A novel sulfonylurea receptor forms with BIR (Kir6.2) a smooth muscle type ATP-sensitive K+ 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.

ATP-sensitive K ϩ (K ATP ) 1 channels, which represent a family of K ϩ channels inhibited by intracellular ATP, have been found in a variety of tissues including heart, pancreatic ␤-cells, skeletal muscle, smooth muscle, and the central nervous system (1)(2)(3)(4). These K ATP channels have been associated with diverse cellular functions, such as shortening of action potential duration and cellular loss of K ϩ ions that occur during metabolic inhibition in heart, insulin secretion from pancreatic ␤-cells, smooth muscle relaxation, regulation of skeletal muscle excitability, and neurotransmitter release (5,6). Furthermore, K ATP channels in different tissues exhibit considerable variation in response to K ϩ channel openers. For example, the pancreatic ␤-cell K ATP channel is activated by diazoxide and only weakly by pinacidil. The cardiac K ATP channel is activated by pinacidil but not by diazoxide. The smooth muscle K ATP channel is activated effectively by both of these compounds (2,5,6). Thus, properties of K ATP channels vary among tissues, having led to the premise that this K ϩ channel family may be composed of heterogeneous K ϩ channel proteins.
Recently, it has been shown that the pancreatic ␤-cell K ATP channel is a complex composed of at least two subunits, a K ϩ channel subunit (BIR/Kir6.2) and the pancreatic sulfonylurea receptor, SUR1 (7,8). Coexpression of these two subunits reconstituted inwardly rectifying ATP-sensitive K ϩ conductance (I K.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 I K.ATP , which was activated by pinacidil and cromakalim but not by diazoxide (9). 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 K ATP channels described in heart and skeletal muscle. The finding that distinct SURs produce different responses of the reconstituted I K.ATP to K ϩ channel openers suggests the existence of other isoforms of SURs that could be responsible for the smooth muscle type of response of I K.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 I K.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 K ATP channel.

EXPERIMENTAL PROCEDURES
Cloning a Sulfonylurea Receptor and an Inwardly Rectifying K ϩ Channel (Kir)-A mouse heart cDNA library (Stratagene, La Jolla, CA) was screened under a mild stringency condition using a 32 P-labeled DNA fragment encoding rat SUR1 (nucleotide positions 3466 -4589) (10), which was obtained by RT-PCR from rat heart RNA. A rat brain cDNA library (Stratagene) was screened for BIR (Kir6.2) using a 32 Plabeled mouse uK ATP -1 (Kir6.1) cDNA probe. Hybridization and DNA sequencing were performed as described previously (10).
Transfection and Electrophysiology-The coding regions of cloned cDNAs were subcloned into the expression vector (pcDNA3, Invitrogen, San Diego, CA). These subcloned plasmids were transfected into human embryonic kidney (HEK) 293T cells fed with Dulbecco's modified Eagle's medium (Nikken, Kyoto, Japan) containing 10% fetal calf serum (Life Technologies, Inc.). Cells (7 ϫ 10 4 /coverslip) were seeded on glass coverslips (15-mm diameter) coated with poly D-lysine (Sigma). After 18 -24 h, the cells were washed once with Opti-MEM (Life Technologies, Inc.) and transfected with the plasmids using lipofectAMINE (Life Technologies, Inc.) in Opti-MEM. 6 h after transfection, fetal calf serum was added to the medium to 10%, and the cells were incubated for 18 -24 h. After this period, the cells were washed and incubated in Dulbecco's modified Eagle's medium containing 10% fetal calf serum for further 24 -48 h before electrophysiological assay.
Single-channel recordings were made at room temperature in the cell-attached or inside-out configuration of patch clamp technique. The pipette had a tip resistance of 6 -7 megaohms when filled with a solution containing (in mM): 140 KCl, 1 CaCl 2 , 1 MgCl 2 , and 5 HEPES-KOH (pH 7.4). Channel activity was measured with a patch clamp amplifier * This work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) D86037, D86038, and D86039.
(Axopatch 200A, Axon Instruments, Inc., Foster City, CA) and continuously monitored with an analog storage oscilloscope (Dual Beam Storage Oscilloscope, Tektronix, Inc., Beaverton, Ore.). Agents applied to cells or inside-out patches were dissolved in the internal solution containing (in mM): 140 KCl, 5 EGTA, 5 HEPES (pH 7.3), and, unless otherwise indicated, 2 MgCl 2 (free Mg 2ϩ concentration, ϳ1.4 mM). ATP was dissolved in the internal solution with free Mg 2ϩ concentration adjusted to 1.4 mM by adding MgCl 2 (referred to as MgATP) or in the internal solution, which contained no MgCl 2 but EDTA (5 mM) instead of EGTA (Mg 2ϩ -free ATP). In both cases, the concentration of ATP is the total ATP concentration unless otherwise indicated. A continuous record of channel currents was stored for subsequent analysis on videocassette tapes through a PCM converter system (VR-10B, Instrutech Corp., Great Neck, NY). For analysis, data were reproduced, low pass filtered at 1 kHz (Ϫ3 decibels) by an 8-pole Bessel filter (Frequency Devices, Harverhill, MA), digitized at 3 or 5 kHz by an AD converter (ITC-16, Instrutech Corp.), and analyzed on a computer (Machintosh Quadra 700, Apple Computer Inc., Cupertino, CA) by using Pulse program (HEKA Electronik, Lambrecht, Germany) and Patch Analyst Pro (MT Corporation, Hyogo, Japan). The channel activity was estimated by measuring the mean current amplitude after subtracting a leak current. Statistical data were expressed as means Ϯ S.D.
RT-PCR Assay for SURs-The cDNAs synthesized from total RNAs extracted from various organs with oligo(dT) primers were used as templates for PCR amplification. The sequences of the primers for amplification of the novel SUR were as follows: 5Ј-ACGGTCGTAAC-CATAGCT-3Ј (forward) and 5Ј-CATGCTAGCAGCCTTAAG-3Ј (reverse), corresponding to nucleotide positions 4486 -4503 and 4847-4864 in MCS10, respectively. The PCR condition was as follows: an initial denaturation at 94°C for 4 min, followed by 30 cycles of denaturation at 94°C for 45 s, annealing at 55°C for 1 min, and extension at 72°C for 2 min, with a final extension step at 72°C for 8 min.

RESULTS
We obtained 49 positive clones after screening approximately 6 ϫ 10 5 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 (7) and 97% identity with that of rat SUR2 (9), 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 (11), 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 uK ATP -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 (8), indicating that it is rat (r-)BIR. Three amino acid residues of r-BIR were divergent from those of m-BIR; Thr 22 , Ser 248 , and Val 337 in r-BIR and Ala 22, Gly 248 , and Ile 337 in m-BIR. In r-BIR, Lys 379 was inserted between the codons for Ala 378 and Pro 379 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 ϳϪ5 pA at Ϫ60 mV occasionally appeared in bursts in the cotransfected cells. Diazoxide (200 M) or pinacidil (100 M) 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 ϳ17 and ϳ27 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 ϩ 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 K ATP 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 insideout patches rapidly ran down in the presence of 100 M intracellular Ca 2ϩ ( 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 ϩ solutions (Fig. 2B, a).
The current-voltage relationship demonstrated weak inward rectification with the single channel conductance of 80.3 pS between Ϫ100 and Ϫ20 mV (Fib. 2B, b).
Intracellular ATP inhibited the channel openings in a concentration-dependent manner in both the absence and the presence of intracellular Mg 2ϩ (Fig. 2C, a). The concentrationresponse relationships could be fitted with the following Hill equation: where [ATP] is the concentration of ATP, K d is the apparent dissociation constant, and n is the Hill coefficient (Fig. 2C, b). The K d and n were estimated as 67.9 M and 1.85 for Mg 2ϩ -free ATP (Fig. 2C, b, closed circles), and 300 M 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 Mg 2ϩ in this solution (Fig. 2C,  b, open diamonds), the apparent K d was estimated as 16.9 M. This value was lower than that found in the absence of Mg 2ϩ (67.9 M). Therefore, these results indicate that both Mg 2ϩ -free ATP and MgATP can inhibit the channel openings.
Intracellular UDP restored the channel openings after rundown of the expressed channel in a concentration-dependent manner (Fig. 2D, a). The concentration-response relationship could be fitted with the following Hill equation: where [UDP] is the concentration of UDP. The K d and n were estimated as 71.7 M 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 5Ј-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 Val 1504 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 (Val 1508 in MCS3 and Met 1507 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.
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.

DISCUSSION
Recent studies have shown that coexpression of SUR (SUR1 or SUR2A) and BIR reconstitutes I K.ATP , but neither of them can express the channel activity on their own (7-9, 11, 12). Likewise, SUR2B only when cotransfected with BIR produced K ATP channel activity. In this study, BIR was used to provide the gating part of K ATP channels. However, it has also been shown that Kir clones other than BIR, such as ROMK1 (Kir1.1) and uK ATP -1 (Kir6.1) can interact with SUR (12). These Kir clones produce K ϩ 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

FIG. 2. Pharmacological 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 ϳ145 mM K ϩ , whereas the bath was perfused with the internal solution which contained ϳ145 mM K ϩ . 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 Ϫ60 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 Ca 2ϩ , 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 Ϫ60 mV. B, currentvoltage 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 Ϫ100 and Ϫ20 mV. types of Kir clones and either produce K ATP 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 K ATP channels (4,13,14).
K ATP channels in different tissues exhibit distinct responses to K ϩ channel openers. The hamster SUR1/m-BIR channel, expressed in COS-1 or HEK 293 cells, is inhibited by 0.1 M glibenclamide or 500 M tolbutamide and activated by 100 M diazoxide (8,11). These properties are characteristic of pancreatic ␤-cell K ATP channels (15,16). In contrast, the r-SUR2A/m-BIR channel requires a high concentration (1 M) of glibenclamide for inhibition and is activated by pinacidil but not by diazoxide. These are the features of cardiac and skeletal muscle K ATP channels (5,17,18). Thus, SUR would appear to be the major determinant of the pharmacological properties of K ATP channels. Because the m-SUR2B/r-BIR channel was activated by both pinacidil and diazoxide, SUR2B corresponds to the smooth muscle K ATP response (4,13,14). 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 K ATP 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 3Ј end. An additional nucleotides of 176 bp specific for m-SUR2A in 3Ј-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 acti-vated K ATP 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 K ATP 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 K ATP channels reconstituted from SUR2A or SUR2B but not from SUR1.
In conclusion, our results indicate that SUR2B forms with BIR a K ATP 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 ϩ channel openers.