Sulfonylurea Stimulation of Secretion Peter Proks,1 Frank Reimann,2 Nick Green,1 Fiona Gribble,2 and Frances Ashcroft1

Sulfonylureas are widely used to treat smooth, and , and some brain neurones. In because they stimulate insulin secretion from pancre- all these tissues, opening of KATP channels in response to atic ␤-cells. They primarily act by binding to the SUR metabolic stress leads to inhibition of electrical activity. subunit of the ATP-sensitive potassium (KATP) channel Thus they are involved in the response to both cardiac and and inducing channel closure. However, the channel is cerebral ischemia (2). They are also important in neuronal still able to open to a limited extent when the is regulation of glucose homeostasis (3), seizure protection bound, so that high-affinity inhibition is not complete, even at saturating drug concentrations. (4), and the control of vascular tone (and, thereby, blood pressure) (5). KATP channels are also found in cardiac, skeletal, and smooth muscle, but in these tissues are composed of The KATP channel is a hetero-octameric complex of two different SUR subunits that confer different drug different types of protein subunits: an inwardly rectifying sensitivities. Thus and block Kϩ channel, Kir6.x, and a sulfonylurea receptor, SUR (6,7). ␤ ϩ channels containing SUR1 ( -cell type), but not SUR2 Kir6.x belongs to the family of inwardly rectifying K (cardiac, smooth muscle types), whereas , (Kir) channels and assembles as a tetramer to form the , , and block both types of channels. This difference has been exploited channel pore (Fig. 1A). Binding of ATP to the intracellular to determine residues contributing to the sulfonyl- domains of this subunit produces channel inhibition (8). urea-binding site. Sulfonylurea block is decreased by SUR is a member of the ABC transporter family, with 17 mutations or agents (e.g., phosphatidylinositol bisphos- transmembrane helices (TMs), arranged as one group of 5 phate) that increase KATP channel open probability. We TMs, and two repeats each of 6 TMs followed by a large now propose a kinetic model that explains this effect in cytosolic loop containing consensus sequences for nucle- terms of changes in the channel open probability and in the transduction between the drug-binding site and the otide binding and hydrolysis. Interaction of Mg nucleotides channel gate. We also clarify the mechanism by which with the nucleotide-binding domains (NBDs) mediates acti- MgADP produces an apparent increase of sulfonylurea vation of the KATP channel (9–12). SUR also endows Kir6.2 efficacy on channels containing SUR1 (but not SUR2). with sensitivity to certain , such as the inhibitory Diabetes 51 (Suppl. 3):S368–S376, 2002 and the stimulatory Kϩ channel openers (6,7). More than one isoform exists for both Kir6.x (Kir6.1, Kir6.2) and SUR (SUR1, SUR2A, SUR2B). In most tissues, ulfonylureas stimulate insulin secretion from Kir6.2 serves as the pore-forming subunit, but it associates pancreatic ␤-cells and are widely used to treat with different SUR subunits; for example, it associates type 2 diabetes. Their principal target is the ATP-sensitive potassium (K ) channel, which with SUR1 in the and brain; SUR2A in heart and S ATP skeletal muscle; and SUR2B in brain and smooth muscle plays a major role in controlling the ␤-cell membrane (13–15). In vascular smooth muscle, the KATP channel is potential. Inhibition of KATP channels by glucose or sulfo- nylureas causes depolarization of the ␤-cell membrane; in composed of Kir6.1 in association with SUR2B. Variation 2ϩ turn, this triggers the opening of voltage-gated Ca chan- in the subunit composition of the KATP channel accounts 2ϩ 2ϩ nels, eliciting Ca influx and a rise in intracellular Ca for the different metabolic and drug sensitivities of KATP which stimulates the exocytosis of insulin-containing se- channels in different cells. cretory granules (1). KATP channels are also found at high Inhibitors of KATP channel activity fall into two groups: density in a variety of other cell types, including cardiac, those that interact with Kir6.2 and those that interact with SUR. Imidazolines (e.g., phentolamine, cibenzoline) and antimalarials (e.g., quinine, mefloquine) block K chan- From the 1University Laboratory of , Oxford University, Oxford, ATP U.K., and the 2Department of Clinical Biochemistry, , nels by binding to Kir6.2 (16–18). In contrast, sulfonyl- Addenbrooke’s Hospital, Cambridge, U.K. ureas (e.g., tolbutamide, gliclazide, glimepiride), and Address correspondence and reprint requests to Prof. Frances M. Ashcroft, University Laboratory of Physiology, Parks Road, Oxford OX1 3PT, U.K. benzamido derivatives (e.g., meglitinide) close KATP chan- E-mail: [email protected]. nels by binding with high affinity to SUR (19–21). Sulfo- Received for publication 12 March 2002 and accepted in revised form 8 May 2002. nylureas also interact with Kir6.2, but with low affinity. All

IC50, half-maximal inhibitory concentration value; KATP channel, ATP- drugs that block K channels stimulate insulin secretion, ϩ ATP sensitive ; Kir channel, inwardly rectifying K channel; but only those that interact with the SUR subunit are used NBD, nucleotide-binding domain; PO, open probability; SUR, sulfonylurea receptor; TM, transmembrane helix. therapeutically to treat type 2 diabetes. Thus, in this P.P., F.G., and F.A. receive honoraria from Servier for speaking engage- review, we focus on how studies of recombinant KATP ments. F.A. is a paid consultant for NovoNordisk. The symposium and the publication of this article have been made possible channels have helped to elucidate the molecular mecha- by an unrestricted educational grant from Servier, Paris. nism of sulfonylurea inhibition.

S368 DIABETES, VOL. 51, SUPPLEMENT 3, DECEMBER 2002 P. PROKS AND ASSOCIATES

inhibit the Kir6.2 subunit are much higher than those found in the plasma of patients treated with the drugs. Thus, in this review, we focus on the high-affinity drug- binding site.

STOICHIOMETRY

Because four SUR subunits contribute to the KATP channel complex, the question arises as to how many subunits must bind drug to close the channel. This topic is currently controversial. Do¨ rschner et al. (22) found that the affinity of sulfonylurea binding to Kir6.2/SUR1 or Kir6.2/SUR2B channels is between three- and sevenfold lower than their potency on channel inhibition (Table 1). Consequently, they concluded that occupation of a single binding site is sufficient to induce channel closure. In contrast, Russ et al. (23) found that the concentration-response relation for [3H]glibenclamide binding to Kir6.1/SUR2B lies to the left of that for glibenclamide block of channel activity, so that the Kd is sevenfold lower than the half-maximal inhibitory concentration on channel activity (IC50) (Table 1). They therefore suggested that glibenclamide can bind indepen- dently to four identical sites on the channel and that occupation of all four sites is required for channel closure (23). Interestingly, the different proposed stoichiometries arise from the different binding affinities measured by these groups (Table 1). It is not clear why the affinities were found to be so different, but possible confounding factors include the nature of the Kir6.x subunit (Kir6.2 or FIG. 1. A: Schematic of the transmembrane topology of Kir6.2 and SUR1. B: Mean relation between gliclazide concentration and inhibi- Kir6.1) and the nucleotide concentrations used. The con- -flicting results suggest that conclusions based on compar ؍ tion of the Kir6.2/SUR1 current, measured in inside-out patches (n 5–8). The conductance in the presence of gliclazide (G) is expressed as ing binding affinities with potencies of channel inhibition a fraction of its amplitude in the absence of the drug (Gc). The line is .؉ ؊ ؉ h1 ؋ ؉ may not be reliable ؍ fit to (equation 2) G/Gc (L (1 L)/(1 ([S]/IC50–1) )) (1/(1 ؍ ؍ ؍ h2 ([S]/IC50–2) )), where IC50–1 51 nmol/l, h1 1.0, IC50–2 3.1 mmol/l, ;S] is the concentration of sulphonylurea] ;(19) 0.40 ؍ and L ,1 ؍ h2

IC50–1 and IC50–2 are the half-maximal inhibitory concentrations at the SULFONYLUREA-BOUND CHANNELS CAN STILL OPEN high- and low-affinity sites, respectively; h1 and h2 are the Hill coefficients for the high- and low-affinity sites, respectively; and L is In the absence of added nucleotides, high-affinity inhibi- the fraction of current that remains unblocked when all high-affinity tion of the KATP current by sulfonylureas is not complete, sites are occupied. but reaches a maximum of 60–80% (Fig. 1B). There are several possible explanations for this finding. One possi- SULFONYLUREAS BLOCK KATP CHANNELS AT TWO bility is that some percentage of the channels are in a state SITES in which they do not bind drug with high affinity. An When measured in excised patches in the absence of alternative possibility is that all channels bind drug with nucleotides, sulfonylurea block of KATP channels is best high affinity, but they are still able to open when drug is described by a two-site model (Fig. 1B). The low-affinity bound, albeit with lower probability. Single-channel cur- site is independent of SUR, as a similar block is seen when rents recorded at sulfonylurea concentrations that satu- Kir6.2 is expressed in the absence of SUR (20). The rate the high-affinity site (but do not interact with Kir6.2) high-affinity site lies on SUR, as it is only present when can be used to distinguish between these possibilities. If SUR is coexpressed with Kir6.2. The presence of both low- some channels exist in a low-affinity state and others in a and high-affinity sites for sulfonylureas on the KATP chan- high-affinity state, and transitions between these states are nel introduces important considerations when interpreting relatively slow, there should be two kinetically distinct experiments with these drugs. First, at high concentra- populations of channels—one with a reduced open prob- tions these drugs may mediate their effects via Kir6.2, and ability (PO) and another with kinetics similar to that channel inhibition therefore cannot be taken to support observed in the absence of drug. On the other hand, if the the idea that they interact with the sulfonylurea receptor. reason for incomplete block by sulfonylureas is because Second, as discussed below, different SURs endow the the channel can still open when the drug is bound at the KATP channel with variable sensitivity to low concentra- high-affinity site(s), then there will be only one type of tions of sulfonylureas, a property that can be used to single-channel kinetics. Under experimental conditions, distinguish the type of sulfonylurea receptor expressed in only a single type of kinetic behavior has been observed in different tissues. Finally, to isolate effects mediated by the the presence of sulfonylureas (24; P.P., F.M.A., unpub- high-affinity site, it is best to use a drug that shows a wide lished observations), supporting the idea that the channel separation between its low- and high-affinity actions, such can still open when drug is bound. Because it is thought as gliclazide or glibenclamide. The low-affinity site is of no that each SUR1 monomer binds drug independently, all clinical relevance, because the concentrations required to four binding sites should be occupied at saturating drug

DIABETES, VOL. 51, SUPPLEMENT 3, DECEMBER 2002 S369 SULFONYLUREA STIMULATION OF INSULIN SECRETION

TABLE 1

Comparison of the IC50 for KATP channel inhibition by various sulfonylureas with the Kd for drug binding SUR1 SUR2A SUR2B

IC50 Kd IC50 Kd IC50 Kd (current) (binding) (current) (binding) (current) (binding) Glibenclamide (nmol/l) 0.13 †[22] Ϸ0.7 *,†[22] 45 †[22] Ϸ300 (*,†)[22] 42 †[22] Ϸ350 *,(†,‡)[22] 4.2 †[21] 7.1 *[32] 27 †[21] 1,200 *,¶[14] 43 ‡[23] 6.3 ‡, 32 *[23] 1.5 ‡, 1.8*[41] 166 †[36] 0.2 {†}[55] (nmol/l) 3.8 †[22] Ϸ17 *,†[22] Ϸ6,000 (*,†)[22] 1,200 †[22] Ϸ6,000 *,(†,‡)[22] Glimepiride (nmol/l) 3.0 †[34] — 5.4 †[34] 7.3 †[34] Tolbutamide (␮mol/l) 4.9 †[22] Ϸ29 *,†[22] 85 †[22] Ϸ270 (*,†)[22] 88 †[22] Ϸ280 *,(†,‡)[22] 5.4 †[21] 140 *[32] Ն nmol/l †[21,54] 10.5 † [52] 9 {†}[55] Ͼ1 mmol/l [55] Gliclazide (nmol/l) 50 †[21] — Ͼ100 ␮mol/l †[21] Meglitinide (␮mol/l) 1.2 †[22] Ϸ7 *[22] 0.53 †[21] Ϸ7 (*)[22] 1.6 †[22] Ϸ8 (*)[22] 0.26 †[21] Mitiglinide (nmol/l) Ϸ60 †[32] 280 *[32] Ͼ 100 ␮mol/l †[32] Ͼ100 ␮mol/l †[32] 3.8 †[33] 3200 †[33] 4,600 †[33] Repaglinide (nmol/l) 5.6 †[53] 1,600 *[32] 2.2 †[53] 2.0 †[53] 21 †[55] 0.6 {†}[55] (␮mol/l) — 8 *[32] 0.8 †[55] 0.2 {†}[55] Only studies on recombinant SUR (indicated) are listed. * SURx expressed in the absence of Kir6.2; †SURx coexpressed with Kir6.2; ‡SURx 3 3 coexpressed with Kir6.1. The Kd was measured by displacement of [ H] glibenclamide or, if no data were available, of [ H]P1075 (indicated by round brackets) or iodo-glibenclamide (¶)or[3H]repaglinide (indicated by curly brackets). Numbers in square brackets indicate references. concentrations. Thus it appears that the KATP channel can This model explains the incomplete block by sulfonyl- still open, even when each of its four SUR subunits has ureas, because, even when drug is bound, the channel bound sulfonylurea. does not inevitably enter the closed states C2B and CT. The maximal extent of block at the high-affinity site can Entry into these closed states is not, therefore, directly be modulated by mutations or truncations in either Kir6.2 proportional to the drug concentration, and channel open- or SUR (25–28), or by membrane phospholipids such as ing is still possible, even at very high concentrations of the phosphatidylinositol bisphosphate (PIP2) (29). All these drug. modulators share the common property that they increase The concentration-response curve for the high-affinity the channel PO (25–27,30,31). We therefore simulated the site is described by the following: effect of PO on sulfonylurea block using the kinetic scheme h h given in Fig. 2B and rate constants calculated from single G/Gc ϭ ͓1 ϩ L ϫ ͓͑S͔/IC50͒ ͔/͓1 ϩ ͓͑S͔/IC50͒ ͔ (1) Kir6.2/SUR1 channel kinetics recorded in the absence and presence of gliclazide. where G is the conductance in the presence of the drug, Gc As Fig. 2A shows, KATP channels exhibit bursts of is the conductance in the absence of drug, L is the fraction openings separated by long closed periods. Gliclazide of current that remains unblocked in the presence of a reduces the duration of the bursts of openings and in- saturating concentration of sulfonylurea, h is the Hill creases the long closed intervals. The burst can be de- coefficient, and IC50 is the drug concentration ([S]) at scribed by one open (O) and one short closed (C1) state, which the fraction of the current inhibitable with high and the long closings between bursts can be lumped affinity is blocked by 50%. The kinetic scheme depicted in together into a single long closed state (C2). We assume Fig. 2B allowed us to derive analytical equations that could that all these states can bind sulfonylurea, so that the be used to calculate the relation between the channel PO channel can exist in both open and closed states when and L (Fig. 2C), and between PO and IC50 (Fig. 2D). It is sulfonylurea is bound. We further assume that when drug clear that both L and IC50 increase with PO. The value of L is bound to SUR1, this either has no effect on channel reaches unity when the PO is maximal, which, in the case ϳ activity (bound but untransduced states) or results in a of the KATP channel, is 0.85, so that the model predicts conformational change that affects the channel kinetics that there will be no channel inhibition when PO is (bound and transduced states). In the former case, the maximal. This has in fact been observed for channels channel flickers between states OB,C1B, and C2B with the containing the mutation C166S, which have a PO close to same rate constants as in the absence of drug. In the maximum (27). As predicted, reducing the PO of Kir6.2- second case, the channel undergoes a conformational C166S/SUR1 channels with ATP partially restored sulfo- change that either results in channel closure (the OB to CT nylurea sensitivity (25). The predicted effect of PO on the transition) or stabilization of the closed state (C2B to C2T). concentration-response curve is illustrated in Fig. 2E, and We made the simplifying assumption that the major con- resembles that found experimentally with increasing con- tribution to the channel PO in the presence of sulfonylurea centrations of PIP2, which also increases PO (29). ⌬ is produced by states with only a single drug molecule Deletion of the NH2-terminus of Kir6.2 (Kir6.2 N) re- bound (i.e., only one SUR in the tetramer is occupied). sults in an increase in the channel PO and a concomitant

S370 DIABETES, VOL. 51, SUPPLEMENT 3, DECEMBER 2002 P. PROKS AND ASSOCIATES

reduction in tolbutamide block when Kir6.2⌬Nisco- expressed with SUR1 (25). The reduction in block pro- duced by deletion of up to nine residues can be accounted for simply by the increase in PO. However, deletion of the first fourteen NH2-terminal residues produced a complete loss of tolbutamide inhibition, an observation that cannot be explained solely by an increase in PO. In addition, decreasing PO with ATP was unable to restore the tolbut- amide sensitivity of this deletion mutant (25). Thus it appears that NH2-terminal residues may be involved in transducing sulfonylurea binding to SUR1 into closure of the Kir6.2 pore.

SPECIFICITY OF HIGH-AFFINITY SULFONYLUREA BLOCK

Studies of cloned channels have revealed that certain KATP channel inhibitors (tolbutamide, gliclazide, and mitiglin- ide) (19–21,32,33) block KATP channels containing SUR1, but not SUR2A or SUR2B, subunits with high affinity. In contrast, meglitinide blocks all three types of cloned KATP channel with similar affinities (21). This finding has been interpreted to indicate that sulfonylureas resembling tol- butamide in structure interact with a binding site that is specific to SUR1, whereas benzamido compounds and their derivatives (like meglitinide) interact with a site that is common to all SUR subtypes. Glibenclamide and glimepiride, which contain both sulfonylurea and nonsul- fonylurea moieties and block both SUR1- and SUR2- containing channels, are postulated to interact with both sites on SUR1, but only a single (benzamido-derivative) site on SUR2 (21,34). The binding affinities and inhibitory potencies of these drugs for the high-affinity sites of different types of KATP channels are illustrated in Table 1.

LOCATION OF THE SULFONYLUREA BINDING SITE ON SUR A single residue within the COOH-terminal set of trans- membrane domains accounts for the difference in tolbut- amide sensitivity of SUR1- and SUR2-containing channels. Mutation of serine 1237 in the cytoplasmic loop between TMs 15 and 16 to tyrosine abolishes the high-affinity block of Kir6.2/SUR1 channels by tolbutamide (35), mitiglinide (33), and nateglinide (55). It is likely that this residue also interacts with the sulfonylurea moiety of drugs such as glibenclamide and glimepiride. This is suggested by the fact that glibenclamide block of Kir6.2/SUR1, or native

؋ ؉␬؋ ؉␬؋ ؉␬؋␬ ؋ ؋ ؉ ؍ Pos PO (1 [S])/(1 [S] TO PO [S] ␬ ؋␬؋␬ ؋ ؋ 2 TC PO [S]), ؍ ␬ ؍ ␬ ؍␬ ؍ ␬ .where 2 k2/k-2; kB/k-B; TO kTO/k؊TO, and TC kTC/k-TC Equating Pos/PO with I/IO leads to ؉␭؋ ؊ ؍ ␬؋؍ FIG. 2. A: Single-channel currents recorded at ؊60 mV in the absence L IC50 1/[1 (Pmax PO)] (top) or presence (bottom) of a saturating concentration of gliclazide ␬؋ ؉␭؋ ؊ ؍ ␮mol/l). B: Kinetic scheme for K channel gating in the presence 10) ATP where IC50 1/{ [1 (Pmax PO)]}, of sulfonylureas. Agents that increase PO usually mediate their effects ؋ ؊⌬ ؋ ؉ ؊⌬ ؋ ؍␭ ,(by increasing the burst duration and reducing the frequency of the k؊2 exp( GS/RT) [1 exp( GC2T,CT/RT)]/(k ؊ TO Pmax interburst closed states (i.e., they reduce k2). The relation between k2, ؊ ␭ ␬ ؍ ⌬ PO, and IC50 was derived as described previously (27). When drug is where GC2T,CT GC2T GCT. For these plots, the values of and bound but untransduced, the channel can exist in states OB,C1B,or were obtained from the IC50 and L (0.4) measured for gliclazide block ؍ C2B. The closed states that result following the conformational change of Kir6.2/SUR1 currents (19), assuming a PO of 0.32 (25). IC50 50 permitted by drug binding are given by CT and CT2. O, open state; C1, nmol/l for the high-affinity site and 3 mmol/l for the low-affinity site. E: intraburst closed state; C2, lumped interburst closed states; gray box, Relation between the inhibition of the KATP current and drug concen- burst state. C and D: Relation between channel PO and the fraction of tration at various values of PO, calculated using the kinetic scheme current that remains unblocked in the presence of a saturating con- given in B and the equations in C and D. The low-affinity block is centration of sulfonylurea (L)(C)orIC50 (D). The PO in the presence assumed to be unchanged by alterations in channel PO, as found of sulfonylurea predicted by the kinetic scheme in B is given by experimentally (25). The curves are fit to equation 2.

DIABETES, VOL. 51, SUPPLEMENT 3, DECEMBER 2002 S371 SULFONYLUREA STIMULATION OF INSULIN SECRETION

␤ -cell KATP channels, is not easily reversed in electrophys- result of the different experimental conditions used by the iological recordings, whereas glibenclamide block is rap- groups or different fractions of uncoupled SUR subunits in idly reversed when SUR1 contains the S1237Y mutation. the presence of Kir6.x. It is clear that additional experi- Kir6.2/SUR2 channels, which already possess a tyrosine at ments are needed to resolve this controversy. this position, also show reversible block by glibenclamide and glimepiride. In addition, glibenclamide binding to MODULATION OF SULFONYLUREA BLOCK BY PIP2 SUR1 is greatly decreased by the S1237Y mutation (35), The phospholipid PIP2 reduces the maximal block pro- whereas glibenclamide binding to SUR2B is enhanced by duced by saturating concentrations of tolbutamide (28) the reverse mutation (36,37). Thus S1237 must contribute and glibenclamide (29). It also increases the IC of the to the binding site for sulfonylureas on SUR1. It is possible 50 drug-sensitive fraction of channels. The effects of PIP2 are that the bulkier volume of the tyrosine side chain, as attenuated by the mutation R176A in Kir6.2 (29), which is compared to serine, sterically hinders the binding of the believed to reduce PIP2 binding to this subunit. Further- sulfonylurea moiety, thereby accounting for its inability to more, negatively charged lipids reduce glibenclamide interact with SUR2. block with similar potency, whereas neutral or weakly The fact that glibenclamide blocks Kir6.2/SUR1-S1237Y charged lipids have no effect. As described above, the channels indicates that residues other than S1237 are effects of PIP on sulfonylurea sensitivity can be largely critical for binding of this drug. Likewise, the block of 2 accounted for by the increase in channel PO produced by Kir6.2/SUR1 by meglitinide and repaglinide, which do not the phospholipid. However, it is also possible that PIP possess a sulfonylurea moiety, is unaltered by the S1237Y 2 (like the NH2-terminal deletions) has additional effects on mutation (35,53), suggesting that these drugs do not inter- sulfonylurea block that are masked by the effect on the act with this residue. Recent studies have shown that two channel PO. nonadjacent cytosolic loops of SUR1, linking TMs 5 and 6 The amount of PIP in the membrane can vary as a and TMs 15 and 16, are critical for [3H]glibenclamide 2 result of altered phospholipid metabolism. Thus PIP2 is binding (38). Because the TM 15–16 loop contains S1237, expected to fall in response to ischemia (because falling this raises the possibility that the TM 5–6 loop may ATP levels reduce its generation from PIP by phosphory- interact with benzamido compounds and their derivatives. lation) and increase when phosphatidylinositol kinases are Interestingly, the drug-binding site of the related ABC stimulated. This means that PIP2 regulation of gliben- transporter MRP1 also involves sites in both NH2- and clamide block will also be subject to regulation. COOH-terminal parts of the protein, and the TM 5–6 loop is essential for high-affinity drug binding (39). These data do not imply that only residue S1237 in the MODULATION OF SULFONYLUREA BLOCK BY MG TM 15–16 loop and unidentified residues in the TM 5–6 NUCLEOTIDES loop interact with sulfonylureas. However, it is clear that As Fig. 1B shows, in the excised patch, the concentration- these residues are critical for high-affinity interactions. response curve for KATP channel inhibition by sulfonyl- This suggests that despite their separate location in the ureas is best fit by assuming both high- and low-affinity primary structure of SUR, the cytosolic loops between sites, with a maximum of 60–70% block at the high-affinity TMs 5–6 and TMs 15–16 lie in close proximity in the site. This is not the case when sulfonylurea block is three-dimensional structure of the channel. The maximal measured in whole cells. Remarkably, sulfonylureas pro- distance between them must be the length of the gliben- duce complete (Ͼ90%) high-affinity inhibition of both the ␤ clamide molecule (17 Å), but because the molecule is very native -cell KATP channel (42,43) and its cloned counter- flexible, they could be much closer. The location of the part, Kir6.2/SUR1 (44), when measured in the whole-cell sulfonylurea binding site(s) within the cytosolic loops of configuration. This difference may arise because experi- SUR is consistent with earlier studies suggesting an intra- ments on recombinant KATP channels have largely been cellular site of action for sulfonylureas (40). carried out in excised patches, whereas experiments on native KATP channels have mainly been carried out in whole cells. We therefore compared the concentration- INTERACTIONS BETWEEN Kir6.2 AND SUR response curve for gliclazide block of Kir6.2/SUR1 cur- It is well established that the sulfonylurea receptor can rents in the intact cell with that in the inside-out patch. modulate the activity of Kir6.2, because binding of sulfo- Figure 3A and B shows that there was no significant nylureas or MgADP to SUR results in a decrease or difference in the gliclazide concentration producing half- increase of channel activity, respectively. There is growing maximal block of Kir6.2/SUR1 channels or native ␤-cell Ϯ evidence that Kir6.x can also affect the properties of SUR. KATP channels when measured in the whole cell (108 3 Co-expression of Kir6.1 with SUR2A, or mutant SUR2B, and 184 Ϯ 30 nmol/l, respectively), and the maximum has been shown to increase the affinity of [3H]gliben- block was similar, being Ͼ90% in both cases. In contrast, clamide binding by fivefold and fourfold, respectively there was a marked difference in the maximal extent of (23,36). An even greater increase in affinity (eightfold) was gliclazide block of Kir6.2/SUR1 currents when measured in found when Kir6.2 was co-expressed with mutant SUR2B the intact cell and in the excised patch, which amounted to (36). Coexpression of Kir6.2 with SUR1 has variously been 93 and 60%, respectively. reported to increase the affinity of [3H]glibenclamide bind- One explanation for the difference in the shape of the ing (32) and to not affect it (22,41). Interestingly, Do¨ r- concentration-response curve in excised patches and schner and colleagues (22) found no change in the whole cells is that it results from the presence of intracel- [3H]glibenclamide binding affinity of SUR1 or SUR2 on lular MgADP, which is always present in the intact cell, but co-expression with Kir6.x. These discrepancies may be a not, unless deliberately added, in excised patches. Intra-

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Gliclazide block of Kir6.2/SUR1 currents in inside-out patches was also much greater in the presence of 100 ␮mol/l MgADP (Fig. 3C), and the concentration-inhibition relation was best fit with a single-site model with an IC50 of 204 Ϯ 7 nmol/l (n ϭ 5). This finding was consistent with the idea that the difference in the maximal block mediated via the high-affinity site in excised patch and whole-cell recordings results from the presence of MgADP in the intact cell. MgADP has both stimulatory and inhibitory effects on the KATP channel, mediated by the SUR and Kir6.2 sub- units, respectively (8). It has previously been hypothesized that the nucleotide produces an apparent enhancement of sulfonylurea block of Kir6.2/SUR1 currents, because the drug abolishes the stimulatory action of MgADP, while leaving the inhibitory effect untouched (20). Consequently, the combined inhibitory effects of sulfonylurea and nucle- otide result in an enhanced inhibition. If this idea is correct, then gliclazide block of mutant KATP channels that show reduced nucleotide inhibition should be unaffected by MgADP. Mutation of the arginine at position 50 of Kir6.2 to glycine (Kir6.2-R50G) strongly reduces nucleotide block at the inhibitory site on Kir6.2 (46). The extent of this reduction can be quantified by examining the effect of ADP in the absence of Mg2ϩ, because, in Mg-free solution, the stimulatory effect of ADP is abolished and the inhibitory effect of ADP is unmasked (20). As Fig. 4A shows, when tested in the absence of Mg2ϩ, ADP potently blocked Kir6.2/SUR1 currents, but blocked Kir6.2-R50G/SUR1 cur- rents much less effectively (Ͻ15%). As a result of this reduction in nucleotide block, ADP produced an enhanced stimulation of Kir6.2-R50G/SUR1 currents (compared with control currents) when applied in the presence of Mg2ϩ. In the absence of nucleotides, the extent of gliclazide block of Kir6.2-R50G/SUR1 was similar to that found for the wild-type channel, whether measured in the presence or absence of Mg2ϩ (Fig. 4B and C). Thus the R50G mutation did not interfere with the action of the drug. However, although MgADP enhanced gliclazide block of wild-type channels, this was not the case for Kir6.2-R50G/ FIG. 3. A: Whole-cell KATP currents recorded using a two-electrode voltage clamp from an intact oocyte expressing Kir6.2/SUR1, in re- SUR1 channels; indeed, the currents were actually slightly sponse to a series of depolarizing and hyperpolarizing 10-mV pulses larger in the presence of the drug (Fig. 4B). These data from a holding potential of ؊10 mV. Azide (3 mmol/l) and gliclazide (1 mmol/l) were added to the extracellular solution, as indicated. Meth- support the idea that gliclazide reduces the stimulatory ods are the same as those described in ref. 42. B: Mean relation action of MgADP on wild-type channels and unmasks the between gliclazide concentration and inhibition of the Kir6.2/SUR1 inhibitory effect of the nucleotide. The fact that Kir6.2- The current evoked by .(6 ؍ current, measured in the intact oocyte (n a voltage step to ؊100 mV in the presence of gliclazide (I) is expressed R50G/SUR1 currents are somewhat greater in the presence as a fraction of its amplitude in the absence of the drug (IO). The solid of both drug and MgADP than in the presence of gliclazide nmol/l 3 ؎ 108 ؍ line is the best fit of equation 1 to the data, with IC 50 alone suggests that 10 ␮mol/l gliclazide markedly reduces, The dotted line shows the fit obtained for .0 ؍ and L ,0.81 ؍ h ,(5 ؍ n) gliclazide block of Kir6.2/SUR1 currents in the excised patch configu- but does not totally abolish, the stimulatory action of the ration (see Fig. 1). The dashed line shows the fit obtained for gliclazide nucleotide. An alternative explanation is that MgADP ␤ block of the native -cell KATP channel in whole-cell recordings from ␮ reduces the potency of gliclazide block, as 10 mol/l ؍ ␤ mouse pancreatic -cells (IC50 184 nmol/l) (42). C: Relation between gliclazide concentration and inhibition of the Kir6.2/SUR1 current, gliclazide would not then saturate the high-affinity site. measured in the inside-out patch in the presence of 100 ␮mol/l MgADP If gliclazide prevents the stimulatory action of MgADP, The current evoked by a voltage step to ؊100 mV in the .(5 ؍ n) presence of gliclazide (I) is expressed as a fraction of its mean and unmasks its inhibitory effect, then the combined block amplitude in the absence of the drug (IO). The solid line is the best fit by gliclazide and ADP on wild-type channels should be 2ϩ ؍ and L ,1.13 ؍ nmol/l, h 204 ؍ of equation 1 to the data, with IC 50 similar in the absence and presence of Mg . This was in 0.065. The dashed line shows the fit obtained in the absence of added 2ϩ nucleotide (adapted from Gribble et al. [19]). fact observed (Fig. 4B and C). When Mg was absent, Kir6.2-R50G/SUR1 currents were of similar amplitude in cellular MgADP has been shown to produce an apparent the presence of gliclazide or gliclazide plus ADP (Fig. 4C). increase in the efficacy of tolbutamide inhibition in both This is presumably because ADP did not cause substantial ␤ native and recombinant -cell KATP channels (20,44,45). block of the mutant channel. Because MgATP is the

DIABETES, VOL. 51, SUPPLEMENT 3, DECEMBER 2002 S373 SULFONYLUREA STIMULATION OF INSULIN SECRETION

FIG. 4. A: Mean Kir6.2/SUR1 or Kir6.2-R50G/ SUR1 conductance in the presence of 100 ␮mol/l ADP (■), 100 ␮mol/l ADP ؉ 2 mmol/l Mg2؉ ( ), or 100 ␮mol/l ATP plus 2 mmol/l Mg2؉ (p). B and C: Mean Kir6.2/SUR1 or Kir6.2-R50G/SUR1 conductance in the pres- ence of 10 ␮mol/l gliclazide (Ⅺ), 10 ␮mol/l gliclazide plus 100 ␮mol/l ADP ( ), or 10 ␮mol/l gliclazide plus 100 ␮mol/l ATP (p), in the presence (B) or absence (C) of 2 mmol/l -Mg2؉. Conductance (G) is expressed as a frac tion of that in the absence of the drug and nucleotide (Gc). The number of patches is given above each bar. The dashed line indi- cates the control conductance level. predominant Mg nucleotide in vivo, we also examined the Kir6.2 and the COOH-terminus of SUR1 in coupling sulfo- effect of gliclazide on MgATP activation. Kir6.2-R50G/ nylurea binding to channel inhibition (25,26). SUR1 currents were activated by MgATP (Fig. 4A), be- cause the inhibitory effect of the nucleotide was impaired CONCLUSION AND PERSPECTIVES by the R50G mutation (10). As with ADP, the stimulatory action of ATP in the presence of Mg2ϩ was abolished by Sulfonylureas have been used for many years to treat type gliclazide (Fig. 4B). 2 diabetes, but it is only now that the details of their The potency of gliclazide block was significantly differ- molecular mechanism of action are beginning to be unrav- ent when measured in the absence and presence of 100 eled. As first proposed by Henquin and colleagues (51), ␮ antidiabetogenic sulfonylureas, benzamido compounds, mol/l MgADP in excised patches (IC50 of 50 and 204 nmol/l, respectively). It is therefore possible that MgADP and their derivatives appear to interact with more than one caused a small reduction in drug potency. This might also part of the KATP channel. This suggests that, like other explain why gliclazide was somewhat less potent in intact ATP-binding cassette transporters, SUR possesses a large oocytes (108 nmol/l). It also would be consistent with the multifaceted drug-binding pocket that can accommodate fact that gliclazide block of Kir6.2-R50G/SUR1 currents is several structurally distinct compounds. Individual com- less in the presence of MgADP than in its absence. pounds differ in the residues within this pocket with which Although the effects of Mg nucleotides on gliclazide bind- they interact. Studies of recombinant KATP channels sug- ing have not been investigated, it is known that [3H]glib- gest that the sulfonylurea moiety interacts with residues in ␤ the TM 15–16 linker of SUR1, whereas benzamido deriva- enclamide binding to native -cell KATP channels is decreased by MgADP (47). It is not clear whether glicla- tives may interact with the TM 5–6 linker in the NH2- zide displaces MgADP from the nucleotide-binding do- terminal part of the protein. How these different parts of mains of SUR1, or if it uncouples MgADP binding from the sulfonylurea receptor contribute to drug binding, and channel activation. However, glibenclamide has been how this is transduced into changes in channel activity, shown to cause unbinding of 8-azido-[32P]ATP to NBD1 of remains far from clear and awaits determination of the SUR1 in the presence of MgADP (48), lending some structure of SUR, and its relation to Kir6.2, at atomic support to the former hypothesis. resolution. Currently, sulfonylureas are classified as first- and TRANSDUCTION second-generation drugs, although there is no structural or functional basis for this classification. We now know that Binding of sulfonylureas to the cytoplasmic domains of some sulfonylureas bind with high-affinity to SUR1, but not SUR1 ultimately results in closure of the pore formed by SUR2, whereas others interact with both types of SUR. We Kir6.2. Precisely how this transduction occurs is unknown. therefore propose that the classification of sulfonylureas, There must be a close physical association between the meglitinide derivatives, and structurally related com- glibenclamide binding site on SUR and the Kir6.2 subunit, 125 pounds be changed to reflect the functional differences as both proteins can be photoaffinity labeled by [ I]glib- among these drugs, and that they be referred to instead as enclamide (7). Five residues within the first TM of Kir6.2 SUR1-specific and non–SUR1-specific. are essential for interaction with SUR (49), and recent studies have indicated that the first five TMs of SUR1 are also involved (50). However, although these regions are ACKNOWLEDGMENTS required for structural interactions, it is not clear whether We thank the and the Beit Memorial Trust they are also involved in functional coupling. Mutagenesis for support. F.A. is the Royal Society GlaxoSmithKline studies, however, have implicated the NH2-terminus of Research Professor, P.P. is a Beit Memorial Trust Fellow,

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F.G. is Wellcome Trust Clinician Scientist Fellow, and F.R. terminus of Kir6.2 in coupling to the sulphonylurea receptor. J Physiol is a Diabetes U.K. R.D. Lawrence Fellow. 518:325–336, 1999 26. Sakura H, Trapp S, Liss B, Ashcroft FM: Altered functional properties of

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