ABCMdb

ABCC8 p.Ser1237Tyr

Predicted by SNAP2:	A: N (53%), C: D (59%), D: D (71%), E: D (75%), F: N (72%), G: D (66%), H: N (61%), I: D (63%), K: D (75%), L: D (53%), M: D (59%), N: D (53%), P: D (85%), Q: D (66%), R: D (75%), T: N (61%), V: D (53%), W: D (59%), Y: N (78%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, T: N, V: N, W: N, Y: N,

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[hide] Coexpression of the type 2 diabetes susceptibility... Diabetes. 2009 Oct;58(10):2419-24. Epub 2009 Jul 8. Hamming KS, Soliman D, Matemisz LC, Niazi O, Lang Y, Gloyn AL, Light PE
Coexpression of the type 2 diabetes susceptibility gene variants KCNJ11 E23K and ABCC8 S1369A alter the ATP and sulfonylurea sensitivities of the ATP-sensitive K(+) channel.
Diabetes. 2009 Oct;58(10):2419-24. Epub 2009 Jul 8., [PMID:19587354]

Abstract [show]
OBJECTIVE: In the pancreatic beta-cell, ATP-sensitive K(+) (K(ATP)) channels couple metabolism with excitability and consist of Kir6.2 and SUR1 subunits encoded by KCNJ11 and ABCC8, respectively. Sulfonylureas, which inhibit the K(ATP) channel, are used to treat type 2 diabetes. Rare activating mutations cause neonatal diabetes, whereas the common variants, E23K in KCNJ11 and S1369A in ABCC8, are in strong linkage disequilibrium, constituting a haplotype that predisposes to type 2 diabetes. To date it has not been possible to establish which of these represents the etiological variant, and functional studies are inconsistent. Furthermore, there have been no studies of the S1369A variant or the combined effect of the two on K(ATP) channel function. RESEARCH DESIGN AND METHODS: The patch-clamp technique was used to study the nucleotide sensitivity and sulfonylurea inhibition of recombinant human K(ATP) channels containing either the K23/A1369 or E23/S1369 variants. RESULTS: ATP sensitivity of the K(ATP) channel was decreased in the K23/A1369 variant (half-maximal inhibitory concentration [IC(50)] = 8.0 vs. 2.5 mumol/l for the E23/S1369 variant), although there was no difference in ADP sensitivity. The K23/A1369 variant also displayed increased inhibition by gliclazide, an A-site sulfonylurea drug (IC(50) = 52.7 vs. 188.7 nmol/l for the E23/S1369 variant), but not by glibenclamide (AB site) or repaglinide (B site). CONCLUSIONS: Our findings indicate that the common K23/A1369 variant K(ATP) channel displays decreased ATP inhibition that may contribute to the observed increased risk for type 2 diabetes. Moreover, the increased sensitivity of the K23/A1369 variant to the A-site sulfonylurea drug gliclazide may provide a pharmacogenomic therapeutic approach for patients with type 2 diabetes who are homozygous for both risk alleles.

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76 The A site is located close to SUR1 transmembrane segments 14-16, and the S1237Y mutation in this region (Fig. 3A) abolishes A-site drug inhibition (18). Login to comment
78 The A site is located close to SUR1 transmembrane segments 14-16, and the S1237Y mutation in this region (Fig. 3A) abolishes A-site drug inhibition (18). Login to comment

[hide] Pharmacological modulation of K(ATP) channels. Biochem Soc Trans. 2002 Apr;30(2):333-9. Gribble FM, Reimann F
Pharmacological modulation of K(ATP) channels.
Biochem Soc Trans. 2002 Apr;30(2):333-9., [PMID:12023875]

Abstract [show]
Pharmacological modulation of ATP-sensitive K+ (K(ATP)) channels is used in the treatment of a number of clinical conditions, including type 2 diabetes and angina. The sulphonylureas and related drugs, which are used to treat type 2 diabetes, stimulate insulin secretion by closing K(ATP) channels in pancreatic beta-cells. Agents used to treat angina, by contrast, act by opening K(ATP) channels in vascular smooth and cardiac muscle. Both the therapeutic K(ATP) channel inhibitors and the K(ATP) channel openers target the sulphonylurea receptor (SUR) subunit of the K(ATP) channel, which exists in several isoforms expressed in different tissues (SUR1 in pancreatic beta-cells, SUR2A in cardiac muscle and SUR2B in vascular smooth muscle). The tissue-specific action of drugs that target the K(ATP) channel is attributed to the properties of these different SUR subtypes. In this review, we discuss the molecular basis of tissue-specific drug action, and its implications for clinical practice.

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94 However, the high-affinity blocking of Kir6.2/SURl channels by nateglinide and mitiglinide, which both show homology with meglitinide, has recently been reported to be abolished by the S1237Y mutation in SURl (see below), rather unexpectedly suggesting that their binding site overlaps with that of the sulphonylureas [37,38]. Login to comment
95 However, the high-affinity blocking of Kir6.2/SURl channels by nateglinide and mitiglinide, which both show homology with meglitinide, has recently been reported to be abolished by the S1237Y mutation in SURl (see below), rather unexpectedly suggesting that their binding site overlaps with that of the sulphonylureas [37,38]. Login to comment

[hide] Toward linking structure with function in ATP-sens... Diabetes. 2004 Dec;53 Suppl 3:S104-12. Bryan J, Vila-Carriles WH, Zhao G, Babenko AP, Aguilar-Bryan L
Toward linking structure with function in ATP-sensitive K+ channels.
Diabetes. 2004 Dec;53 Suppl 3:S104-12., [PMID:15561897]

Abstract [show]
Advances in understanding the overall structural features of inward rectifiers and ATP-binding cassette (ABC) transporters are providing novel insight into the architecture of ATP-sensitive K+ channels (KATP channels) (KIR6.0/SUR)4. The structure of the K(IR) pore has been modeled on bacterial K+ channels, while the lipid-A exporter, MsbA, provides a template for the MDR-like core of sulfonylurea receptor (SUR)-1. TMD0, an NH2-terminal bundle of five alpha-helices found in SURs, binds to and activates KIR6.0. The adjacent cytoplasmic L0 linker serves a dual function, acting as a tether to link the MDR-like core to the KIR6.2/TMD0 complex and exerting bidirectional control over channel gating via interactions with the NH2-terminus of the KIR. Homology modeling of the SUR1 core offers the possibility of defining the glibenclamide/sulfonylurea binding pocket. Consistent with 30-year-old studies on the pharmacology of hypoglycemic agents, the pocket is bipartite. Elements of the COOH-terminal half of the core recognize a hydrophobic group in glibenclamide, adjacent to the sulfonylurea moiety, to provide selectivity for SUR1, while the benzamido group appears to be in proximity to L0 and the KIR NH2-terminus.

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132 Substitution of Ser1237 with Tyr, the analogous residue in SUR2, reduced the apparent affinity of SUR1 for tolbutamide and glibenclamide (82). Login to comment

[hide] Differential selectivity of insulin secretagogues:... J Diabetes Complications. 2003 Mar-Apr;17(2 Suppl):11-5. Gribble FM, Reimann F
Differential selectivity of insulin secretagogues: mechanisms, clinical implications, and drug interactions.
J Diabetes Complications. 2003 Mar-Apr;17(2 Suppl):11-5., [PMID:12623163]

Abstract [show]
The sulphonylurea receptor (SUR) subunits of K(ATP) channels are the targets for several classes of therapeutic drugs. Sulphonylureas close K(ATP) channels in pancreatic beta-cells and are used to stimulate insulin release in type 2 diabetes, whereas the K(ATP) channel opener nicorandil acts as an antianginal agent by opening K(ATP) channels in cardiac and vascular smooth muscle. The predominant type of SUR varies between tissues: SUR1 in beta-cells, SUR2A in cardiac muscle, and SUR2B in smooth muscle. Sulphonylureas and related drugs exhibit differences in tissue specificity, as the drugs interact to varying degrees with different types of SUR. Gliclazide and tolbutamide are beta-cell selective and reversible. Glimepiride, glibenclamide, and repaglinide, however, inhibit cardiac and smooth muscle K(ATP) channels in addition to those in beta-cells and are only slowly reversible. Similar properties have been observed by recording K(ATP) channel activity in intact cells and in Xenopus oocytes expressing cloned K(ATP) channel subunits. While K(ATP) channels in cardiac and smooth muscle are largely closed under physiological conditions (but open during ischaemia), they are activated by antianginal agents such as nicorandil. Under these conditions, they may be inhibited by sulphonylureas that block SUR2-type K(ATP) channels (e.g., glibenclamide). Care should, therefore, be taken when choosing a sulphonylurea if potential interactions with cardiac and smooth muscle K(ATP) channels are to be avoided.

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67 The difference between SUR1 and SUR2 that accounts for the different sensitivities to KATP channel inhibitors has been localised to the TM15-16 loop, in which a single amino acid substitution (S1237Y) in SUR1 is sufficient to abolish the action of tolbutamide on channel activity and to prevent binding of [3 H]glibenclamide (Ashfield et al., 1999). Login to comment
69 The actions of meglitinide and repaglinide on Kir6.2/SUR1 currents are not affected by the S1237Y mutation (Ashfield et al., 1999; Dabrowski et al., 2001), supporting the idea that a different region of the binding site (e.g., the TMs 5-6 loop) is involved. Login to comment
71 Inability to bind to the TMs 15-16 loop, either in SUR2 or SUR1-S1237Y, accounts for the rapid reversibility of glibenclamide and glimepiride in these cases. Login to comment
72 The slow reversibility of repaglinide cannot be explained on the same basis, as it is unaffected by the SUR type or the S1237Y mutation in SUR1, and is likely to be attributable to its greater hydrophobicity. Login to comment

[hide] Analysis of the differential modulation of sulphon... J Physiol. 2003 Feb 15;547(Pt 1):159-68. Epub 2003 Jan 10. Reimann F, Dabrowski M, Jones P, Gribble FM, Ashcroft FM
Analysis of the differential modulation of sulphonylurea block of beta-cell and cardiac ATP-sensitive K+ (K(ATP)) channels by Mg-nucleotides.
J Physiol. 2003 Feb 15;547(Pt 1):159-68. Epub 2003 Jan 10., [PMID:12562963]

Abstract [show]
Sulphonylureas stimulate insulin secretion by binding with high-affinity to the sulphonylurea receptor (SUR) subunit of the ATP-sensitive potassium (K(ATP)) channel and thereby closing the channel pore (formed by four Kir6.2 subunits). In the absence of added nucleotides, the maximal block is around 60-80 %, indicating that sulphonylureas act as partial antagonists. Intracellular MgADP modulated sulphonylurea block, enhancing inhibition of Kir6.2/SUR1 (beta-cell type) and decreasing that of Kir6.2/SUR2A (cardiac-type) channels. We examined the molecular basis of the different response of channels containing SUR1 and SUR2A, by recording currents from inside-out patches excised from Xenopus oocytes heterologously expressing wild-type or chimeric channels. We used the benzamido derivative meglitinide as this drug blocks Kir6.2/SUR1 and Kir6.2/SUR2A currents, reversibly and with similar potency. Our results indicate that transfer of the region containing transmembrane helices (TMs) 8-11 and the following 65 residues of SUR1 into SUR2A largely confers a SUR1-like response to MgADP and meglitinide, whereas the reverse chimera (SUR128) largely endows SUR1 with a SUR2A-type response. This effect was not specific for meglitinide, as tolbutamide was also unable to prevent MgADP activation of Kir6.2/SUR128 currents. The data favour the idea that meglitinide binding to SUR1 impairs either MgADP binding or the transduction pathway between the NBDs and Kir6.2, and that TMs 8-11 are involved in this modulatory response. The results provide a basis for understanding how beta-cell K(ATP) channels show enhanced sulphonylurea inhibition under physiological conditions, whereas cardiac K(ATP) channels exhibit reduced block in intact cells, especially during metabolic inhibition.

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124 The binding sites for these drugs are not, however, believed to be identical, as the mutation S1237Y in SUR1 abolishes tolbutamide, but not meglitinide, inhibition (Ashfield et al. 1999). Login to comment
158 The cytosolic loop linking TMs 15 and 16 has been shown to play a role in sulphonylurea binding to SUR1, as mutation of S1237Y abolished tolbutamide block and [3 H]glibenclamide binding, whilst inhibition by meglitinide(whichresemblesthenon-sulphonylureamoiety of glibenclamide) was unaffected (Ashfield et al. 1999). Login to comment

[hide] Nateglinide, a D-phenylalanine derivative lacking ... J Pharmacol Exp Ther. 2003 Mar;304(3):1025-32. Chachin M, Yamada M, Fujita A, Matsuoka T, Matsushita K, Kurachi Y
Nateglinide, a D-phenylalanine derivative lacking either a sulfonylurea or benzamido moiety, specifically inhibits pancreatic beta-cell-type K(ATP) channels.
J Pharmacol Exp Ther. 2003 Mar;304(3):1025-32., [PMID:12604678]

Abstract [show]
A novel antidiabetic agent, nateglinide, is a D-phenylalanine derivative lacking either a sulfonylurea or benzamido moiety. We examined with the patch-clamp method the effect of nateglinide on recombinant ATP-sensitive K(+) (K(ATP)) channels expressed in human embryonic kidney 293T cells transfected with a Kir6.2 subunit and either of a sulfonylurea receptor (SUR) 1, SUR2A, and SUR2B. In inside-out patches, nateglinide reversibly inhibited the spontaneous openings of all three types of SUR/Kir6.2 channels. Nateglinide inhibited SUR1/Kir6.2 channels with high and low affinities (K(i) = 75 nM and 114 microM) but SUR2A/Kir6.2 and SUR2B/Kir6.2 channels only with low affinity (K(i) = 105 and 111 microM, respectively). Nateglinide inhibited the K(ATP) current mediated by Kir6.2 lacking C-terminal 26 amino acids only with low affinity (K(i) = 290 microM) in the absence of SUR. Replacement of serine at position 1237 of SUR1 to tyrosine [SUR1(S1237Y)] specifically abolished the high-affinity inhibition of SUR1/Kir6.2 channels by nateglinide. MgADP or MgUDP (100 microM) augmented the inhibitory effect of nateglinide on SUR1/Kir6.2 but not SUR1(S1237Y)/Kir6.2 or SUR2A/Kir6.2 channels. This augmenting effect of MgADP was also observed with the SUR1/Kir6.2(K185Q) channel, which was not inhibited by MgADP, but not with the SUR1(K1384A)/Kir6.2 channel, which was not activated by MgADP. These results indicate that therapeutic concentrations of nateglinide (approximately 10 microM) may selectively inhibit pancreatic type SUR1/Kir6.2 channels through SUR1, especially when the channel is activated by intracellular MgADP, even though the agent does not contain either a sulfonylurea or benzamido moiety.

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6 Replacement of serine at position 1237 of SUR1 to tyrosine [SUR1(S1237Y)] specifically abolished the high-affinity inhibition of SUR1/Kir6.2 channels by nateglinide. Login to comment
7 MgADP or MgUDP (100 ␮M) augmented the inhibitory effect of nateglinide on SUR1/Kir6.2 but not SUR1(S1237Y)/Kir6.2 or SUR2A/Kir6.2 channels. Login to comment
18 In preparation of this manuscript, Hansen et al. (2002) reported that nateglinide inhibits SUR1/Kir6.2 channels with the half-maximum inhibitory concentration of 800 nM and that this inhibition is abolished by the S1237Y mutation of SUR1, consistent with our present observations. Login to comment
36 SUR1 whose serine at position 1237 was substituted with tyrosine [SUR1(S1237Y)], SUR1 whose lysine at position 1384 was substituted with alanine [SUR1(K1384A)], and Kir6.2 whose lysine at position 185 was substituted with glutamine [Kir6.2(K185Q)] were constructed using the GeneEditor in vitro site-directed mutagenesis system (Promega, Madison, WI). Login to comment
93 Inhibitory Effect of Nateglinide on Kir6.2⌬C26 and SUR1(S1237Y)/Kir6.2 Channels. Login to comment
102 Inhibitory effect of nateglinide on Kir6.2⌬C26 and SUR1(S1237Y)/Kir6.2 channel currents. Login to comment
103 Left column, inhibitory effects of nateglinide on spontaneously opening Kir6.2⌬C26 (A) and SUR1(S1237Y)/Kir6.2 (B) channels were measured at -60 mV in inside-out patches. ATP and nateglinide were added to the bath solution as indicated by bars. Right column, concentration-response relationships for inhibition of Kir6.2⌬C26 (A) and SUR1(S1237Y)/Kir6.2 (B) channel currents by nateglinide. Login to comment
106 The lines were fit with the single-site model with Ki2 ϭ 290 ␮M and h2 ϭ 0.9 for Kir6.2⌬C26 channels, and Ki2 ϭ 72 ␮M and h2 ϭ 0.7 for SUR1(S1237Y)/Kir6.2 channels. Login to comment
108 [SUR1(S1237Y)] abolishes the high-affinity inhibition of SUR1/Kir6.2 channels by tolbutamide (Ashfield et al., 1999). Login to comment
110 Nateglinide inhibited SUR1(S1237Y)/ Kir6.2 channel currents only with low-affinity (Ki2 ϭ 72 ␮M), indicating that the same amino acid residue in SUR1 mediates the high-affinity inhibition of SUR1/Kir6.2 channels by nateglinide and tolbutamide. Login to comment
117 To examine whether the potentiation by MgADP of the effect of nateglinide upon SUR1/Kir6.2 was related to the high-affinity site for the drug, we repeated the experiment using SUR1(S1237Y)/Kir6.2 channels (Fig. 3B). Login to comment
124 Effect of nateglinide on SUR1/Kir6.2, SUR1(S1237Y)/Kir6.2, and SUR2A/Kir6.2 channels in the presence and absence of 100 ␮M MgADP. Login to comment
125 The inhibitory effects of nateglinide on SUR1/Kir6.2 (A), SUR1(S1237Y)/Kir6.2 (B), and SUR2A/Kir6.2 (C) channels were measured in inside-out patches. ATP, ADP, and nateglinide were added to the bath solution as indicated by bars. Login to comment
179 The high-affinity inhibition of the channels by nateglinide was eliminated by the S1237Y mutation of SUR1 (Fig. 2A). Login to comment
185 Mitiglinide is another antidiabetic agent that lacks either a sulfonylurea or benzamido moiety but causes high-affinity inhibition of SUR1/Kir6.2 channels that is abolished by the S1237Y mutation (Reimann et al., 2001). Login to comment
193 Although nateglinide lacks a sulfonylurea moiety, its effect on SUR1/ Kir6.2 channels resembles that of tolbutamide with respect to the S1237Y mutation and the interaction with intracellular MgADP. Login to comment
92 Inhibitory Effect of Nateglinide on Kir6.2èc;C26 and SUR1(S1237Y)/Kir6.2 Channels. Login to comment
101 Inhibitory effect of nateglinide on Kir6.2èc;C26 and SUR1(S1237Y)/Kir6.2 channel currents. Login to comment
105 The lines were fit with the single-site model with Ki2 afd; 290 òe;M and h2 afd; 0.9 for Kir6.2èc;C26 channels, and Ki2 afd; 72 òe;M and h2 afd; 0.7 for SUR1(S1237Y)/Kir6.2 channels. Login to comment
107 [SUR1(S1237Y)] abolishes the high-affinity inhibition of SUR1/Kir6.2 channels by tolbutamide (Ashfield et al., 1999). Login to comment
109 Nateglinide inhibited SUR1(S1237Y)/ Kir6.2 channel currents only with low-affinity (Ki2 afd; 72 òe;M), indicating that the same amino acid residue in SUR1 mediates the high-affinity inhibition of SUR1/Kir6.2 channels by nateglinide and tolbutamide. Login to comment
116 To examine whether the potentiation by MgADP of the effect of nateglinide upon SUR1/Kir6.2 was related to the high-affinity site for the drug, we repeated the experiment using SUR1(S1237Y)/Kir6.2 channels (Fig. 3B). Login to comment
123 Effect of nateglinide on SUR1/Kir6.2, SUR1(S1237Y)/Kir6.2, and SUR2A/Kir6.2 channels in the presence and absence of 100 òe;M MgADP. Login to comment
178 The high-affinity inhibition of the channels by nateglinide was eliminated by the S1237Y mutation of SUR1 (Fig. 2A). Login to comment
184 Mitiglinide is another antidiabetic agent that lacks either a sulfonylurea or benzamido moiety but causes high-affinity inhibition of SUR1/Kir6.2 channels that is abolished by the S1237Y mutation (Reimann et al., 2001). Login to comment
192 Although nateglinide lacks a sulfonylurea moiety, its effect on SUR1/ Kir6.2 channels resembles that of tolbutamide with respect to the S1237Y mutation and the interaction with intracellular MgADP. Login to comment

[hide] Sulphonylurea action revisited: the post-cloning e... Diabetologia. 2003 Jul;46(7):875-91. Epub 2003 Jun 18. Gribble FM, Reimann F
Sulphonylurea action revisited: the post-cloning era.
Diabetologia. 2003 Jul;46(7):875-91. Epub 2003 Jun 18., [PMID:12819907]

Abstract [show]
Hypoglycaemic agents such as sulphonylureas and the newer group of "glinides" stimulate insulin secretion by closing ATP-sensitive potassium (K(ATP)) channels in pancreatic beta cells, but have varying cross-reactivity with related channels in extrapancreatic tissues such as heart, vascular smooth and skeletal muscle. Experiments on the structure-function relationships of recombinant K(ATP) channels and the phenotypes of mice deficient in different K(ATP) channel subunits have provided important insights into the mechanisms underlying sulphonylurea selectivity, and the potential consequences of K(ATP) channel blockade outside the pancreatic beta cell. The different pharmacological properties of K(ATP) channels from beta cells compared with those from cardiac, smooth and skeletal muscle, are accounted for by the expression of alternative types of sulphonylurea receptor, with non-identical drug binding sites. The sulphonylureas and glinides are found to fall into two groups: one exhibiting selectivity for beta cell sulphonylurea receptors (SUR1), and the other blocking cardiovascular and skeletal muscle sulphonylurea receptors (SUR2) with potencies similar to their action on SUR1. In seeking potential side effects of K(ATP) channel inhibitors in humans, it is essential to take these drug differences into account, along with the probability (suggested by the studies on K(ATP) channel knockout mice) that the effects of extrapancreatic K(ATP) channel inhibition might be either subtle or rare. Further studies are still required before a final decision can be made on whether non-selective agents are appropriate for the therapy of Type 2 diabetes.

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130 The binding site for sulphonylureas and glinides could therefore be envisaged as a pocket with at least two binding motifs, one (exclusive to SUR1 and abolished by the S1237Y mutation) favouring sulphonylurea groups, and the other (common to SUR1 and SUR2) preferring meglitinide-like molecules [4, 44, 91]. Login to comment
135 In line with this idea, mutation of S1237Y in SUR1 increased glibenclamide reversibility in patch clamp experiments and impaired the binding of [3H]glibenclamide [49, 91], whereas the reverse mutation (Y1206S) in SUR2B increased the [3H]glibenclamide binding affinity [92]. Login to comment
136 Neither repaglinide reversibility, nor binding of [3H]repaglinide, were affected by the S1237Y mutation in SUR1 [47, 49]. Login to comment
154 Thus, they both inhibited Kir6.2/SUR1 with higher affinity than Kir6.2/SUR2 currents, and inhibition of SUR1-type channels was impaired by the S1237Y mutation [48, 49, 50, 97]. Login to comment

[hide] Kir6.2-dependent high-affinity repaglinide binding... Br J Pharmacol. 2005 Feb;144(4):551-7. Hansen AM, Hansen JB, Carr RD, Ashcroft FM, Wahl P
Kir6.2-dependent high-affinity repaglinide binding to beta-cell K(ATP) channels.
Br J Pharmacol. 2005 Feb;144(4):551-7., [PMID:15678092]

Abstract [show]
1. The beta-cell K(ATP) channel is composed of two types of subunit - the inward rectifier K(+) channel (Kir6.2) which forms the channel pore, and the sulphonylurea receptor (SUR1), which serves as a regulatory subunit. The N-terminus of Kir6.2 is involved in transduction of sulphonylurea binding into channel closure, and deletion of the N-terminus (Kir6.2DeltaN14) results in functional uncoupling of the two subunits. In this study, we investigate the interaction of the hypoglycaemic agents repaglinide and glibenclamide with SUR1 and the effect of Kir6.2 on this interaction. We further explore how the binding properties of repaglinide and glibenclamide are affected by functional uncoupling of SUR1 and Kir6.2 in Kir6.2DeltaN14/SUR1 channels. All binding experiments are performed on membranes in ATP-free buffer at 37 degrees C. 2. Repaglinide was found to bind with low affinity (K(D)=59+/-16 nM) to SUR1 alone, but with high affinity (increased approximately 150-fold) when SUR1 was co-expressed with Kir6.2 (K(D)=0.42+/-0.03 nM). Glibenclamide, tolbutamide and nateglinide all bound with marginally lower affinity to SUR1 than to Kir6.2/SUR1. 3. Repaglinide bound with low affinity (K(D)=51+/-23 nM) to SUR1 co-expressed with Kir6.2DeltaN14. In contrast, the affinity for glibenclamide, tolbutamide and nateglinide was only mildly changed as compared to wild-type channels. 4. In whole-cell patch-clamp experiments inhibition of Kir6.2DeltaN14/SUR1 currents by both repaglinide and nateglinde is abolished. 5. The results suggest that Kir6.2 causes a conformational change in SUR1 required for high-affinity repaglinide binding, or that the high-affinity repaglinide-binding site includes contributions from both SUR1 and Kir6.2. Glibenclamide, tolbutamide and nateglinide binding appear to involve only SUR1.

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28 Recent studies from our laboratory suggested that the binding site for repaglinide is not identical to that of sulphonylureas, since repaglinide binding and channel inhibition by this drug were unaffected by mutation S1237Y in SUR1, which abolishes KATP channel inhibition by tolbutamide (Hansen et al., 2002). Login to comment

[hide] Sulfonylurea stimulation of insulin secretion. Diabetes. 2002 Dec;51 Suppl 3:S368-76. Proks P, Reimann F, Green N, Gribble F, Ashcroft F
Sulfonylurea stimulation of insulin secretion.
Diabetes. 2002 Dec;51 Suppl 3:S368-76., [PMID:12475777]

Abstract [show]
Sulfonylureas are widely used to treat type 2 diabetes because they stimulate insulin secretion from pancreatic beta-cells. They primarily act by binding to the SUR subunit of the ATP-sensitive potassium (K(ATP)) channel and inducing channel closure. However, the channel is still able to open to a limited extent when the drug is bound, so that high-affinity sulfonylurea inhibition is not complete, even at saturating drug concentrations. K(ATP) channels are also found in cardiac, skeletal, and smooth muscle, but in these tissues are composed of different SUR subunits that confer different drug sensitivities. Thus tolbutamide and gliclazide block channels containing SUR1 (beta-cell type), but not SUR2 (cardiac, smooth muscle types), whereas glibenclamide, glimepiride, repaglinide, and meglitinide block both types of channels. This difference has been exploited to determine residues contributing to the sulfonylurea-binding site. Sulfonylurea block is decreased by mutations or agents (e.g., phosphatidylinositol bisphosphate) that increase K(ATP) channel open probability. We now propose a kinetic model that explains this effect in terms of changes in the channel open probability and in the transduction between the drug-binding site and the channel gate. We also clarify the mechanism by which MgADP produces an apparent increase of sulfonylurea efficacy on channels containing SUR1 (but not SUR2).

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124 The curves are fit to equation 2. beta-cell KATP channels, is not easily reversed in electrophysiological recordings, whereas glibenclamide block is rapidly reversed when SUR1 contains the S1237Y mutation. Login to comment
126 In addition, glibenclamide binding to SUR1 is greatly decreased by the S1237Y mutation (35), whereas glibenclamide binding to SUR2B is enhanced by the reverse mutation (36,37). Login to comment
129 The fact that glibenclamide blocks Kir6.2/SUR1-S1237Y channels indicates that residues other than S1237 are critical for binding of this drug. Login to comment
130 Likewise, the block of Kir6.2/SUR1 by meglitinide and repaglinide, which do not possess a sulfonylurea moiety, is unaltered by the S1237Y mutation (35,53), suggesting that these drugs do not interact with this residue. Login to comment
123 The curves are fit to equation 2. beta-cell KATP channels, is not easily reversed in electrophysiological recordings, whereas glibenclamide block is rapidly reversed when SUR1 contains the S1237Y mutation. Login to comment
125 In addition, glibenclamide binding to SUR1 is greatly decreased by the S1237Y mutation (35), whereas glibenclamide binding to SUR2B is enhanced by the reverse mutation (36,37). Login to comment
128 The fact that glibenclamide blocks Kir6.2/SUR1-S1237Y channels indicates that residues other than S1237 are critical for binding of this drug. Login to comment

[hide] Sulfonylureas correct trafficking defects of disea... J Biol Chem. 2006 Nov 3;281(44):33403-13. Epub 2006 Sep 6. Yan FF, Casey J, Shyng SL
Sulfonylureas correct trafficking defects of disease-causing ATP-sensitive potassium channels by binding to the channel complex.
J Biol Chem. 2006 Nov 3;281(44):33403-13. Epub 2006 Sep 6., [PMID:16956886]

Abstract [show]
ATP-sensitive potassium (K(ATP)) channels mediate glucose-induced insulin secretion by coupling metabolic signals to beta-cell membrane potential and the secretory machinery. Reduced K(ATP) channel expression caused by mutations in the channel proteins: sulfonylurea receptor 1 (SUR1) and Kir6.2, results in loss of channel function as seen in congenital hyperinsulinism. Previously, we reported that sulfonylureas, oral hypoglycemic drugs widely used to treat type II diabetes, correct the endoplasmic reticulum to the plasma membrane trafficking defect caused by two SUR1 mutations, A116P and V187D. In this study, we investigated the mechanism by which sulfonylureas rescue these mutants. We found that glinides, another class of SUR-binding hypoglycemic drugs, also markedly increased surface expression of the trafficking mutants. Attenuating or abolishing the ability of mutant SUR1 to bind sulfonylureas or glinides by the following mutations: Y230A, S1238Y, or both, accordingly diminished the rescuing effects of the drugs. Interestingly, rescue of the trafficking defects requires mutant SUR1 to be co-expressed with Kir6.2, suggesting that the channel complex, rather than SUR1 alone, is the drug target. Observations that sulfonylureas also reverse trafficking defects caused by neonatal diabetes-associated Kir6.2 mutations in a way that is dependent on intact sulfonylurea binding sites in SUR1 further support this notion. Our results provide insight into the mechanistic and structural basis on which sulfonylureas rescue K(ATP) channel surface expression defects caused by channel mutations.

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105 If sulfonylureas rescue the A116P and V187D trafficking mutants by binding to the channel protein, then introducing the S1237Y mutation should also reduce or abolish the ability of sulfonylureas to correct the trafficking defect. Login to comment

[hide] Differential interactions of nateglinide and repag... Diabetes. 2002 Sep;51(9):2789-95. Hansen AM, Christensen IT, Hansen JB, Carr RD, Ashcroft FM, Wahl P
Differential interactions of nateglinide and repaglinide on the human beta-cell sulphonylurea receptor 1.
Diabetes. 2002 Sep;51(9):2789-95., [PMID:12196472]

Abstract [show]
Repaglinide and nateglinide represent a new class of insulin secretagogues, structurally unrelated to sulphonylureas, that were developed for the treatment of type 2 diabetes. The inhibitory effect of these drugs was investigated on recombinant wild-type and mutant Kir6.2/SUR1 channels expressed in HEK293 cells. Nateglinide and repaglinide dose-dependently inhibited whole-cell Kir6.2/SUR1 currents with half-maximal inhibitory concentration (IC(50)) values of 800 and 21 nmol/l, respectively. Mutation of serine 1237 in SUR1 to tyrosine (S1237Y) abolished tolbutamide and nateglinide block, suggesting that these drugs share a common point of interaction on the SUR1 subunit of the ATP-sensitive K(+) channel. In contrast, repaglinide inhibition was unaffected by the S1237Y mutation (IC(50) = 23 nmol/l). Radioligand binding studies revealed a single high-affinity binding site for [(3)H]repaglinide on membranes prepared from HEK293 cells expressing wild-type (equilibrium dissociation constant [K(D)] = 0.40 nmol/l) or mutant (K(D) = 0.31 nmol/l) Kir6.2/SUR1 channels. Nateglinide and tolbutamide displaced [(3)H]repaglinide binding to wild-type channels with IC(50) values of 0.7 and 26 micro mol/l, respectively, but produced <10% displacement of [(3)H]repaglinide bound to mutant channels. This is consistent with the idea that binding of nateglinide and tolbutamide, but not repaglinide, is abolished by the SUR1[S1237Y] mutation and that the binding site for repaglinide is not identical to that of nateglinde/tolbutamide. These results are discussed in terms of a conformational analysis of the drug molecules.

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3 Mutation of serine 1237 in SUR1 to tyrosine (S1237Y) abolished tolbutamide and nateglinide block, suggesting that these drugs share a common point of interaction on the SUR1 subunit of the ATP-sensitive K؉ channel. Login to comment
4 In contrast, repaglinide inhibition was unaffected by the S1237Y mutation (IC50 ‫؍‬ 23 nmol/l). Login to comment
7 This is consistent with the idea that binding of nateglinide and tolbutamide, but not repaglinide, is abolished by the SUR1[S1237Y] mutation and that the binding site for repaglinide is not identical to that of nateglinde/tolbutamide. Login to comment
34 SUR1 currents by the nonsulphonylurea mitiglinide, as with tolbutamide, is abolished by the S1237Y mutation (16). Login to comment
45 The point mutation SUR1[S1237Y] was constructed by standard molecular biology techniques and confirmed by DNA sequencing. Login to comment
48 Cells were seeded at 50% confluency and transfected with Kir6.2 and SUR1[S1237Y] at a plasmid ratio of 1:3 on the next day. Login to comment
63 Binding experiments were performed in triplicate (Kir6.2/SUR1) or duplicate (Kir6.2/SUR1[S1237Y]). Login to comment
92 RESULTS Electrophysiology. Whole-cell currents were recorded from HEK293 cells coexpressing Kir6.2 and either SUR1 or SUR1[S1237Y]. Login to comment
93 After establishment of the whole-cell configuration and dialysis with intracellular solution, there was a gradual increase in both Kir6.2/SUR1 and Kir6.2/ SUR1[S1237Y] currents due to opening of KATP channels. Login to comment
95 In contrast, tolbutamide had very little effect on Kir6.2/SUR1[S1237Y] currents (Fig. 1B). Login to comment
97 Although glibenclamide also inhibited Kir6.2/SUR1[S1237Y] channels (by 67 Ϯ 6%; n ϭ 3), in this case, inhibition was largely reversed on wash-out of the drug (Fig. 1B). Login to comment
101 A similar extent of block was observed for Kir6.2/ SUR1[S1237Y] (94 Ϯ 1%, n ϭ 4) (Fig. 2B), suggesting that S1237 is not required for repaglinide inhibition. Login to comment
103 In contrast, nateglinide (100 ␮mol/l) produced reversible inhibition of Kir6.2/SUR1 currents (96 Ϯ 2%, n ϭ 4) (Fig. 2A) but was without significant effect on Kir6.2/SUR1[S1237Y] channels (Fig. 2B). Login to comment
105 Repaglinide blocked Kir6.2/SUR1 and Kir6.2/SUR1[S1237Y] currents with similar potency: IC50 ϭ 21 nmol/l (95% CI 17-26) and IC50 ϭ 23 nmol/l (18-28), respectively. Login to comment
114 Similarly, binding of [3 H]repaglinide to Kir6.2/SUR1[S1237Y] revealed a single binding site (Fig. 4B) with a KD of 0.31 Ϯ 0.02 nmol/l and a Bmax of 1.6 Ϯ 0.2 pmol/mg protein (n ϭ 3). Login to comment
118 HEK 293 cells expressing Kir6.2/SUR1 (A) or Kir6.2/SUR1[S1237Y] (B) channels were clamped at -70 mV. Login to comment
122 HEK 293 cells expressing Kir6.2/SUR1 (A) or Kir6.2/ SUR1[S1237Y] (B) channels were clamped at -70 mV. Login to comment
125 Concentration-response curves for inhibition of Kir6.2/SUR1 (f/F) and Kir6.2/SUR1[S1237Y] (Ⅺ/E) channels by repaglinide (f/Ⅺ) or nateglinide (F/E). Login to comment
130 We therefore examined the ability of unlabelled nateglinide to displace [3 H]repaglinide binding to membranes isolated from HEK293 cells expressing Kir6.2/SUR1 or Kir6.2/SUR1[S1237Y]. Login to comment
137 In the case of Kir6.2/SUR1[S1237Y], repaglinide displaced [3 H]repaglinide binding with a Ki of 0.4 Ϯ 0.2 nmol/l (n ϭ 3), which is similar to that found for the wild-type channel. Login to comment
141 The data are consistent with the idea that the nateglinide binding, as with that of tolbutamide, is abolished by the S1237Y mutation in SUR1. Login to comment
147 Saturation binding of [3 H]repaglinide to membranes prepared from HEK 293 cells expressing Kir6.2/SUR1 (A) or Kir6.2/ SUR1[S1237Y] (B). Login to comment
148 Data are from a single representative experiment in which data points were collected in triplicate (Kir6.2/SUR1) or duplicate (Kir6.2/SUR1[S1237Y]). Login to comment
150 Competition binding to membranes from HEK 293 cells expressing Kir6.2/SUR1 (A) or Kir6.2/SUR1[S1237Y] (B). Login to comment
153 Data are from a single representative experiment in which data points were collected in triplicate (Kir6.2/SUR1) or duplicate (Kir62/SUR1[S1237Y]). Login to comment
179 TABLE 1 Comparison of [3 H]repaglinide binding data on Kir6.2/SUR1 and Kir6.2/SUR1[S1237Y] Compound Kir6.2/SUR1 Kir6.2/SUR1[S1237Y] IC50 (nmol/l) nH Ki (nmol/l) IC50 (nmol/l) nH Ki (nmol/l) Repaglinide 1.9 Ϯ 0.8 -1.19 Ϯ 0.10 0.6 Ϯ 0.3 1.2 Ϯ 0.7 -1.05 Ϯ 0.14 0.4 Ϯ 0.2 Glibenclamide 0.7 Ϯ 0.2 -1.14 Ϯ 0.13 0.2 Ϯ 0.1 105 Ϯ 17 -0.90 Ϯ 0.25 36 Ϯ 6 Nateglinide 679 Ϯ 121 -0.98 Ϯ 0.05 235 Ϯ 42 Ͼ30,000 N/A N/A Tolbutamide 26,000 Ϯ 9,300 -0.91 Ϯ 0.12 9,000 Ϯ 3,220 Ͼ300,000 N/A N/A Data are means Ϯ SD (n ϭ 3). Login to comment
181 Glibenclamide produced a reversible block of Kir6.2/ SUR1[S1237Y]. Login to comment
184 Thus, glibenclamide displaced [3 H]repaglinide binding to the mutant channel with a much lower potency than for the wild-type channel, consistent with a larger dissociation rate constant for glibenclamide binding to Kir6.2/SUR1[S1237Y]. Login to comment
188 This suggests that the binding affinity of repaglinide is enhanced by interaction with additional residues in SUR1 and that this interaction is not disrupted by the S1237Y mutation. Login to comment
189 The effect of the S1237Y mutation on nateglinide-induced KATP channel inhibition could be due to either reduced drug binding or an impaired ability of SUR1 to transduce drug binding into channel closure. Login to comment
190 However, because Kir6.2/SUR1[S1237Y] retains the ability to be blocked fully by repaglinide and glibenclamide, the transduction mechanism does not appear to be compromised by the mutation. Login to comment
35 SUR1 currents by the nonsulphonylurea mitiglinide, as with tolbutamide, is abolished by the S1237Y mutation (16). Login to comment
46 The point mutation SUR1[S1237Y] was constructed by standard molecular biology techniques and confirmed by DNA sequencing. Login to comment
49 Cells were seeded at 50% confluency and transfected with Kir6.2 and SUR1[S1237Y] at a plasmid ratio of 1:3 on the next day. Cells to be used for electrophysiological experiments were also cotransfected with green fluorescent protein (GFP) to enable visual identification of transfected cells. Login to comment
64 Binding experiments were performed in triplicate (Kir6.2/SUR1) or duplicate (Kir6.2/SUR1[S1237Y]). Login to comment
94 After establishment of the whole-cell configuration and dialysis with intracellular solution, there was a gradual increase in both Kir6.2/SUR1 and Kir6.2/ SUR1[S1237Y] currents due to opening of KATP channels. Login to comment
96 In contrast, tolbutamide had very little effect on Kir6.2/SUR1[S1237Y] currents (Fig. 1B). Login to comment
98 Although glibenclamide also inhibited Kir6.2/SUR1[S1237Y] channels (by 67 afe; 6%; n afd; 3), in this case, inhibition was largely reversed on wash-out of the drug (Fig. 1B). Login to comment
102 A similar extent of block was observed for Kir6.2/ SUR1[S1237Y] (94 afe; 1%, n afd; 4) (Fig. 2B), suggesting that S1237 is not required for repaglinide inhibition. Login to comment
104 In contrast, nateglinide (100 òe;mol/l) produced reversible inhibition of Kir6.2/SUR1 currents (96 afe; 2%, n afd; 4) (Fig. 2A) but was without significant effect on Kir6.2/SUR1[S1237Y] channels (Fig. 2B). Login to comment
106 Repaglinide blocked Kir6.2/SUR1 and Kir6.2/SUR1[S1237Y] currents with similar potency: IC50 afd; 21 nmol/l (95% CI 17-26) and IC50 afd; 23 nmol/l (18-28), respectively. Login to comment
115 Similarly, binding of [3 H]repaglinide to Kir6.2/SUR1[S1237Y] revealed a single binding site (Fig. 4B) with a KD of 0.31 afe; 0.02 nmol/l and a Bmax of 1.6 afe; 0.2 pmol/mg protein (n afd; 3). Login to comment
119 HEK 293 cells expressing Kir6.2/SUR1 (A) or Kir6.2/SUR1[S1237Y] (B) channels were clamped at d1a;70 mV. Login to comment
123 HEK 293 cells expressing Kir6.2/SUR1 (A) or Kir6.2/ SUR1[S1237Y] (B) channels were clamped at d1a;70 mV. Login to comment
126 Concentration-response curves for inhibition of Kir6.2/SUR1 (f/F) and Kir6.2/SUR1[S1237Y] (ǧa;/E) channels by repaglinide (f/ǧa;) or nateglinide (F/E). Login to comment
131 We therefore examined the ability of unlabelled nateglinide to displace [3 H]repaglinide binding to membranes isolated from HEK293 cells expressing Kir6.2/SUR1 or Kir6.2/SUR1[S1237Y]. Login to comment
138 In the case of Kir6.2/SUR1[S1237Y], repaglinide displaced [3 H]repaglinide binding with a Ki of 0.4 afe; 0.2 nmol/l (n afd; 3), which is similar to that found for the wild-type channel. Login to comment
142 The data are consistent with the idea that the nateglinide binding, as with that of tolbutamide, is abolished by the S1237Y mutation in SUR1. Login to comment
149 Data are from a single representative experiment in which data points were collected in triplicate (Kir6.2/SUR1) or duplicate (Kir6.2/SUR1[S1237Y]). Login to comment
151 Competition binding to membranes from HEK 293 cells expressing Kir6.2/SUR1 (A) or Kir6.2/SUR1[S1237Y] (B). Login to comment
154 Data are from a single representative experiment in which data points were collected in triplicate (Kir6.2/SUR1) or duplicate (Kir62/SUR1[S1237Y]). Login to comment
180 TABLE 1 Comparison of [3 H]repaglinide binding data on Kir6.2/SUR1 and Kir6.2/SUR1[S1237Y] Compound Kir6.2/SUR1 Kir6.2/SUR1[S1237Y] IC50 (nmol/l) nH Ki (nmol/l) IC50 (nmol/l) nH Ki (nmol/l) Repaglinide 1.9 afe; 0.8 afa;1.19 afe; 0.10 0.6 afe; 0.3 1.2 afe; 0.7 afa;1.05 afe; 0.14 0.4 afe; 0.2 Glibenclamide 0.7 afe; 0.2 afa;1.14 afe; 0.13 0.2 afe; 0.1 105 afe; 17 afa;0.90 afe; 0.25 36 afe; 6 Nateglinide 679 afe; 121 afa;0.98 afe; 0.05 235 afe; 42 b0e;30,000 N/A N/A Tolbutamide 26,000 afe; 9,300 afa;0.91 afe; 0.12 9,000 afe; 3,220 b0e;300,000 N/A N/A Data are means afe; SD (n afd; 3). Login to comment
182 Glibenclamide produced a reversible block of Kir6.2/ SUR1[S1237Y]. Login to comment
185 Thus, glibenclamide displaced [3 H]repaglinide binding to the mutant channel with a much lower potency than for the wild-type channel, consistent with a larger dissociation rate constant for glibenclamide binding to Kir6.2/SUR1[S1237Y]. Login to comment
191 However, because Kir6.2/SUR1[S1237Y] retains the ability to be blocked fully by repaglinide and glibenclamide, the transduction mechanism does not appear to be compromised by the mutation. Login to comment

[hide] Role of two adjacent cytoplasmic tyrosine residues... Biochem Pharmacol. 2005 Feb 1;69(3):451-61. Epub 2004 Dec 16. Conseil G, Deeley RG, Cole SP
Role of two adjacent cytoplasmic tyrosine residues in MRP1 (ABCC1) transport activity and sensitivity to sulfonylureas.
Biochem Pharmacol. 2005 Feb 1;69(3):451-61. Epub 2004 Dec 16., [PMID:15652236]

Abstract [show]
The human ATP-binding cassette (ABC) protein MRP1 causes resistance to many anticancer drugs and is also a primary active transporter of conjugated metabolites and endogenous organic anions, including leukotriene C(4) (LTC(4)) and glutathione (GSH). The sulfonylurea receptors SUR1 and SUR2 are related ABC proteins with the same domain structure as MRP1, but serve as regulators of the K(+) channel Kir6.2. Despite their functional differences, the activity of both SUR1/2 and MRP1 can be blocked by glibenclamide, a sulfonylurea used to treat diabetes. Residues in the cytoplasmic loop connecting transmembrane helices 15 and 16 of the SUR proteins have been implicated as molecular determinants of their sensitivity to glibenclamide and other sulfonylureas. We have now investigated the effect of mutating Tyr(1189) and Tyr(1190) in the comparable region of MRP1 on its transport activity and sulfonylurea sensitivity. Ala and Ser substitutions of Tyr(1189) and Tyr(1190) caused a > or =50% decrease in the ability of MRP1 to transport different organic anions, and a decrease in LTC(4) photolabeling. Kinetic analyses showed the decrease in GSH transport was attributable primarily to a 10-fold increase in K(m). In contrast, mutations of these Tyr residues had no major effect on the catalytic activity of MRP1. Furthermore, the mutant proteins showed no substantial differences in their sensitivity to glibenclamide and tolbutamide. We conclude that MRP1 Tyr(1189) and Tyr(1190), unlike the corresponding residues in SUR1, are not involved in its differential sensitivity to sulfonylureas, but nevertheless, may be involved in the transport activity of MRP1, especially with respect to GSH.

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41 Binding of [3 H]glibenclamide to membranes expressing SUR1-S1237Y is abolished concomitantly with the loss of high-affinity tolbutamide block. Login to comment

[hide] Effects of mitiglinide (S 21403) on Kir6.2/SUR1, K... Br J Pharmacol. 2001 Apr;132(7):1542-8. Reimann F, Proks P, Ashcroft FM
Effects of mitiglinide (S 21403) on Kir6.2/SUR1, Kir6.2/SUR2A and Kir6.2/SUR2B types of ATP-sensitive potassium channel.
Br J Pharmacol. 2001 Apr;132(7):1542-8., [PMID:11264248]

Abstract [show]
1. We have investigated the mechanism of action of the novel anti-diabetic agent mitiglinide (S 21403) on Kir6.2/SUR1, Kir6.2/SUR2A and Kir6.2/SUR2B types of ATP-sensitive potassium (K(ATP)) channel. These possess a common pore-forming subunit, Kir6.2, and different regulatory sulphonylurea receptor (SUR) subunits. It is believed that they correspond to native K(ATP) channels in pancreatic beta-cells, heart and non-vascular smooth muscle, respectively. 2. Kir6.2 was coexpressed with SUR1, SUR2A or SUR2B in Xenopus oocytes and macroscopic currents were recorded in giant inside-out membrane patches. Mitiglinide was added to the intracellular membrane surface. 3. Mitiglinide inhibited Kir6.2/SUR currents at two sites: a low-affinity site on Kir6.2 and a high-affinity site on SUR. Low-affinity inhibition was similar for all three types of K(ATP) channel but high-affinity inhibition was greater for Kir6.2/SUR1 currents (IC(50), 4 nM) than for Kir6.2/SUR2A or Kir6.2/SUR2B currents (IC(50), 3 and 5 microM, respectively). 4. Inhibition of Kir6.2/SUR1 currents was only slowly reversible on the time scale of electrophysiological experiments. 5. Kir6.2/SUR1-S1237Y currents, which previously have been shown to lack high affinity tolbutamide inhibition, resembled Kir6.2/SUR2 currents in being unaffected by 100 nM but blocked by 10 microM mitiglinide. 6. Our results show that mitiglinide is a high-affinity drug that shows a 1000 fold greater affinity for the beta-cell type than the cardiac and smooth muscle types of K(ATP) channel, when measured in excised patches.

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8 5 Kir6.2/SUR1-S1237Y currents, which previously have been shown to lack high anity tolbutamide inhibition, resembled Kir6.2/SUR2 currents in being unaected by 100 nM but blocked by 10 mM mitiglinide. Login to comment
45 A mutant form of SUR1 (SUR1-S1237Y) and a truncated form of Kir6.2 (Kir6.2DC36), which lacks the C-terminal 36 amino acids and forms functional channels in the absence of SUR, were prepared as described previously (Ash®eld et al., 1999; Tucker et al., 1997). Login to comment
110 We next investigated whether mitiglinide binds to the same (or an overlapping) site on SUR1 as tolbutamide, by examining the eect of mitiglinide on channels containing a mutant form of SUR1, SUR1-S1237Y. Login to comment
112 As shown in Figure 5, Kir6.2/ SUR1-S1237Y currents were unaected by 100 nM mitiglinide but were blocked by 65.5+3.0% (n=9) in the presence of 10 mM of the drug. Login to comment
113 There was no signi®cant dierence in the extent of inhibition of Kir6.2/SUR1, Kir6.2/SUR1-S1237Y or KIR6.2/SUR2A currents produced by 10 mM mitiglinide. Login to comment
114 However, the block of Kir6.2/SUR1-S1237Y currents was readily reversible, which is in contrast to the block of Kir6.2/SUR1 currents but similar that of Kir6.2/ SUR2-type currents. Login to comment
123 SUR1 containing the mutation S1237Y lacks both high-anity tolbutamide block and [3 H]-glibenclamide binding (Ash®eld et al., 1999). Login to comment
124 We found that Kir6.2/SUR1-S1237Y channels were blocked by less than 10% by 100 nM mitiglinide, a concentration that saturates the high-anity site on SUR1. Login to comment
126 In contrast, 10 mM mitiglinide blocked Kir6.2/SUR1-S1237Y channels as much as Kir6.2/SUR1 and Kir6.2/SUR2A channels, but Kir6.2DC channels were not aected by this drug concentration. Login to comment
127 This further suggests that mitiglinide binds with intermediate anity to a site that is common to SUR1-S1237Y and SUR2. Login to comment
129 In contrast, the intermediate-anity block of Kir6.2/SUR2A, Kir6.2/SUR2B and Kir6.2/SUR1-S1237Y currents was readily reversible. Login to comment
144 In contrast to the wild-type channel, Kir6.2/SUR1-S1237Y channels are blocked to a similar extent by 10 mM meglitinide and 10 mM mitiglinide (*65%; this study and Ash®eld et al., 1999). Login to comment
158 This conclusion is based on experiments in which the drug was applied to the intracellular surface of excised membrane patches and therefore should not be extrapolated directly to the whole-cell condition, because cytosolic substances may modify the drug properties (e.g. Ventakesh et al., 1991; Kir6.2/SUR1-S1237Y Figure 5 Inhibition of Kir6.2/SUR1-S1237Y currents by S21403. Login to comment
159 (A) Macroscopic currents recorded from inside-out patches in response to a series of voltage ramps from 7110 to +100 mV from oocytes expressing Kir6.2 and SUR1-S1237Y. Login to comment

[hide] Testing the bipartite model of the sulfonylurea re... J Pharmacol Exp Ther. 2007 Aug;322(2):701-8. Epub 2007 May 10. Winkler M, Stephan D, Bieger S, Kuhner P, Wolff F, Quast U
Testing the bipartite model of the sulfonylurea receptor binding site: binding of A-, B-, and A + B-site ligands.
J Pharmacol Exp Ther. 2007 Aug;322(2):701-8. Epub 2007 May 10., [PMID:17495126]

Abstract [show]
ATP-sensitive K(+) (K(ATP)) channels are composed of pore-forming subunits (Kir6.x) and of regulatory subunits, the sulfonylurea receptors (SURx). Subtypes of K(ATP) channels are expressed in different organs. The sulfonylureas and glinides (insulinotropes) close the K(ATP) channel in pancreatic beta-cells and stimulate insulin secretion. The insulinotrope binding site of the pancreatic channel (Kir6.2/SUR1) consists of two overlapping (sub)-sites, site A, located on SUR1 and containing Ser1237 (which in SUR2 is replaced by Tyr1206), and site B, formed by SUR1 and Kir6.2. Insulinotropes bind to the A-, B-, or A + B-site(s) and are grouped accordingly. A-ligands are highly selective in closing the pancreatic channel, whereas B-ligands are nonselective and insensitive to the mutation S1237Y. We have examined the binding of insulinotropes representative of the three groups in [(3)H]glibenclamide competition experiments to determine the contribution of Kir6.x to binding affinity, the effect of the mutation Y1206S in site A of SUR2, and the subtype selectivity of the compounds. The results show that the bipartite nature of the SUR1 binding site applies also to SUR2. Kir6.2 as part of the B-site may interact directly or allosterically with structural elements common to all insulinotropes, i.e., the negative charge and/or the adjacent phenyl ring. The B-site confers a moderate subtype selectivity on B-ligands. The affinity of B-ligands is altered by the mutation SUR2(Y1206S), suggesting that the mutation affects the binding chamber of SUR2 as a whole or subsite A, including the region where the subsites overlap.

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5 A-ligands are highly selective in closing the pancreatic channel, whereas B-ligands are nonselective and insensitive to the mutation S1237Y. Login to comment
33 In addition, their potency in closing the pancreatic channel was not affected by the mutation S1237Y in SUR1 (Ashfield et al., 1999; Hansen et al., 2002). Login to comment
39 We evaluated the effect of coexpression with Kir6.x on the affinity of SURx for the insulinotropes, the effect of the mutation SUR2(Y1206S), i.e., the reverse of the mutation SUR1(S1237Y), and the selectivity in binding to the recombinant pancreatic, myocardial, and vascular KATP channels. Login to comment

[hide] Identification of the high-affinity tolbutamide si... Diabetes. 1999 Jun;48(6):1341-7. Ashfield R, Gribble FM, Ashcroft SJ, Ashcroft FM
Identification of the high-affinity tolbutamide site on the SUR1 subunit of the K(ATP) channel.
Diabetes. 1999 Jun;48(6):1341-7., [PMID:10342826]

Abstract [show]
ATP-sensitive potassium channels (K(ATP)) are formed from four pore-forming Kir6.2 subunits complexed with four regulatory sulfonylurea receptor subunits (SUR1 in pancreatic beta-cells, SUR2A in heart). The sensitivity of the channel to different sulfonylureas depends on the SUR isoform. In particular, Kir6.2-SUR1 but not Kir6.2-SUR2A channels are blocked by tolbutamide with high affinity. We made chimeras between SUR1 and SUR2A to identify the region of the protein involved in high-affinity tolbutamide block. Chimeric SURs were coexpressed with Kir6.2 in Xenopus oocytes, and macroscopic currents were measured in inside-out membrane patches. High-affinity tolbutamide inhibition could be conferred on SUR2A by replacing transmembrane domains (TMs) 14-16 with the corresponding region of SUR1. Conversely, high-affinity tolbutamide inhibition of SUR1 was abolished by replacing TMs 13-16 with the corresponding SUR2A sequence, or by mutating a single serine residue within this region to tyrosine (S1237Y). Binding of [3H]glibenclamide to membranes expressing SUR1 was abolished concomitantly with the loss of high-affinity tolbutamide block. These results suggest that a site in the COOH-terminal set of TMs of the SUR1 subunit of the K(ATP) channel is involved in the binding of tolbutamide and glibenclamide.

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4 High-affinity tolbutamide inhibition could be conferred on SUR2A by replacing transmembrane domains (TMs) 14-16 with the corresponding region of SUR1. Conversely, high-affinity tolbutamide inhibition of SUR1 was abolished by replacing TMs 13-16 with the corresponding SUR2A sequence, or by mutating a single serine residue within this region to tyrosine (S1237Y). Login to comment
32 alent region of SUR2A or by a mutation within this region (S1237Y). Login to comment
138 While MgADP did not promote tolbutamide inhibition of Kir6.2-SUR1(S1237Y) currents, the nucleotide was able to enhance meglitinideblock(Fig. 4). Login to comment
145 The dashed lines indicate the data for Kir6.2-SUR1 and Kir6.2-SUR2A given in A and B. D: Kir6.2-SUR1(S1237Y) currents were fit with equation 4: Ki1 = 1.3 mmol/l, h1 = .9, L = 0 (n = 5). Login to comment
153 We comparedthe binding of [3 H]glibenclamide to SUR1, SUR12-e, and SUR1(S1237Y) expressed in Cos7 cells, and evaluatedprotein expression by immunoblotting with the M2 anti-FLAG antibody (Fig. 6). Login to comment
154 Binding of [3 H]glibenclamide to SUR12-e or SUR1(S1237Y) was not significantly greater than that found inuntransfected cells. Login to comment
158 High-affinity tolbutamide inhibition could be conferred on SUR2A by replacing TMs 14-16 with the equivalent region of SUR1. Conversely, the reverse chimera, or a mutation (S1237Y) within this region of SUR1, abolished both high-affinity tolbutamide block and [3 H]glibenclamide binding. Login to comment
169 Effects of tolbutamide (A) or meglitinide (B) in the presence or absence of 100 µmol/l MgADP, for channels comprising Kir6.2 and either SUR1, SUR2A, or SUR1(S1237Y). Login to comment
179 Oocytes were coinjected with mRNAs encoding Kir6.2 and either SUR1, SUR2A, SUR12-e, SUR21-x, or SUR1(S1237Y). Login to comment
194 This can be most simply explained if the S1237Y mutation specifically impaired tolbutamide binding, without interfering with either meglitinide binding or the mechanism by which sulfonylureas interfere with channel activation by MgADP. Login to comment
198 Thus, when SUR1 was rendered insensitive to tolbutamide [SUR12-e or SUR1(S1237Y)], glibenclamide block became reversible, while endowment of SUR2A with tolbutamide sensitivity (SUR21-x) was accompanied by irreversible glibenclamide inhibition. Login to comment
206 A: Membranes from Cos7 cells expressing SUR1, SUR12-e, or SUR1(S1237Y) were separated by SDS polyacrylamide gel electrophoresis, and probed with the anti-FLAG monoclonal antibody M2. Login to comment
208 B: [3 H]glibenclamide binding to membranes from Cos7 cells transfected with SUR1, SUR12-e, or SUR1(S1237Y) or to untransfected cells. Login to comment
220 In support of this view, we found that SUR12-e and SUR1(S1237Y), which form KATP channels that are blocked reversibly by glibenclamide, did not exhibit significant [3 H]glibenclamide binding. Login to comment
224 Conversely, high-affinity tolbutamide block and [3 H]glibenclamide binding were abolished by the reverse chimera or by mutation of a single amino acid, S1237Y, in the intracellular loop between TMs 15 and 16 of SUR1. Login to comment