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A Novel Sulfonylurea Receptor Forms with BIR (Kir6.2) a Smooth Muscle Type ATP-sensitive K+ Channel

1996; Elsevier BV; Volume: 271; Issue: 40 Linguagem: Inglês

10.1074/jbc.271.40.24321

1083-351X

Shojiro Isomoto, Chikako Kondo, Mitsuhiko Yamada, Shigeto Matsumoto, Omi Higashiguchi, Yoshiyuki Horio, Yūji Matsuzawa, Yoshihisa Kurachi,

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We have isolated a cDNA encoding a novel isoform of the sulfonylurea receptor from a mouse heart cDNA library. Coexpression of this isoform and BIR (Kir6.2) in a mammalian cell line elicited ATP-sensitive K+ (KATP) channel currents. The channel was effectively activated by both diazoxide and pinacidil, which is the feature of smooth muscle KATP channels. Sequence analysis indicated that this clone is a variant of cardiac type sulfonylurea receptor (SUR2). The 42 amino acid residues located in the carboxyl-terminal end of this novel sulfonylurea receptor is, however, divergent from that of SUR2 but highly homologous to that of the pancreatic one (SUR1). Therefore, this short part of the carboxyl terminus may be important for diazoxide activation of KATP channels. The reverse transcription-polymerase chain reaction analysis showed that mRNA of this clone was ubiquitously expressed in diverse tissues, including brain, heart, liver, urinary bladder, and skeletal muscle. These results suggest that this novel isoform of sulfonylurea receptor is a subunit reconstituting the smooth muscle KATP channel. We have isolated a cDNA encoding a novel isoform of the sulfonylurea receptor from a mouse heart cDNA library. Coexpression of this isoform and BIR (Kir6.2) in a mammalian cell line elicited ATP-sensitive K+ (KATP) channel currents. The channel was effectively activated by both diazoxide and pinacidil, which is the feature of smooth muscle KATP channels. Sequence analysis indicated that this clone is a variant of cardiac type sulfonylurea receptor (SUR2). The 42 amino acid residues located in the carboxyl-terminal end of this novel sulfonylurea receptor is, however, divergent from that of SUR2 but highly homologous to that of the pancreatic one (SUR1). Therefore, this short part of the carboxyl terminus may be important for diazoxide activation of KATP channels. The reverse transcription-polymerase chain reaction analysis showed that mRNA of this clone was ubiquitously expressed in diverse tissues, including brain, heart, liver, urinary bladder, and skeletal muscle. These results suggest that this novel isoform of sulfonylurea receptor is a subunit reconstituting the smooth muscle KATP channel. INTRODUCTIONATP-sensitive K+ (KATP) 1The abbreviations used are: KATPATP-sensitive K+ channelIK.ATPKATP conductanceSURsulfonylurea receptorRTreverse transcriptionPCRpolymerase chain reactionHEKhuman embryonic kidneybpbase pairs; m-, mouse; r-, rat. channels, which represent a family of K+ channels inhibited by intracellular ATP, have been found in a variety of tissues including heart, pancreatic β-cells, skeletal muscle, smooth muscle, and the central nervous system (1Noma A. Nature. 1983; 305: 147-148Google Scholar, 2Ashcroft S.J.H. Ashcroft F.M. Cell. Signalling. 1990; 2: 197-214Google Scholar, 3Spruce A.E. Standen N.B. Stanfield P.R. J. Physiol. (Lond.). 1987; 382: 213-236Google Scholar, 4Standen N.B. Quayle J.M. Davis N.W. Brayden J.E. Huang Y. Nelson M.T. Science. 1989; 245: 177-180Google Scholar). These KATP channels have been associated with diverse cellular functions, such as shortening of action potential duration and cellular loss of K+ ions that occur during metabolic inhibition in heart, insulin secretion from pancreatic β-cells, smooth muscle relaxation, regulation of skeletal muscle excitability, and neurotransmitter release (5Terzic A. Jahangir A. Kurachi Y. Am. J. Physiol. 1995; 269: C525-C545Google Scholar, 6Nelson M.T. Quayle J.M. Am. J. Physiol. 1995; 268: C799-C822Google Scholar). Furthermore, KATP channels in different tissues exhibit considerable variation in response to K+ channel openers. For example, the pancreatic β-cell KATP channel is activated by diazoxide and only weakly by pinacidil. The cardiac KATP channel is activated by pinacidil but not by diazoxide. The smooth muscle KATP channel is activated effectively by both of these compounds (2Ashcroft S.J.H. Ashcroft F.M. Cell. Signalling. 1990; 2: 197-214Google Scholar, 5Terzic A. Jahangir A. Kurachi Y. Am. J. Physiol. 1995; 269: C525-C545Google Scholar, 6Nelson M.T. Quayle J.M. Am. J. Physiol. 1995; 268: C799-C822Google Scholar). Thus, properties of KATP channels vary among tissues, having led to the premise that this K+ channel family may be composed of heterogeneous K+ channel proteins.Recently, it has been shown that the pancreatic β-cell KATP channel is a complex composed of at least two subunits, a K+ channel subunit (BIR/Kir6.2) and the pancreatic sulfonylurea receptor, SUR1 (7Aguilar-Bryan L. Nichols C.G. Wechsler S.W. Clement IV, J.P. Boyd III, A.E. Gonzalez G. Herrera-Sosa H. Nguy K. Bryan J. Nelson D.A. Science. 1995; 268: 423-426Google Scholar, 8Inagaki N. Gonoi T. Clement IV, J.P. Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1170Google Scholar). Coexpression of these two subunits reconstituted inwardly rectifying ATP-sensitive K+ conductance (IK.ATP), which was inhibited by sulfonylureas and activated by diazoxide. It was also reported that coexpression of BIR and an isoform of SUR isolated from a rat brain cDNA library, designated SUR2, elicited IK.ATP, which was activated by pinacidil and cromakalim but not by diazoxide (9Inagaki N. Gonoi T. Clement IV, J.P. Wang C.-Z. Aguilar-Bryan L. Bryan J. Seino S. Neuron. 1996; 16: 1011-1017Google Scholar). SUR2 mRNA was expressed at high levels in heart and skeletal muscle as assessed by Northern blot analysis. Thus, the complex of BIR and SUR2 may reconstitute KATP channels described in heart and skeletal muscle. The finding that distinct SURs produce different responses of the reconstituted IK.ATP to K+ channel openers suggests the existence of other isoforms of SURs that could be responsible for the smooth muscle type of response of IK.ATP.In this study, we have tried to find SURs by screening a mouse heart cDNA library and obtained a novel isoform. Coexpression of BIR and this novel SUR reconstituted IK.ATP activated by both pinacidil and diazoxide. The reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that mRNA for this SUR was expressed in various tissues, including brain, heart, lung, liver, urinary bladder, and skeletal muscle. These findings suggest that this novel SUR is a subunit that could represent part of the smooth muscle KATP channel.RESULTSWe obtained 49 positive clones after screening approximately 6 × 105 plaques of the mouse heart cDNA library. Two of these clones, named MCS3 and MCS10, were further analyzed by sequencing. The nucleotide sequence of MCS10 revealed a single open reading frame encoding a protein of 1546 amino acid residues (Fig. 1). The amino acid sequence of MCS10 had 67% identity with that of rat SUR1 (7Aguilar-Bryan L. Nichols C.G. Wechsler S.W. Clement IV, J.P. Boyd III, A.E. Gonzalez G. Herrera-Sosa H. Nguy K. Bryan J. Nelson D.A. Science. 1995; 268: 423-426Google Scholar) and 97% identity with that of rat SUR2 (9Inagaki N. Gonoi T. Clement IV, J.P. Wang C.-Z. Aguilar-Bryan L. Bryan J. Seino S. Neuron. 1996; 16: 1011-1017Google Scholar), indicating that MCS10 is homologous to SUR2. The hydropathy profile of MCS10 was similar to those of SUR1 and SUR2, suggesting that this clone has a similar topology with 13 putative transmembrane regions and the amino- and carboxyl-terminal regions located extracellularly and intracellularly, respectively. MCS10 had two potential nucleotide binding folds with Walker A and B consensus motifs (11Sakura H. Ämmälä C. Smith P.A. Gribble F.M. Ashcroft F.M. FEBS Lett. 1995; 377: 338-344Google Scholar), two potential N-linked glycosylation sites, cyclic-AMP-dependent protein kinase phosphorylation sites, and protein kinase C-dependent phosphorylation sites.The screening of a rat brain cDNA library with mouse uKATP-1 as a probe resulted in isolation of one clone, of which nucleotide sequencing revealed a single open reading frame encoding a protein of 390 amino acid residues. The amino acid sequence of this clone was 99% identical to that of mouse (m-)BIR (8Inagaki N. Gonoi T. Clement IV, J.P. Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1170Google Scholar), indicating that it is rat (r-)BIR. Three amino acid residues of r-BIR were divergent from those of m-BIR; Thr22, Ser248, and Val337 in r-BIR and Ala22, Gly248, and Ile337 in m-BIR. In r-BIR, Lys379 was inserted between the codons for Ala378 and Pro379 in m-BIR.By using patch clamp technique, we analyzed the channels expressed in HEK 293T cells cotransfected with MCS10 and r-BIR (Fig. 2). In the cell-attached configuration, unitary currents of ~−5 pA at −60 mV occasionally appeared in bursts in the cotransfected cells. Diazoxide (200 μM) or pinacidil (100 μM) added to the bathing solution markedly increased the channel activity as shown in Fig. 2A, a; diazoxide and pinacidil increased the mean channel current amplitude by ~17 and ~27 times, respectively, in this particular cell-attached patch where such a large number of channels were activated that individual single channel current levels could not be distinguished. Such responses to the K+ channel openers were not observed in the cells transfected with either MCS10 or r-BIR alone (not shown). Both diazoxide- and pinacidil-induced channel activity were inhibited by tolbutamide or glibenclamide, specific blockers of KATP channels (Fig. 2A, a). On patch excision, maximal channel activity appeared promptly and was almost completely inhibited by 1 mM of intracellular MgATP. Similar data were obtained from five other patches in the cotransfected cells. Spontaneous openings of the MCS10/r-BIR channels in the inside-out patches rapidly ran down in the presence of 100 μM intracellular Ca2+ (Fig. 2A, b). The channel could be easily reactivated after treating the patch with 1 mM MgATP. MgUDP (10 mM) restored the channel activity after run-down (Fig. 2A, b). Single channel recordings of the MCS10/r-BIR channels in a cell-attached patch are shown in Fig. 2B. The channels opened in bursts at all membrane potentials examined. The currents flowing through the channels reversed around 0 mV under the symmetrical K+ solutions (Fig. 2B, a). The current-voltage relationship demonstrated weak inward rectification with the single channel conductance of 80.3 pS between −100 and −20 mV (Fib. 2B, b).Fig. 2Pharmacological and electrophysiological properties of MCS10/r-BIR channels expressed in HEK 293T cells. MCS10 and r-BIR were heterologously expressed in HEK 293T cells and analyzed in the cell-attached and inside-out configurations of the patch clamp technique. The pipette solution contained ~145 mM K+, whereas the bath was perfused with the internal solution which contained ~145 mM K+. A, pharmacological properties of MCS10/r-BIR channels. a, the response of the channels to diazoxide, pinacidil, tolbutamide, or glibenclamide in a cell-attached patch. The membrane potential was −60 mV. At the end of this record, the patch was excised from the cell (at IO). The zero current level is indicated as a thin horizontal line. b, run-down of the channels by intracellular Ca2+, reactivation by MgATP, and restoration by UDP in an inside-out patch. This patch was different from that shown in a. The membrane potential was −60 mV. B, current-voltage relationship of the MCS10/r-BIR channels in a cell-attached patch. a, current traces obtained at various membrane potentials. Membrane potentials are indicated at the left on the traces. Arrowheads indicate the zero current level at each potential. b, the single channel current-voltage relationship estimated from a current amplitude histogram obtained from the data shown in a. The straight line is the regression line for the data obtained between −100 and −20 mV. C, concentration-dependent inhibition of the channel openings by ATP in the presence or the absence of Mg2+. a, effects of intracellular ATP in the absence of Mg2+ (upper panel) and in the presence of 1.4 mM of free Mg2+ (lower panel). The ATP concentrations indicated were those of total ATP. These two traces were obtained from the same patch, and the scales shown under the lower trace were also applicable to the upper trace. The membrane potential was −60 mV. The zero current level is indicated as a thin horizontal line. b, the concentration-response relationships in the presence of 1.4 mM free Mg2+ (open circles and diamonds) and in the absence of Mg2+ (filled circles). The data obtained at various concentrations of ATP are expressed as percentages of the value obtained in the absence of ATP. The number of observations at each point was three. Open and filled circles are the plots against the total ATP concentrations, whereas open diamonds are the estimated concentrations of ATP not complexed with Mg2+ in this solution. D, concentration-dependent restoration of the MCS10/r-BIR channels by intracellular UDP after run-down by Ca2+ as in A, b. a, various concentrations of UDP was applied to the intracellular surface of an inside-out patch membrane. The membrane potential was −60 mV. b, the concentration-response relationship. The number of observations at each point was three. The data at each concentration of UDP were expressed as percentages of the value in the presence of 1 mM UDP, which always gave the maximum activation. Symbols and bars indicate the means ± S.D.View Large Image Figure ViewerDownload (PPT)Intracellular ATP inhibited the channel openings in a concentration-dependent manner in both the absence and the presence of intracellular Mg2+ (Fig. 2C, a). The concentration-response relationships could be fitted with the following Hill equation: %Inhibitation=100/1+ATP/Kdn(Eq. 1) where [ATP] is the concentration of ATP, Kd is the apparent dissociation constant, and n is the Hill coefficient (Fig. 2C, b). The Kd and n were estimated as 67.9 μM and 1.85 for Mg2+-free ATP (Fig. 2C, b, closed circles), and 300 μM and 1.43 for MgATP (Fig. 2C, b, open circles), respectively. When the inhibition evoked by MgATP was replotted by calculating the concentration of ATP not complexed with Mg2+ in this solution (Fig. 2C, b, open diamonds), the apparent Kd was estimated as 16.9 μM. This value was lower than that found in the absence of Mg2+ (67.9 μM). Therefore, these results indicate that both Mg2+-free ATP and MgATP can inhibit the channel openings.Intracellular UDP restored the channel openings after run-down of the expressed channel in a concentration-dependent manner (Fig. 2D, a). The concentration-response relationship could be fitted with the following Hill equation: Relative activation=1001+Kd/UDPn(Eq. 2) where [UDP] is the concentration of UDP. The Kd and n were estimated as 71.7 μM and 1.74, respectively. Thus, UDP may activate this channel in a positive cooperative manner.The sequence analysis of another clone obtained, MCS3, showed that this clone was essentially the same as rat SUR2. MCS3, which lacked 5′-untranslated and coding regions, has a sequence identical to MCS10 and possessed an additional 176-bp insertion in the COOH terminus between nucleotide positions 4505 and 4506 of MCS10. The insertion of these 176 bp generated divergent amino acid sequences in the COOH termini between MCS3 and MCS10 (Fig. 3A). Thus, MCS3 had an amino acid sequence identical to MCS10 through Val1504 and then diverged in the COOH-terminal ends. These findings indicated that MCS10 and MCS3 may be formed by alternative splicing of a single mouse gene. A comparison of amino acid sequences in the COOH termini of MCS10, MCS3, r-SUR1, and r-SUR2 is shown in Fig. 3B. The amino acid sequence of MCS3 was identical to that of r-SUR2 except for one amino acid residue (Val1508 in MCS3 and Met1507 in r-SUR2), suggesting that MCS3 is a mouse homolog of r-SUR2. Based on these results, we designated r-SUR2, MCS3, and MCS10 as r-SUR2A, m-SUR2A, and m-SUR2B, respectively. For the alternative regions composed of 42 amino acid residues located in the ends of their sequences, m-SUR2B showed 74% identity with r-SUR1 and 33% identity with m-SUR2A.Fig. 3The nucleotide and deduced amino acid sequences in the 3′ ends of SURs. A, aligned nucleotide and deduced amino acid sequences of m-SUR2A (MCS3) and m-SUR2B (MCS10). The nucleotides of m-SUR2B are numbered to the right of each sequence. Termination codons are underlined. The nucleotide sequence of m-SUR2A possesses an insertion of 176 bp, which is shaded, between positions 4505 and 4506 of m-SUR2B. B, a comparison of amino acid sequences in the COOH termini of SURs. The amino acid residues of SURs are numbered to the right of each sequence. Identical residues are boxed.View Large Image Figure ViewerDownload (PPT)To determine tissue distributions of m-SUR2A and m-SUR2B mRNAs, the RT-PCR assay was performed. The specific primers for amplification of both m-SUR2A and m-SUR2B were designed to produce cDNA fragments of 555 and 379 bp, respectively. As shown in Fig. 4, m-SUR2A mRNA was expressed in cerebellum, eye, atrium, ventricle, urinary bladder, and skeletal muscle. The m-SUR2B mRNA distributed not only in these tissues but in all other tissues examined: forebrain, lung, liver, pancreas, kidney, spleen, stomach, small intestine, colon, uterus, ovary, and fat tissue.Fig. 4RT-PCR detection of m-SUR2A and m-SUR2B. The RT-PCR assay was performed as described under “Experimental Procedures.” The RT-PCR yielded visible amplified product (555 bp) of m-SUR2A in mRNA of cerebellum, eye, atrium, ventricle, urinary bladder, and skeletal muscle, and that (379 bp) of m-SUR2B in mRNA of all tissues examined in this study.View Large Image Figure ViewerDownload (PPT)DISCUSSIONRecent studies have shown that coexpression of SUR (SUR1 or SUR2A) and BIR reconstitutes IK.ATP, but neither of them can express the channel activity on their own (7Aguilar-Bryan L. Nichols C.G. Wechsler S.W. Clement IV, J.P. Boyd III, A.E. Gonzalez G. Herrera-Sosa H. Nguy K. Bryan J. Nelson D.A. Science. 1995; 268: 423-426Google Scholar, 8Inagaki N. Gonoi T. Clement IV, J.P. Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1170Google Scholar, 9Inagaki N. Gonoi T. Clement IV, J.P. Wang C.-Z. Aguilar-Bryan L. Bryan J. Seino S. Neuron. 1996; 16: 1011-1017Google Scholar, 11Sakura H. Ämmälä C. Smith P.A. Gribble F.M. Ashcroft F.M. FEBS Lett. 1995; 377: 338-344Google Scholar, 12Ämmälä C. Moorhouse A. Gribble F. Ashfield R. Proks P. Smith P.A. Sakura H. Coles B. Ashcroft S.J.H. Ashcroft F.M. Nature. 1996; 379: 545-548Google Scholar). Likewise, SUR2B only when cotransfected with BIR produced KATP channel activity. In this study, BIR was used to provide the gating part of KATP channels. However, it has also been shown that Kir clones other than BIR, such as ROMK1 (Kir1.1) and uKATP-1 (Kir6.1) can interact with SUR (12Ämmälä C. Moorhouse A. Gribble F. Ashfield R. Proks P. Smith P.A. Sakura H. Coles B. Ashcroft S.J.H. Ashcroft F.M. Nature. 1996; 379: 545-548Google Scholar). These Kir clones produce K+ channel activity for themselves but become sensitive to glibenclamide when the SUR1 clone is cotransfected. Therefore, SUR2B may also be able to couple to several types of Kir clones and either produce KATP channel activity and/or induce glibenclamide sensitivity. This might be the mechanism responsible for the reported diversity of the gating and conductance properties of smooth muscle KATP channels (4Standen N.B. Quayle J.M. Davis N.W. Brayden J.E. Huang Y. Nelson M.T. Science. 1989; 245: 177-180Google Scholar, 13Kajioka S. Kitamura K. Kuriyama H. J. Physiol. 1991; 444: 397-418Google Scholar, 14Beech D.J. Zhang H. Nakao K. Bolton T.B. Br. J. Pharmacol. 1993; 110: 573-582Google Scholar).KATP channels in different tissues exhibit distinct responses to K+ channel openers. The hamster SUR1/m-BIR channel, expressed in COS-1 or HEK 293 cells, is inhibited by 0.1 μM glibenclamide or 500 μM tolbutamide and activated by 100 μM diazoxide (8Inagaki N. Gonoi T. Clement IV, J.P. Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1170Google Scholar, 11Sakura H. Ämmälä C. Smith P.A. Gribble F.M. Ashcroft F.M. FEBS Lett. 1995; 377: 338-344Google Scholar). These properties are characteristic of pancreatic β-cell KATP channels (15Cook D.L. Hales C.N. Nature. 1984; 311: 271-273Google Scholar, 16Findlay I. Dunne M.J. Petersen O.H. J. Membr. Biol. 1985; 88: 165-172Google Scholar). In contrast, the r-SUR2A/m-BIR channel requires a high concentration (1 μM) of glibenclamide for inhibition and is activated by pinacidil but not by diazoxide. These are the features of cardiac and skeletal muscle KATP channels (5Terzic A. Jahangir A. Kurachi Y. Am. J. Physiol. 1995; 269: C525-C545Google Scholar, 17Faivre J.F. Findlay I. Biochim. Biophys. Acta. 1989; 984: 1-5Google Scholar, 18Findlay I. J. Pharmacol. Exp. Ther. 1992; 261: 540-545Google Scholar). Thus, SUR would appear to be the major determinant of the pharmacological properties of KATP channels. Because the m-SUR2B/r-BIR channel was activated by both pinacidil and diazoxide, SUR2B corresponds to the smooth muscle KATP response (4Standen N.B. Quayle J.M. Davis N.W. Brayden J.E. Huang Y. Nelson M.T. Science. 1989; 245: 177-180Google Scholar, 13Kajioka S. Kitamura K. Kuriyama H. J. Physiol. 1991; 444: 397-418Google Scholar, 14Beech D.J. Zhang H. Nakao K. Bolton T.B. Br. J. Pharmacol. 1993; 110: 573-582Google Scholar). The ubiquitous expression of SUR2B mRNA in all of the tissues that we have tested is consistent with the notion that SUR2B is a subunit of the smooth muscle KATP channel.We have identified two mouse homologs of the second class of SUR, designated m-SUR2A and m-SUR2B. Analysis of the nucleotide sequences of these isoforms showed that the molecular diversity of SUR2 transcripts may be due to alternative splicing at the 3′ end. An additional nucleotides of 176 bp specific for m-SUR2A in 3′-coding region generated divergent transcripts in the COOH termini. Comparative analysis of the amino acid sequences in the COOH termini (Fig. 3) showed that m-SUR2A and m-SUR2B are highly homologous to r-SUR2A and r-SUR1, respectively. Because diazoxide activated KATP channels reconstituted from SUR1 or SUR2B but not from SUR2A, this alternative COOH-terminal end may be the functional domain important for diazoxide activation of KATP channels. On the other hand, binding sites of pinacidil to SURs may be in the regions different from the COOH-terminal end, because pinacidil activated KATP channels reconstituted from SUR2A or SUR2B but not from SUR1.In conclusion, our results indicate that SUR2B forms with BIR a KATP channel with a pharmacology representative of smooth muscle, whereas SUR1/BIR and SUR2A/BIR channels form pancreatic and cardiac types, respectively. The successful cloning of this novel SUR should open a novel approach to elucidate the molecular mechanism of smooth muscle regulation and also to develop new vasorelaxants belonging to K+ channel openers. INTRODUCTIONATP-sensitive K+ (KATP) 1The abbreviations used are: KATPATP-sensitive K+ channelIK.ATPKATP conductanceSURsulfonylurea receptorRTreverse transcriptionPCRpolymerase chain reactionHEKhuman embryonic kidneybpbase pairs; m-, mouse; r-, rat. channels, which represent a family of K+ channels inhibited by intracellular ATP, have been found in a variety of tissues including heart, pancreatic β-cells, skeletal muscle, smooth muscle, and the central nervous system (1Noma A. Nature. 1983; 305: 147-148Google Scholar, 2Ashcroft S.J.H. Ashcroft F.M. Cell. Signalling. 1990; 2: 197-214Google Scholar, 3Spruce A.E. Standen N.B. Stanfield P.R. J. Physiol. (Lond.). 1987; 382: 213-236Google Scholar, 4Standen N.B. Quayle J.M. Davis N.W. Brayden J.E. Huang Y. Nelson M.T. Science. 1989; 245: 177-180Google Scholar). These KATP channels have been associated with diverse cellular functions, such as shortening of action potential duration and cellular loss of K+ ions that occur during metabolic inhibition in heart, insulin secretion from pancreatic β-cells, smooth muscle relaxation, regulation of skeletal muscle excitability, and neurotransmitter release (5Terzic A. Jahangir A. Kurachi Y. Am. J. Physiol. 1995; 269: C525-C545Google Scholar, 6Nelson M.T. Quayle J.M. Am. J. Physiol. 1995; 268: C799-C822Google Scholar). Furthermore, KATP channels in different tissues exhibit considerable variation in response to K+ channel openers. For example, the pancreatic β-cell KATP channel is activated by diazoxide and only weakly by pinacidil. The cardiac KATP channel is activated by pinacidil but not by diazoxide. The smooth muscle KATP channel is activated effectively by both of these compounds (2Ashcroft S.J.H. Ashcroft F.M. Cell. Signalling. 1990; 2: 197-214Google Scholar, 5Terzic A. Jahangir A. Kurachi Y. Am. J. Physiol. 1995; 269: C525-C545Google Scholar, 6Nelson M.T. Quayle J.M. Am. J. Physiol. 1995; 268: C799-C822Google Scholar). Thus, properties of KATP channels vary among tissues, having led to the premise that this K+ channel family may be composed of heterogeneous K+ channel proteins.Recently, it has been shown that the pancreatic β-cell KATP channel is a complex composed of at least two subunits, a K+ channel subunit (BIR/Kir6.2) and the pancreatic sulfonylurea receptor, SUR1 (7Aguilar-Bryan L. Nichols C.G. Wechsler S.W. Clement IV, J.P. Boyd III, A.E. Gonzalez G. Herrera-Sosa H. Nguy K. Bryan J. Nelson D.A. Science. 1995; 268: 423-426Google Scholar, 8Inagaki N. Gonoi T. Clement IV, J.P. Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1170Google Scholar). Coexpression of these two subunits reconstituted inwardly rectifying ATP-sensitive K+ conductance (IK.ATP), which was inhibited by sulfonylureas and activated by diazoxide. It was also reported that coexpression of BIR and an isoform of SUR isolated from a rat brain cDNA library, designated SUR2, elicited IK.ATP, which was activated by pinacidil and cromakalim but not by diazoxide (9Inagaki N. Gonoi T. Clement IV, J.P. Wang C.-Z. Aguilar-Bryan L. Bryan J. Seino S. Neuron. 1996; 16: 1011-1017Google Scholar). SUR2 mRNA was expressed at high levels in heart and skeletal muscle as assessed by Northern blot analysis. Thus, the complex of BIR and SUR2 may reconstitute KATP channels described in heart and skeletal muscle. The finding that distinct SURs produce different responses of the reconstituted IK.ATP to K+ channel openers suggests the existence of other isoforms of SURs that could be responsible for the smooth muscle type of response of IK.ATP.In this study, we have tried to find SURs by screening a mouse heart cDNA library and obtained a novel isoform. Coexpression of BIR and this novel SUR reconstituted IK.ATP activated by both pinacidil and diazoxide. The reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that mRNA for this SUR was expressed in various tissues, including brain, heart, lung, liver, urinary bladder, and skeletal muscle. These findings suggest that this novel SUR is a subunit that could represent part of the smooth muscle KATP channel.