragment length polymorphism, unique to the GCF2 gene, was used to score fragments as GCF2-positive. Scoring data were transferred electronically to the Whitehead Institute/MIT Center for Genome Research or the Stanford Human Genome Center for analysis with the LOD score cutoff set to 15. The β3 gene was also mapped by fluorescence in situhybridization (FISH) in experiments performed by Genome Systems. Briefly, BAC DNA was isolated from clones B716 (β3c and conserved core), B766 (β3a–c unique exons), and B767 (β3b-core) and labeled with digoxigenin-dUTP by nick translation. Labeled probe was combined with a biotin-conjugated probe specific for centromeric sequences of chromosome 3 and hybridized to normal metaphase chromosomes derived from phytohemagglutinin-stimulated peripheral blood lymphocytes in a solution containing 50% formamide, 10% dextran sulfate, and 2% SSC. Probe detection was accomplished by incubating the slides with fluoresceinated antidigoxigenin antibodies and Texas Red avidin, followed by counterstaining with 4′,6-diamino-2-phenylindole (Molecular Probes). First strand cDNAs, prepared from human heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, thymus, prostate, testes, ovary, small intestine, colon, and peripheral blood leukocyte mRNAs, were purchased from CLONTECH and used as templates for PCR amplification reactions using subunit- and splice variant-specific primer pairs. To enable differentiation of fragments amplified from cDNA and contaminating genomic DNA (or incompletely spliced RNA), primer pairs were designed to span at least one intron. To improve specificity, β3 sense primers were designed to anneal in the splice variant-specific domains, which prevented amplification of cDNAs derived from chromosome 22 transcripts. Additionally, antisense primers were designed to anneal within the core domain, which prevented amplification of GCF2 fragments. PCR reactions were assembled according to the Advantage cDNA Polymerase Mix protocol (CLONTECH) and cycled 23 times for 30 s at 94 °C and 4 min at 68 °C. Positive controls for cDNA integrity were performed with primers derived from β-actin (the actin primers did not span an intron and, therefore, amplify fragments from both cDNA and genomic DNA). Products were identified by Southern analysis of blots that were probed with randomly primed,32P-labeled cDNAs specific for β2 (nucleotides 268–1080), β3a (nucleotides 70–384), β3b (nucleotides 463–797), or the core region common to all the β3 splice variants (nucleotides 1158–1450 of β3c). Hybridization was carried out overnight at 42 °C in 0.25 m NaPO4, 0.5 mNaCl, 1.0 mm EDTA, 7% SDS, and 1% bovine serum albumin. Blots were washed twice in 5× SSC, 0.1% SDS at 42 °C for 30 min and twice in 1× SSC, 0.1% SDS at 42 °C for 30 min each and then exposed to x-ray film. Positives were scored as tissues exhibiting a specifically amplified cDNA fragment of the expected size that also hybridized to the cognate probe. Expression analysis was repeated once using a different lot of template cDNAs with consistent results. In situ hybridization experiments were performed using an oligonucleotide probe derived from the sequence unique to β3c. The antisense probe corresponded to nucleotides 907–857, and the control sense probe to nucleotides 825–875 of the β3c sequence. Probes were labeled at their 3′ ends using the DIG oligonucleotide tailing kit (Roche Molecular Biochemicals) with biotin-16-dUTP (Roche) substituted for digoxigenin-dUTP. Formalin fixed human pancreas specimens (National Disease Research Interchange) were processed to paraffin, sectioned at 8 μm, and mounted on Superfrost plus slides (Fisher). In situ hybridization was carried out as described previously using 2 pmol of labeled probe/ml of hybridization buffer (24Lynch K.R. O'Neill G.P. Liu Q. Im D.S. Sawyer N. Metters K.M. Coulombe N. Abramovitz M. Figueroa D.J. Zeng Z. Connolly B.M. Bai C. Austin C.P. Chateauneuf A. Stocco R. Greig G.M. Kargman S. Hooks S.B. Hosfield E. Williams D.L.J. Ford H.A. Caskey C.T. Evans J.F. Nature. 1999; 399: 789-793Crossref PubMed Scopus (892) Google Scholar). The hybridization signal was amplified using the TSA Direct Red FISH tyramide reagent (NEN Life Science Products) according to manufacturer's directions. Immunohistochemistry was performed sequentially following in situ hybridization. Sections demonstrating optimal β3c mRNA signal were incubated with either guinea pig anti-human insulin sera (Dako) or rabbit anti-human glucagon sera (Dako) for 1 h at room temperature. Bound antibody was detected with fluorescein isothiocyanate-conjugated donkey anti-guinea pig IgG (Jackson Immunoresearch; 15 μg/ml in phosphate-buffered saline) or donkey anti-rabbit IgG (Jackson Immunoresearch; 15 μg/ml in phosphate-buffered saline), respectively. Sections were counterstained with 4′,6-diamino-2-phenylindole, and images were obtained and processed using a Nikon E1000 microscope, Micromax CCD camera (Princeton Instruments), and Metamorph imaging program (Universal Imaging). The cDNA encoding the BK α-subunit was a kind gift from Ligia Toro (identical in sequence to U11058 with one exception: this clone contains the conservative R1112K mutation) (25Wallner M. Meera P. Ottolia M. Kaczorowski G.J. Latorre R. Garcia M.L. Stefani E. Toro L. Receptors Channels. 1995; 3: 185-199PubMed Google Scholar). Plasmids encoding channel subunit cDNAs were linearized with appropriate restriction enzymes and cRNA synthesized by standard procedures (26Goldin A.L. Sumikawa K. Methods Enzymol. 1992; 207: 279-297Crossref PubMed Scopus (58) Google Scholar, 27Swanson R. Folander K. Methods Enzymol. 1992; 207: 310-319Crossref PubMed Scopus (14) Google Scholar). cRNAs were injected into Xenopusoocytes using 1.5 ng of α-subunit RNA/oocyte ± β-subunit RNA at equimolar concentration, 5-fold, or 10-fold molar excess. The molar ratio of the β/α RNAs in coinjection experiments was varied from 1 to 10 in attempts to maximize stoichiometric assembly of the two subunits. Preliminary comparisons of the magnitudes of functional effects induced by β-subunits demonstrated saturation of effects at 95% nucleotide identity between this region of chromosome 22 and exons 3, 4, and the intervening intron of the β3 gene (Fig. 2). However, there is no further homology to any of the other introns or exons of the β3 gene in the additional >100 kilobase pairs of chromosome 22 sequence available in AP000365 or AP000547. Analysis of the structure of the gene encoding β3a–d demonstrated that this family of subunits arises from alternative splicing of a single gene. A search of the high throughput genomic sequence data base revealed a single entry (AC007823) that contains the unique (5′) sequences of the β3a, β3b, β3c, and β3d cDNAs. All of these sequences, as well as the first 191 bp of the conserved core domain, are present on a 37-kilobase pair fragment of human genomic DNA in the following order: β3a-β3b-β3c/d-core(1–191) (Fig. 2). The sequences unique to the β3a and β3b subunits are present on distinct exons (1a and 1b), whereas the unique β3c and β3d sequences arise by
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