1 The binding of modulators of the ATP-sensitive K + channel (K ATP channel) to the murine sulphonylurea receptor, SUR2B, was investigated. SUR2B, a proposed subunit of the vascular K ATP channel, was expressed in HEK 293 cells and binding assays were performed in membranes at 378C using the tritiated K ATP channel opener, .4 mM at 3 mM and 1 mM free Mg 2+ , respectively. Glibenclamide enhanced the dissociation of the [ 3 H]-P1075-SUR2B complex suggesting a negative allosteric coupling between the binding sites for P1075 and the sulphonylureas. 6 It is concluded that an MgATP site on SUR2B with mM a nity must be occupied to allow opener binding whereas Mg 2+ concentrations 510 mM decrease the a nities for openers and glibenclamide. The properties of the [ 3 H]-P1075 site strongly suggest that SUR2B represents the drug receptor of the openers in vascular smooth muscle.

ATP-dependent K(+) channels are composed of pore-forming subunits of the Kir6.x family and of sulfonylurea receptors (SURs). SUR1, expressed in pancreatic beta-cells, has a higher affinity for sulfonylureas, such as glibenclamide, than SUR2B, expressed in smooth muscle. This difference is mainly caused by serine 1237 in SUR1 corresponding to tyrosine 1206 in SUR2B. To increase the affinity of SUR2B for glibenclamide, the mutant SUR2B(Y1206S) was constructed. In whole-cell patch-clamp experiments, glibenclamide inhibited the channel formed by coexpression of mutant SUR2B with Kir6.1 or 6.2 in human embryonic kidney cells with IC(50) values of 2.7 and 13 nM, respectively (wild-type, 43 and 167 nM). In intact cells, [(3)H]glibenclamide bound to mutant SUR2B with a K(D) value of 4.7 nM (wild-type, 32 nM); coexpression with Kir6.1 or 6.2 increased affinity by 4- and 8-fold, respectively. Binding of the opener [(3)H]P1075 to SUR2B(Y1206S) was the same as to wild-type and was unaffected by coexpression. In cells, the ratio of glibenclamide:P1075 sites was approximately 1:1; in membranes, it varied with the MgATP concentration. Heterologous competition curves were generally biphasic; the shape of the curve depended on the Kir-subtype. The effects of coexpression were weakened or abolished when binding assays were conducted in membranes. It is concluded that the mutation Y1206S increases the affinity of SUR2B for and the channel sensitivity toward glibenclamide by 7- to 15-fold. The interaction of glibenclamide (but not opener) with mutant SUR2B is modified by coexpression with Kir6.x in a manner depending on the Kir subtype and on the integrity of the cell.

1 ATP-sensitive K + channels are composed of pore-forming subunits Kir6.2 and of sulphonylurea receptors (SURs); the latter are the target of the hypoglycaemic sulphonylureas like glibenclamide. Here, we report on the negative allosteric modulation by MgATP and MgADP of glibenclamide binding to SUR1 and to SUR2 mutants with high glibenclamide anity, SUR2A(Y1206S) and SUR2B(Y1206S). 2 ATP, in the presence of an ATP-regenerating system to oppose hydrolysis during incubation, inhibited glibenclamide binding to SUR1 and SUR2B(Y1206S) by *60%, to SUR2A(Y1206S) by 21%). Inhibition curves for the SUR2(Y1206S) isoforms were monophasic with IC 50 values of 5 ± 10 mM; the curve for SUR1 was biphasic (IC 50 values 4.7 and 1300 mM). 3 Glibenclamide inhibition curves for ADP, performed in the presence of an ATP-consuming system to oppose ATP formation from ADP, were generally shifted rightwards and showed positive cooperativity, in particular with the SUR2(Y1206S) isoforms. 4 In the absence of the coupled enzyme systems, inhibition curves of MgATP or MgADP were generally shifted leftwards. This indicated synergy of MgATP and MgATP in acting together. 5 Coexpression of SUR1 and SUR2B(Y1206S) with Kir6.2 reduced both potency and ecacy of ATP in inhibiting glibenclamide binding; this was particularly marked for Kir6.2/SUR1. 6 The data show (a) that the inhibitory eects of ATP and ADP on glibenclamide binding dier from one another, (b) that they depend on the SUR subtype, and (c) that they are weakened by coexpression with Kir6.2.

ATP-sensitive K ϩ channels are closed by the hypoglycemic sulfonylureas like glibenclamide (GBC) and activated by a class of vasorelaxant compounds, the K ϩ channel openers. These channels are octamers of Kir6.x and sulfonylurea receptor (SUR) subunits with 4:4 stoichiometry. The properties of the opener-sensitive K ϩ channel in the vasculature are well matched by the SUR2B/Kir6.1 channel; however, the GBC sensitivity of the recombinant channel is unknown. In binding experiments we have determined the affinity of GBC for SUR2B and the SUR2B/Kir6.1 channel and compared the results with the channel blocking potency of GBC. All experiments were performed in whole transfected human embryonic kidney cells at 37°C. The equilibrium dissociation constants (K D ) of GBC binding to SUR2B and to the SUR2B/Kir6.1 complex were determined to be 32 and 6 nM, respectively; the K D value of the opener P1075 (N-cyano-NЈ-(1,1-dimethylpropyl)-NЈЈ-3-pyridylguanidine) (Ϸ5 nM) was, however, not affected by cotransfection. In whole cell voltage-clamp experiments, GBC inhibited the SUR2B/Kir6.1 channel with IC 50 Ϸ 43 nM. The data show that, in the intact cell: 1) SUR2B, previously considered to be a low-affinity SUR, has a rather high affinity for GBC; 2) coexpression with the inward rectifier Kir6.1 increases the affinity of SUR2B for GBC; 3) the recombinant channel exhibits the same GBC affinity as the opener-sensitive K ϩ channel in vascular tissue; and 4) the K D value of GBC binding to the octameric channel is 7 times lower than the IC 50 value for channel inhibition. The latter finding suggests that occupation of all four GBC sites per channel is required for channel closure.

Sulfonylurea receptors (SURs) constitute the regulatory subunits of ATP-sensitive K ؉ channels (K ATP channels). SUR binds nucleotides and synthetic K ATP channel modulators, e.g. the antidiabetic sulfonylurea glibenclamide, which acts as a channel blocker. However, knowledge about naturally occurring ligands of SUR is very limited. In this study, we show that the plant phenolic compound trans-resveratrol can bind to SUR and displace binding of glibenclamide. Electrophysiological measurements revealed that resveratrol is a blocker of pancreatic SUR1/ K IR 6.2 K ATP channels. We further demonstrate that, like glibenclamide, resveratrol induces enhanced apoptosis. This was shown by analyzing different apoptotic parameters (cell detachment, nuclear condensation and fragmentation, and activities of different caspase enzymes). The observed apoptotic effect was specific to cells expressing the SUR1 isoform and was not mediated by the electrical activity of K ATP channels, as it was observed in human embryonic kidney 293 cells expressing SUR1 alone. Enhanced susceptibility to resveratrol was not observed in pancreatic ␤-cells from SUR1 knock-out mice or in cells expressing the isoform SUR2A or SUR2B or the mutant SUR1(M1289T). Resveratrol was much more potent than glibenclamide in inducing SUR1-specific apoptosis. Treatment with etoposide, a classical inducer of apoptosis, did not result in SUR isoform-specific apoptosis. In conclusion, resveratrol is a natural SUR ligand that can induce apoptosis in a SUR isoform-specific manner. Considering the tissue-specific expression patterns of SUR isoforms and the possible effects of SUR mutations on susceptibility to apoptosis, these observations could be important for diabetes and/or cancer research.Sulfonylurea receptors (SURs) 2 are members of the ATPbinding cassette protein family (subfamily C). SURs are known to be the important regulatory subunits of ATP-sensitive K ϩ channels (K ATP channels). These channels are heteromeric complexes composed of four SUR subunits that surround a central pore formed by four subunits from the K IR 6.x family. K ATP channels found in various tissues exhibit distinct physiological and pharmacological properties because of the combination of different subunit isoforms (reviewed in Ref. 1). In addition to two nucleotide-binding domains, SUR possesses binding sites for synthetic K ATP channel modulators. The binding sites for blockers and openers are different, but they are linked via complex allosteric interactions (2, 3).Because of their nucleotide sensitivity, K ATP channels couple the energy metabolism of a cell to the membrane potential. This is important in the pancreatic ␤-cell, in which closure of K ATP channels triggers insulin secretion via membrane depolarization in response to changes in blood glucose levels (4 -6). K ATP channel-blocking drugs such as the sulfonylureas and the glinides can promote insulin secretion and are used in the treatment of diabetes type 2. Insulin secretion is also modulated and amplified by other pathways...

Sulfonylurea receptors (SURs) are subunits of ATP-sensitive K ϩ channels (K ATP channels); they mediate the channel-closing effect of sulfonylureas such as glibenclamide and the channelactivating effect of K ATP channel openers such as the pinacidil analog P1075. We investigated the inhibition by MgATP and P1075 of glibenclamide binding to SUR2B, the SUR subtype in smooth muscle. To increase specific binding, experiments were also performed using SUR2B(Y1206S), a mutant with higher affinity for glibenclamide than for the wild-type (K D ϭ 4 versus 22 nM, respectively) but otherwise exhibiting similar pharmacological properties. (1 mM), inhibition was biphasic with one K i value resembling the true affinity of P1075 for SUR2B (2-6 nM) and the other resembling K i in the absence of MgATP (Ϸ1 M). The data show that (1) MgATP induces heterogeneity in the glibenclamide sites; (2) the high-affinity glibenclamide sites remaining with MgATP are linked to two classes of P1075 sites; and (3) P1075 interacts specifically with SUR2B also in the absence of MgATP. The data are discussed with the assumption that SUR2B, expressed alone, forms tetramers; that MgATP induces allosteric interactions between the subunits; and that mixed SUR2B-glibenclamide-P1075 complexes can exist at equilibrium.

We have demonstrated a functional coupling between the beta(3)-adrenoceptor and ion channel function in the mammalian heart. Our findings point to a potential role for beta(3)-adrenoceptors in cardiac electrophysiology and pathophysiology.

Sulfonylurea receptor 1 (SUR1) is the regulatory subunit of the pancreatic ATP-sensitive K ϩ channel (K ATP channel), which is essential for triggering insulin secretion via membrane depolarization. Sulfonylureas, such as glibenclamide and tolbutamide, act as K ATP channel blockers and are widely used in diabetes treatment. These antidiabetic substances are known to induce apoptosis in pancreatic ␤-cells or ␤-cell lines under certain conditions. However, the precise molecular mechanisms of this sulfonylurea-induced apoptosis are still unidentified. To investigate the role of SUR in apoptosis induction, we tested the effect of glibenclamide on recombinant human embryonic kidney 293 cells expressing either SUR1, the smooth muscular isoform SUR2B, or the mutant SUR1(M1289T) at which a single amino acid in transmembrane helix 17 (TM17) was exchanged by the corresponding amino acid of SUR2. By analyzing cell detachment, nuclear condensation, DNA fragmentation, and caspase-3-like activity, we observed a SUR1-specific enhancement of glibenclamide-induced apoptosis that was not seen in SUR2B, SUR1(M1289T), or control cells. Coexpression with the pore-forming Kir6.2 subunit did not significantly alter the apoptotic effect of glibenclamide on SUR1 cells. In conclusion, expression of SUR1, but not of SUR2B or SUR1(M1289T), renders cells more susceptible to glibenclamide-induced apoptosis. Therefore, SUR1 as a pancreatic protein could be involved in specific variation of ␤-cell mass and might also contribute to the regulation of insulin secretion at this level. According to our results, TM17 is essentially involved in SUR1-mediated apoptosis. This effect does not require the presence of functional Kir6.2-containing K ATP channels, which points to additional, so far unknown functions of SUR.Sulfonylurea receptors (SUR) are members of the ATPbinding cassette protein family and form important regulatory subunits of ATP-sensitive K ϩ channels (K ATP channels). These channels are heterotetrameric complexes formed by SUR and the pore-forming Kir6.x subunit. Different combinations of these subunits (SUR1, SUR2A, or SUR2B and Kir6.1 or Kir6.2) form channels in various tissues with distinct pharmacological and electrophysiological properties. SUR2 is encoded by a different gene than SUR1. Alternative splicing of the SUR2 gene leads to expression of either SUR2A, predominantly found in heart and skeletal muscle, or SUR2B, typically occurring in smooth vascular muscle (Gribble and Reimann, 2003). To some extent, SUR1 and SUR2 show inverse pharmacological profiles; SUR1 exhibits high affinity for several sulfonylureas but low affinity for most K ATP channel openers, whereas SUR2 shows lower affinity for sulfonylureas and high affinity for openers (Schwanstecher et al., 1998;Hambrock et al., 2002).In the pancreatic ␤-cell, K ATP channels (constituted by SUR1 and Kir6.2) are essential for triggering insulin secretion via membrane depolarization. High blood glucose concentrations lead to an elevated ATP/ADP ratio and result in clo...