Structural Basis for the Interference Between Nicorandil and Sulfonylurea Action Frank Reimann,1,2 Frances M

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Structural Basis for the Interference Between Nicorandil and Sulfonylurea Action Frank Reimann,1,2 Frances M. Ashcroft,2 and Fiona M. Gribble1

Nicorandil is a new antianginal agent that potentially may be used to treat the cardiovascular side effects of diabetes. It is both a nitric oxide donor and an opener of icorandil is currently undergoing clinical trials ؉ ATP-sensitive K (KATP) channels in muscle and there- and is being increasingly used for the treatment by causes vasodilation of the coronary vasculature. The of angina (1). It has a dual action, operating aim of this study was to investigate the domains of the both as a nitric oxideϪgenerating agent and as K channel involved in nicorandil activity and to de- N ϩ ATP an opener of ATP-sensitive K (K ) channels in vascular termine whether nicorandil interacts with hypoglycemic ATP smooth and cardiac muscle (2). The KATP channels in sulfonylureas that target KATP channels in pancreatic ␤ ␤ these tissues are closely related to those that regulate -cells. KATP channels in muscle and -cells share a com- ␤ mon pore-forming subunit, Kir6.2, but possess alterna- insulin secretion from pancreatic -cells (3). Like the tive sulfonylurea receptors (SURs; SUR1 in ␤-cells, ␤-cell channel, they are blocked by many of the sulfonyl- SUR2A in cardiac muscle, and SUR2B in smooth muscle). ureas that are widely used to treat type 2 diabetes (4). We expressed recombinant KATP channels in Xenopus Because type 2 diabetes increases the risk of cardiovascu- oocytes and measured the effects of drugs and nucleo- lar disease, many diabetic patients might benefit from tides by recording macroscopic currents in excised mem- therapy with both sulfonylureas and nicorandil. The cur- brane patches. Nicorandil activated Kir6.2/SUR2A and rent study was undertaken to investigate the molecular Kir6.2/SUR2B but not Kir6.2/SUR1 currents, consistent basis for the previously reported tissue specificity of with its specificity for cardiac and smooth muscle KATP nicorandil and to determine whether significant interac- channels. Drug activity depended on the presence of tions might occur between nicorandil and sulfonylureas intracellular nucleotides and was impaired when the Walker A lysine residues were mutated in either nucle- when both drugs are administered concurrently. otide-binding domain of SUR2. Chimeric studies showed KATP channels respond to alterations in the metabolic that the COOH-terminal group of transmembrane helices activity of the cell and thereby act as sensors of glucose ␤ (TMs), especially TM 17, is responsible for the specific- and oxygen availability. In pancreatic -cells, KATP chan- ity of nicorandil for channels containing SUR2. The nels are open at low glucose concentrations; their closure splice variation between SUR2A and SUR2B altered the in response to an increase in glucose metabolism leads to off-rate of the nicorandil response. Finally, we showed membrane depolarization, thereby triggering the entry of that nicorandil activity was unaffected by gliclazide, calcium and release of insulin (5). The opening of KATP which specifically blocks SUR1-type KATP channels, but channels in vascular smooth muscle results in muscle was severely impaired by glibenclamide and glimepiride, relaxation and vasodilation (6). In cardiac muscle, the which target both SUR1 and SUR2-type KATP channels. Diabetes 50:2253–2259, 2001 KATP channels are believed to be closed under normal physiological conditions; their opening during hypoxia results in a protective shortening of the cardiac action potential that reduces cardiac work (7). In all these tissues, the KATP channels are believed to respond to changes in the concentrations of adenine nucleotides. The molecular composition of KATP channels differs among tissue types and provides a basis for targeting drugs to a specific organ. The pore of the channel is formed from four inwardly rectifying potassium channel subunits (Kir6.2, but Kir6.1 in some smooth muscle channels) (8–10), and ATP inhibits KATP channels by direct interaction with Kir6.2 (11). An additional regulatory subunit, the SUR, endows the channels with sensitivity to Mg From the 1Department of Clinical Biochemistry, Addenbrooke’s Hospital, nucleotides (which enhance channel activity) and thera- Cambridge, U.K.; and the 2University Laboratory of Physiology, Parks Road, Oxford, U.K. peutic drugs such as the sulfonylureas and KATP channel Address correspondence and reprint requests to Dr. Fiona M. Gribble, openers (3,12). The SUR, a member of the ATP-binding Department of Clinical Biochemistry, Addenbrooke’s Hospital, Box 232, Hills Rd., Cambridge CB2 2QR, U.K. E-mail: [email protected]. cassette (ABC) transporter family, is a large membrane Received for publication 27 March 2001 and accepted in revised form 5 July protein containing 17 transmembrane helices (TMs), ar- 2001. ranged in three groups of five, six, and six (13). It also ABC, ATP-binding cassette; ANOVA, analysis of variance; KATP channel, ATP-sensitive Kϩ channel; NBD, nucleotide-binding domain; SUR, sulfonyl- possesses two intracellular nucleotide-binding domains urea receptor; TM, transmembrane helix. (NBDs). Each KATP channel contains four Kir and four

DIABETES, VOL. 50, OCTOBER 2001 2253 NICORANDIL ACTION ON KATP CHANNELS

SUR subunits (3). Two genes encoding SURs have been Roche) and manually defolliculated. Oocytes were injected with ϳ0.1 ng identified. SUR1 is predominantly expressed in pancreatic Kir6.2 mRNA and ϳ2 ng mRNA encoding SUR1, SUR2A, SUR2B, or the ␤ indicated SUR chimera. The final injection volume was 50 nl/oocyte. Isolated -cells and some neurones (14,15). Alternative splicing of oocytes were maintained in Barth’s solution and studied 1–7 days after SUR2 produces SUR2A (in cardiac muscle) and SUR2B (in injection (24). smooth muscle) (16,17). SUR1 and SUR2 share 71% iden- Electrophysiology. Patch pipettes were pulled from borosilicate glass and tity; SUR2A and SUR2B differ only in their COOH-terminal had resistances of 250–500 k⍀ when filled with pipette solution. Macroscopic currents were recorded from giant excised inside-out patches at a holding 42 amino acids. potential of 0 mV and at 20–24°C using an Axopatch 200B patch-clamp In recent years, a number of sites on SURs critical for amplifier (Axon Instruments, Foster City, CA) (24). The pipette (external) the binding and regulatory activity of nucleotides, sulfo- solution contained (in mmol/l) 140 KCl, 1.2 MgCl2, 2.6 CaCl2, and 10 HEPES (pH 7.4 with KOH). The bath (intracellular) solution contained (in mmol/l) 107 nylureas, and KATP channel openers have been identified. KCl, 1.2 MgCl , 1 CaCl , 10 EGTA, and 10 HEPES (pH 7.2 with KOH; final [Kϩ] The effect of nucleotides is dependent on their interaction 2 2 ϳ140 mmol/l). Nicorandil was prepared as a 100 mmol/l stock solution in with Walker A and Walker B motifs in the NBDs (18,19). DMSO. Glibenclamide and glimepiride were prepared as 1,000ϫ stock solu- By contrast, the most important domain for drug bind- tions in DMSO. Gliclazide was prepared as a 100 mmol/l stock in 100 mmol/l ing appears to be the COOH-terminal group of six TMs. KOH. Nucleotides were added with equivalent concentrations of MgCl2, and Binding sites for glibenclamide and the K channel the pH was readjusted as required. Rapid exchange of solutions was achieved ATP by positioning the patch in the mouth of one of a series of adjacent inflow opener P1075 (an analog of pinacidil) have been mapped pipes placed in the bath. to this area (20,21); differences between SUR1 and SUR2 In most experiments, currents were recorded in response to repetitive 3-s within this region account for the tissue-specific effects of voltage ramps from Ϫ110 mV to ϩ100 mV. They were filtered at 5 kHz, digitized using a Digidata 1320A Interface, sampled at 0.4 kHz, and analyzed sulfonylureas and some KATP channel openers. For exam- ple, a single residue (S1237 of SUR1) that is essential for using pClamp software (Axon Instruments). The slope conductance was measured by fitting a straight line to the current-voltage relation between Ϫ20 the selective inhibition of SUR1-containing channels by mV and Ϫ100mV; the average response to five consecutive ramps was calcu- tolbutamide has been identified in the cytoplasmic loop lated in each solution. In time-course experiments, macroscopic currents connecting TMs 15 and 16 (20). Two additional amino were recorded at a fixed potential of –50 mV, and the off-rate of nicorandil was acids in TM 17 (T1253 and L1249 of SUR2) have been measured by fitting an exponential function to the decay in current after removal of the drug. implicated in the SUR2-selective action of cromakalim Statistical analysis. Data are presented as means Ϯ 1 SE. Statistical analogs (22). However, homologous residues of SUR1 and significance was tested using analysis of variance (ANOVA) or t test (paired, SUR2 are also involved in drug binding, as diazoxide and unpaired, or single sample), as indicated in the text. the nonsulfonylurea moiety of glibenclamide exhibit similar potencies on KATP channels containing the different SUR subtypes (4,23). Domains important for the action of RESULTS ␤ nicorandil have not yet been identified. KATP channels Kir6.2/SUR1 ( -cell type), Kir6.2/SUR2A In this study, we investigated the effects of nicorandil on (cardiac type), and Kir6.2/SUR2B (smooth muscle type) recombinant KATP channels by expressing the different were heterologously expressed in Xenopus oocytes. In all channel subtypes in Xenopus oocytes. Currents were cases, the currents were small in cell-attached membrane recorded in inside-out membrane patches in response to patches because of the inhibitory effect of cytoplasmic the addition of nucleotides and drugs to the intracellular nucleotides but increased when the patches were excised membrane surface. Our results confirmed previous studies into nucleotide-free solution. that have shown that nicorandil action is dependent on the Nucleotide dependence of nicorandil activation. Fig- presence of intracellular nucleotides and further sug- ure 1 shows that nicorandil had very little effect on gested how this might be mediated by the NBDs of SURs. Kir6.2/SUR2A or Kir6.2/SUR2B currents in the absence of We used SUR chimeras to show that the last TM of SUR2 added nucleotide, as previously reported for native KATP is critical for nicorandil action, whereas the splice varia- channels (25). In this respect, therefore, the action of tion between SUR2A and SUR2B modifies the potency of nicorandil contrasts with that of pinacidil and its analog the drug by altering its off-rate. Finally, we showed that P1075, which are effective even in the absence of added sulfonylureas are not homogenous in their ability to inter- nucleotide (26,27). Nicorandil (100 ␮mol/l) activated both fere with nicorandil activation: although glibenclamide and types of KATP channel in the presence of ATP, and the glimepiride abolished the stimulatory effect of nicorandil, relative activation at different ATP concentrations was this was not the case for gliclazide. Our data therefore similar for both SUR2 isoforms (Table 1). However, in the suggest that for patients requiring both nicorandil and presence of high nucleotide concentrations, which them- sulfonylurea therapy, the most appropriate choice of sul- selves produced strong current inhibition, the absolute fonylurea might be one that, like gliclazide, preferentially increase in current induced by nicorandil was correspond- inhibits SUR1-type KATP channels. ingly smaller. The effect of nicorandil on Kir6.2/SUR2A currents in the presence of 300 ␮mol/l ATP plus 100 ␮mol/l ADP was not significantly different from that in 100 ␮mol/l RESEARCH DESIGN AND METHODS of ATP alone. The smaller relative activation of Kir6.2/ Molecular biology. Mouse Kir6.2 (Genbank D50581) (10), rat SUR1 (Gen- SUR2B currents in 300 ␮mol/l ATP plus 100 ␮mol/l ADP bank L40624) (14), rat SUR2A (Genbank D83598) (16), and rat SUR2B (Genbank D86038) (17) cDNAs were cloned into the pBF vector. Chimeras may have reflected the fact that the currents reached a between SUR1 and SUR2A were constructed as previously described (20). maximum level of activation. Capped mRNA was prepared using the mMESSAGE mMACHINE large scale in Effects of Walker mutations. The nucleotide require- vitro transcription kit (Ambion, Austin, TX) as previously described (24). ment for the action of other K-channel openers is deter- Oocyte collection. Female Xenopus laevis were anesthetized with MS222 (2 g/l added to the water). One ovary was removed via a mini-laparotomy, the mined by the NBDs of the SUR subunit (18,19,26–28). incision was sutured, and the animal was allowed to recover. Immature stage Mutation of the Walker A lysine residue in NBD1 or NBD2 V–VI oocytes were incubated for 60 min with 1.0 mg/ml collagenase (type A; of SUR1 impairs nucleotide binding and hydrolysis, as in

2254 DIABETES, VOL. 50, OCTOBER 2001 F. REIMANN, F.M. ASHCROFT, AND F.M. GRIBBLE

FIG. 1. Effects of nicorandil on Kir6.2/SUR2A and Kir6.2/SUR2B currents. Macroscopic currents recorded from oocytes coexpressing Kir6.2 and either SUR2A or SUR2B in response to a series of voltage ramps from –110 to ؉100 mV. ATP (300 or 100 ␮mol/l), ADP (100 ␮mol/l), and nicorandil (100 ␮mol/l) were added to the intracellular solution, as indicated by the bars. other ABC transporters (29–33). When the Walker A lysine domains were swapped between SUR1 and SUR2A, were was mutated in NBD1 of SUR2A or SUR2B, pinacidil acti- coexpressed with Kir6.2 in Xenopus oocytes. We tested vation became dependent on the presence of added nucle- the effect of nicorandil in the presence of 300 ␮mol/l ATP otide (26,27). However, mutation of the equivalent residue alone or 300 ␮mol/l ATP plus 100 ␮mol/l ADP, and ex- in NBD2 of SUR2A or SUR2B did not impair the amplitude pressed the results relative to the conductance in nucleo- of pinacidil activation. tide-free solution. We therefore investigated the role of the Walker A Figure 3 shows that nicorandil sensitivity could be lysines in the action of nicorandil. Figure 2 shows that introduced into SUR1 by transferring TMs 13–17 from mutating the Walker A lysine in NBD1 (K707A) of SUR2A SUR2 to SUR1 (chimera SUR1212). The last TM (TM 17) or SUR2B abolished nicorandil activation in the presence was crucial for this effect, as nicorandil activity was of 100 ␮mol/l ATP. By contrast, mutating the Walker A not transferred by swapping TMs 13–16 alone (chimera lysine in NBD2 (K1348A) of SUR2A or SUR2B impaired, SUR129). The reverse chimera, in which TMs 13–16 were but did not completely eliminate, the effect of nicorandil. swapped from SUR1 to SUR2 (chimera SUR219), was also Nicorandil action requires the last TM of SUR2. The action of nicorandil is speciﬁc for KATP channels containing one of the SUR2 isoforms, as Kir6.2/SUR1 currents were unaffected by 100 ␮mol/l nicorandil in solutions containing ATP alone (300 ␮mol/l) or ATP plus ADP (300 and 100 ␮mol/l, respectively) (Fig. 3). This difference between SUR1 and SUR2 enabled us to use a chimeric approach in identifying regions of SUR2 critical for the activity of nicorandil. Chimeric SURs, in which different

TABLE 1 Relative current activation by nicorandil in the presence of different nucleotide concentrations Kir6.2/SUR2A Kir6.2/SUR2B ␮ Ϯ Ϯ 100 mol/l ATP 4.5 0.5 (28)* 3.7 0.7 (18)* FIG. 2. Effect of nicorandil on KATP currents containing Walker A 300 ␮mol/l ATP 3.3 Ϯ 0.9 (7)* 5.1 Ϯ 0.6 (17)* mutations. Mean KATP conductance for Kir6.2 coexpressed with SUR2A 300 ␮mol/l ATP ϩ or SUR2B wild-type (WT), K707A, and K1348A, as indicated, recorded in the presence of 100 ␮mol/l ATP (f)or100␮mol/l ATP ؉ 100 ␮mol/l 100 ␮mol/l ADP 4.4 Ϯ 0.3 (6)* 1.3 Ϯ 0.1 (6) nicorandil (Ⅺ). The slope conductance (G) is expressed as a fraction of

the mean (Gc) of that obtained in control (nucleotide and drug-free) Data are means Ϯ SE (n). Current in the presence of nicorandil plus solution before and after exposure to ATP and nicorandil. The number nucleotide is expressed relative to the current in nucleotide alone. of patches is given above each bar. *P < 0.05; **P < 0.01; ***P < 0.001; *Not signiﬁcantly different by ANOVA. NS, nonsigniﬁcant (using a paired t test).

DIABETES, VOL. 50, OCTOBER 2001 2255 NICORANDIL ACTION ON KATP CHANNELS

FIG. 3. Effects of chimeras. A: Cartoon of chimeras. Grey regions indicate SUR1 and black regions indicate SUR2A. B: Macroscopic conductance recorded from patches excised from oocytes coexpressing Kir6.2 and SUR1, SUR2A, SUR2B, or indicated SUR chimera, in the presence of 300 ␮mol/l ATP (Ⅺ)or300␮mol/l ATP plus 100 ␮mol/l nicorandil (f). Mean conductance in the presence of the test solution (G) is expressed relative to the mean conductance in the control (nucleotide and drug-free) solution before and after the addition of agents (Gc). The dashed line indicates the relative conductance in control solution. The number of patches is given above each bar. C: Macroscopic conductance recorded from patches excised from oocytes coexpressing Kir6.2 and SUR1, SUR2A, SUR2B, or indicated SUR chimera in the presence of 300 ␮mol/l ATP plus 100 ␮mol/l ADP (Ⅺ)or300␮mol/l ATP plus 100 ␮mol/l ADP plus 100 ␮mol/l nicorandil (f). The slope conductance (G) is expressed as a fraction of the mean

(Gc) of that obtained in control (nucleotide and drug-free) solution before and after exposure to nucleotides and nicorandil. The dashed line indicates the relative conductance in control solution. The number of patches is given above each bar. *P < 0.05; **P < 0.01; ***P < 0.001; NS, nonsignificant (using a single-sample t test of the percent increase by nicorandil). activated by nicorandil, but to a lesser extent than wild- nicorandil off-rates (Fig. 4B). Mean data are given in Fig. type SUR2A. This was more clearly seen when ADP was 4C. added. These data suggest that regions within both TMs Effects of different sulfonylureas on nicorandil acti- 13–16 and TM 17 are essential for the full effect of nico- vation. We have previously shown that glibenclamide randil. Similar regions have been reported as essential for blocks both Kir6.2/SUR1 and Kir6.2/SUR2A channels with 3 the binding of another KATP channel opener, [ H]P1075 similar potency at a high-affinity site, whereas gliclazide (21), and for the activity of cromakalim analogs (22,34,35). blocks only KATP channels containing the SUR1 subunit Nicorandil is more effective on SUR2B than SUR2A. with high potency (23,37). Because many patients require Previous studies have shown that the reversal rates of concurrent therapy for angina and diabetes, we next in- pinacidil and P1075 activation are dramatically slowed by vestigated whether the different sulfonylureas interfere nucleotide interaction with the NBDs of SUR, and that this with nicorandil activation. effect is greater for Kir6.2/SUR2B than Kir6.2/SUR2A chan- Figure 5 compares the effect of nicorandil in the absence nels (27). It was postulated that the slower off-rate of and presence of three different sulfonylureas. The sulfo- P1075 accounts for the higher affinity of the drug for nylureas were tested at concentrations sufficient to SUR2B reported in binding studies, which are routinely saturate the high-affinity site on the SUR subunit. All the performed in the presence of ATP. The affinity of nicoran- solutions contained ATP, and the conductance in the dil was also greater for SUR2B than SUR2A when mea- presence of the drug was expressed relative to that in ATP 3 ϳ ϳ ␮ sured by the displacement of [ H]P1075 (Kd 8 and 19 alone. The stimulatory effect of 100 mol/l nicorandil on ␮mol/l, respectively) (36). Because SUR2A and SUR2B Kir6.2/SUR2A and Kir6.2/SUR2B channels was not altered differ only in their final 42 amino acids, a region that does by the presence of a therapeutic concentration of glicla- not include the domains that have been implicated in zide (10 ␮mol/l) but was severely impaired by both glib- P1075 binding (21), we investigated whether the different enclamide (100 nmol/l) and glimepiride (100 nmol/l). binding affinities of nicorandil might be associated with differences in the off-rate of the drug. Figure 4A shows that in the presence of 100 ␮mol/l ATP, DISCUSSION the off-rate of nicorandil was considerably slower for In this study, we identified regions of the sulfonylurea Kir6.2/SUR2B currents (time constant, ␶ϳ25 s) than for receptor SUR2 that are responsible for the tissue-specific Kir6.2/SUR2A currents (␶ϳ4 s). This would be consistent effects of nicorandil, and showed that the greater potency with the higher affinity of the drug for SUR2B. The further of nicorandil on Kir6.2/SUR2B (smooth muscle type) as addition of 100 ␮mol/l ADP did not significantly alter the compared with Kir6.2/SUR2A (cardiac muscle type) chan-

2256 DIABETES, VOL. 50, OCTOBER 2001 F. REIMANN, F.M. ASHCROFT, AND F.M. GRIBBLE

underlying mechanism to explain the nucleotide depen- dency of nicorandil action. Previous studies have con- cluded that ATP remains bound at NBD1 for several minutes after patch excision and that this is prevented by mutation of the Walker A lysine (26,39). In the current study, mutating the Walker A lysine in NBD1 abolished nicorandil activation, even in the presence of added ATP, suggesting that nucleotide binding at this NBD is crucial for activity of the drug. Because nicorandil was ineffective in the absence of added nucleotide, even when applied immediately after patch excision, it appears that ATP binding at NBD1 is not sufficient to support nicorandil activation. Mutation of the Walker A lysine in NBD2 impaired but did not completely abolish nicorandil activation. Other studies have shown that NBD2 of SUR2 is able to hydro- lyze ATP and that mutating the Walker A lysine to alanine reduced, but did not completely prevent, nucleotide hydrolysis at NBD2 (33,40). Our results therefore suggest that binding and/or hydrolysis of nucleotide at NBD2, in addition to that at NBD1, is required for nicorandil action. The nucleotide requirements of nicorandil differ from those of pinacidil, which appears to be effective even when nucleotide is bound only at NBD1 (26). In this respect, nicorandil action more closely resembles that of diazoxide, which, in the case of Kir6.2/SUR2A currents, is also enhanced in the presence of added ADP (41). Measuring the nicorandil off-rate. The splice variation between SUR2A and SUR2B alters only the COOH-terminal 42 amino acids and results in channels that exhibit different responses to nucleotides (17,27,42). The principal FIG. 4. Effect of nucleotides on the time course of nicorandil reversal. effect of the splice variation on the action of nicorandil A and B: Kir6.2/SUR2A and Kir6.2/SUR2B currents recorded at a holding potential of –50 mV. Nicorandil and MgATP (100 ␮mol/l each; was to modify the drug off-rate. Previous studies have A) and nicorandil, MgATP, and MgADP (100 ␮mol/l each; B) were shown that pinacidil activation of Kir6.2/SUR2B currents added as indicated by the horizontal bars. The dashed line indicates the zero current level. C: Mean time constants for the recovery from reverses more slowly than that of Kir6.2/SUR2A currents, nicorandil activation. The recovery from drug activation in the pres- and that this rate is dependent on nucleotide interactions ence or absence of ADP was fitted with a single exponential function of with both NBDs (26,27). The different nicorandil off-rates ␶ ؉ ؍ the form I Io exp – (t/ ) c (where I is the current at time t, Io is the and ␶ is the time constant). The number of patches is measured for channels containing SUR2A and SUR2B may ,0 ؍ current at t given above the bars. No significant difference (NS) was observed when therefore be explained by an interaction between nucleo- the time constants in the absence and presence of ADP for a given channel type were compared using an unpaired t test. tide binding at the NBDs and a drug-binding site in the COOH-terminal group of TMs of SUR2. The slower nicorandil off-rate recorded for Kir6.2/SUR2B compared with nels may, at least in part, be attributable to a slower Kir6.2/SUR2A currents is reflected in the greater affinity of reversal of channel activation when the drug is removed. SUR2B for nicorandil measured in binding studies (36). Also important is that we demonstrated that the ability The affinities of SUR2A and SUR2B for nicorandil, of nicorandil to open Kir6.2/SUR2A and Kir6.2/SUR2B measured in binding studies by the displacement of channels is unaffected by concurrent application of gli- [3H]P1075, were 19 and 8 ␮mol/l, respectively (36). Similar clazide but is severely impaired by both glibenclamide values have been reported for native cardiac and smooth and glimepiride. muscle tissues (36). Although the potency of nicorandil on Nucleotide requirement for nicorandil action. The Kir6.2/SUR2B channels expressed in HEK293 cells was ␮ effects of several sulfonylureas and KATP channel openers also in this range (EC50,10 mol/l), this was not the case Ͼ ␮ are modified by the interaction of Mg nucleotides with the for Kir6.2/SUR2A channels (EC50 500 mol/l) (43). Our NBDs of SURs (18,19,26–28,38). Nicorandil is no excep- results are more compatible with those obtained in bind- tion, and its stimulatory effect on Kir6.2/SUR2A and Kir6.2/ ing studies, as we did not observe any further increase in SUR2B channels requires the presence of ATP at the the response of Kir6.2/SUR2A currents to nicorandil when intracellular membrane surface. Previous studies on na- the drug concentration was increased from 100 ␮mol/l to 1 tive KATP channels in cardiac muscle have suggested that mmol/l (data not shown). We also found that the splice nicorandil action is also dependent on the presence of variation between SUR2A and SUR2B altered the nicoran- MgADP (25). Our data show that although ADP enhances dil off-rate approximately fivefold, consistent with the the absolute current increase induced by the drug, ATP is moderate difference in the affinity between SUR2A and sufficient to support nicorandil action. SUR2B found in binding studies. A possible explanation The effects of the Walker A mutations suggest an for the greater EC50 of nicorandil measured for Kir6.2/

DIABETES, VOL. 50, OCTOBER 2001 2257 NICORANDIL ACTION ON KATP CHANNELS

FIG. 5. Interference of sulfonylurea and nicorandil action. Macroscopic: currents were recorded from oocytes coexpressing Kir6.2 and either SUR2A or SUR2B. Nicorandil (100 ␮mol/l) was applied in the presence of ATP (100 ␮mol/l, but 300 ␮mol/l in the case of Kir6.2/SUR2B currents with glimepiride). After activation reached steady state, gliclazide (10 ␮mol/l), glibenclamide (100 nmol/l), or glimepiride (100 nmol/l) was also added. The mean conductance in the presence of nicorandil or nicorandil plus sulfonylurea (G) is expressed relative to the mean conductance measured in the presence of ATP alone (GATP). The conductance in the presence of ATP (and ATP plus nicorandil) is given as the average of conductances measured before and after application of the sulfonylurea. The number of patches is given above each bar. *P < 0.01; **P < 0.001; NS, nonsigniﬁcant (using a paired t test to compare the conductance in ATP plus nicorandil and ATP plus nicorandil plus sulfonylurea).

SUR2A currents in HEK293 cells is the presence of the that glibenclamide displaces the binding of [3H]P1075 from high (3 mmol/l) intracellular ATP concentration used in SUR2 (36). these experiments (43), as our data showed that nicorandil Conclusion. The differential potency of nicorandil be- activation is modulated by the nucleotide concentration. tween tissues can be explained by sequence variations in The intracellular solution in whole cell experiments may TMs 13–17 and the last 42 amino acids of the SUR. The also contain other factors that modulate channel activity. effect of nicorandil is impaired by concomitant adminis- Identification of domains critical for nicorandil ac- tration of glibenclamide or glimepiride, but is not affected by gliclazide. The results thus clearly indicate that care tivity. The specificity of nicorandil for KATP channels containing SUR2 isoforms enabled us to use a chimeric should be taken in clinical practice when prescribing approach to identifying regions of SURs important for combinations of KATP channel openers and sulfonylureas, activity of the drug. We showed that the final TM (TM 17) because drugs like glibenclamide may impair the clinical of SUR2 is essential for nicorandil activity but that the effectiveness of nicorandil. preceding four TMs are also required to confer a full drug response. The chimeric approach has been previously ACKNOWLEDGMENTS used to show that the COOH-terminal group of TMs is also This work was supported by the Wellcome Trust. involved in the binding of radiolabeled glibenclamide to We thank Dr. M. Conway (Kilkenny, Ireland) for discus- SUR1 and of P1075 to SUR2 (20,21). The final TM helix was sion and Dr. S. Seino (Chiba University, Chiba, Japan) for shown to be crucial for both [3H]P1075 binding and SUR2A. cromakalim activity (21,22,35), although it was not required for pinacidil activity (12). REFERENCES Interaction between nicorandil and sulfonylureas. 1. Patel DJ, Purcell HJ, Fox KM: Cardioprotection by opening of the K(ATP) Our results clearly showed that nicorandil action on channel in unstable angina: is this a clinical manifestation of myocardial preconditioning? Results of a randomized study with nicorandil: CESAR 2 Kir6.2/SUR2A and Kir6.2/SUR2B channels is severely im- investigation: clinical European studies in angina and revascularization. paired by glibenclamide and glimepiride but is unaffected Eur Heart J 20:51–57, 1999 by gliclazide. The lack of interference by gliclazide is not 2. Cogolludo AL, Perez-Vizcaino F, Fajardo S, Ibarra M, Tamargo J: Effects of surprising, as this drug blocks ␤-cell but not cardiac or nicorandil as compared to mixtures of sodium nitroprusside and levcro- smooth muscle types of K channel (37). In contrast, makalim in isolated rat aorta. Br J Pharmacol 126:1025–1033, 1999 ATP 3. Aguilar-Bryan L, Bryan J: Molecular biology of ATP-sensitive potassium glibenclamide and glimepiride block all three types of channels. Endocr Rev 2:101–135, 1999 ϩ KATP channels with high affinity (23,44). Although gliben- 4. Ashcroft FM, Gribble FM: ATP-sensitive K channels in health and disease. clamide and glimepiride can reverse the stimulatory effect Diabetologia 42:903–919, 1999 of nicorandil, we cannot distinguish whether this is be- 5. Rorsman P: The pancreatic beta-cell as a fuel sensor: an electrophysiolo- gist’s viewpoint. Diabetologia 40:487–495, 1997 cause the drugs impair nicorandil binding or interfere with 6. Quayle JM, Nelson MT, Standen NB: ATP-sensitive and inwardly-rectifying the transduction of nicorandil binding into channel open- potassium channels in smooth muscle. Physiol Rev 77:1165–1232, 1997 ing. The former idea, however, is supported by the fact 7. Nichols CG, Lederer WJ: Adenosine triphosphate-sensitive potassium

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