17beta-Estradiol modulates apoptosis in pancreatic beta-cells by specific involvement of the sulfonylurea receptor (SUR) isoform SUR1.

Apoptosis of pancreatic beta-cells is an important factor in the pathophysiology of diabetes. Previously, we have shown that the "phytoestrogen" resveratrol can induce beta-cell apoptosis dependent on the expression of sulfonylurea receptor (SUR) 1, the regulatory subunit of pancreatic ATP-sensitive K(+) channels. Here, we investigate whether 17beta-estradiol also influences beta-cell apoptosis in a SUR1-dependent manner. Therefore, islets from wild type or SUR1 knock-out mice, clonal beta-cells, or HEK293 cells expressing different SUR forms were treated with 17beta-estradiol or estrone. Different apoptotic parameters were determined and estrogen binding to SUR was analyzed. In murine islets, 17beta-estradiol treatment resulted in significant apoptotic changes, which in their nature (either apoptotic or anti-apoptotic) were dependent on the age of the animal. These effects were not observed in SUR1 knock-out mice. Furthermore, 17beta-estradiol, which specifically binds to SUR, induced enhanced apoptosis in SUR1-expressing HEK293 cells and clonal beta-cells, whereas apoptosis in recombinant cells expressing SUR2A or SUR2B (cardiac or vascular SUR-isoforms) or sham-transfected control cells was significantly lower. The apoptotic potency of 17beta-estradiol was much higher than that of resveratrol or estrone. SUR1-specific 17beta-estradiol-induced apoptosis was either abolished by the mutation M1289T in transmembrane helix 17 of SUR1 or clearly enhanced by two mutations in nucleotide binding fold 2 (R1379C, R1379L). In conclusion, 17beta-estradiol treatment modulates beta-cell apoptosis under specific involvement of SUR1 in an age-dependent manner. 17beta-Estradiol-induced apoptosis can be influenced by certain SUR1 mutations. These findings may contribute to the understanding of pathophysiological changes in beta-cell mass and could, for instance, provide interesting aspects concerning the etiology of gestational diabetes.

Resveratrol binds to the sulfonylurea receptor (SUR) and induces apoptosis in a SUR subtype-specific manner.

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 beta-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.

Glibenclamide-induced apoptosis is specifically enhanced by expression of the sulfonylurea receptor isoform SUR1 but not by expression of SUR2B or the mutant SUR1(M1289T).

Sulfonylurea receptor 1 (SUR1) is the regulatory subunit of the pancreatic ATP-sensitive K+ 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 beta-cells or beta-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 beta-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.

The mutation Y1206S increases the affinity of the sulphonylurea receptor SUR2A for glibenclamide and enhances the effects of coexpression with Kir6.2.

1. ATP-sensitive K(+) channels (K(ATP) channels) are tetradimeric complexes of inwardly rectifying K(+) channels (Kir6.x) and sulphonylurea receptors (SURs). The SURs SUR2A (cardiac) and SUR2B (smooth muscle) differ only in the last 42 amino acids. In SUR2B, the mutation Y1206S, located at intracellular loop 8, increases the affinity for glibenclamide (GBC) about 10-fold. Here, we examined whether the mutation Y1206S in SUR2A had effects similar to those in SUR2B.2. GBC bound to SUR2A with K(D)=20 nM; the mutation increased affinity approximately 5 x. 3. In cells, coexpression of SUR2A with Kir6.2 increased the affinity for GBC approximately 3 x; with the mutant, the increase was 9 x. 4. The mutation did not affect the affinity of SUR2A for openers; coexpression with Kir6.2 reduced opener affinity of wild-type and mutant SUR2A by about 2 x. 5. The negative allosteric interaction between the opener, P1075, and GBC at wild-type and mutant SUR2A was markedly affected by the presence of MgATP and by coexpression with Kir6.2. 6. In inside-out patches, GBC inhibited the wild-type Kir6.2/SUR2A and 2B channels with IC(50) values of 27 nM; the mutation shifted the IC(50) values to approximately 1 nM. 7. The data show that the mutation Y1206S increased the affinity of SUR2A for GBC and modulated the effects of coexpression. Overall, the changes were similar to those observed with SUR2B(Y1206S), suggesting that the differences in the last 42 carboxy-terminal amino acids of SUR2A and 2B are of limited influence on the binding of GBC and P1075 to the SUR2 isoforms.

Effect of two amino acids in TM17 of Sulfonylurea receptor SUR1 on the binding of ATP-sensitive K+ channel modulators.

The sulfonylurea receptor (SUR) is the important regulatory subunit of ATP-sensitive K+ channels. It is an ATP-binding cassette protein comprising 17 transmembrane helices. SUR is endowed with binding sites for channel blockers like the antidiabetic sulfonylurea glibenclamide and for the chemically very heterogeneous channel openers. SUR1, the typical pancreatic SUR isoform, shows much higher affinity for glibenclamide but considerably lower affinity for most openers than SUR2. In radioligand binding assays, we investigated the role of two amino acids, T1285 and M1289, located in transmembrane helix (TM)-17, in opener binding to SUR1. These amino acids were exchanged for the corresponding amino acids of SUR2. In competition experiments using [3H]glibenclamide as radioligand, SUR1(T1285L, M1289T) showed much higher affinity toward the cyanoguanidine openers pinacidil and P1075 than SUR1 wild type. The affinity for the thioformamide aprikalim was also markedly increased. In contrast, the affinity for the benzopyrans rilmakalim and levcromakalim was unaffected; however, the amount of displaced [3H]glibenclamide binding was nearly doubled. The binding properties of the opener diazoxide and the blocker glibenclamide were unchanged. In conclusion, mutation of two amino acids in TM17 of SUR1, especially of M1289, leads to class-specific effects on opener binding by increasing opener affinity or by changing allosteric coupling between opener and glibenclamide binding.

Interaction of a novel dihydropyridine K+ channel opener, A-312110, with recombinant sulphonylurea receptors and KATP channels: comparison with the cyanoguanidine P1075.

1. ATP-sensitive K(+) channels (K(ATP) channels) are composed of pore-forming subunits (Kir6.x) and of regulatory subunits, the sulphonylurea receptors (SURx). Synthetic openers of K(ATP) channels form a chemically heterogeneous class of compounds that are of interest in several therapeutic areas. We have investigated the interaction of a novel dihydropyridine opener, A-312110 ((9R)-9-(4-fluoro-3-iodophenyl)-2,3,5,9-tetrahydro-4H-pyrano[3,4-b]thieno [2,3-e]pyridin-8(7H)-one-1,1-dioxide), with SURs and Kir6/SUR channels in comparison to the cyanoguanidine opener P1075. 2. In the presence of 1 mM MgATP, A-312110 bound to SUR2A (the SUR in cardiac and skeletal muscle) and to SUR2B (smooth muscle) with K(i) values of 14 and 18 nM; the corresponding values for P1075 were 16 and 9 nM, respectively. Decreasing the MgATP concentration reduced the affinity of A312110 binding to SUR2A significantly more than that to SUR2B; for P1075, the converse was true. At SUR1 (pancreatic beta-cell), both openers showed little binding up to 100 microM. 3. In the presence of MgATP, both openers inhibited [(3)H]glibenclamide binding to the SUR2 subtypes in a biphasic manner. In the absence of MgATP, the high-affinity component of the inhibition curves was absent. 4. In inside-out patches, the two openers activated the Kir6.2/SUR2A and Kir6.2/SUR2B channels with similar potency (approximately 50 nm). Both were almost 2 x more efficacious in opening the Kir6.2/SUR2B than the Kir6.2/SUR2A channel. 5. The results show that the novel dihydropyridine A-312110 is a potent K(ATP) channel opener with binding and channel-opening properties similar to those of P1075.

Binding and effect of K ATP channel openers in the absence of Mg2+.

1 Openers of ATP-sensitive K(+) channels (K(ATP) channels) are thought to act by enhancing the ATPase activity of sulphonylurea receptors (SURs), the regulatory channel subunits. At higher concentrations, some openers activate K(ATP) channels also in the absence of MgATP. Here, we describe binding and effect of structurally diverse openers in the absence of Mg(2+) and presence of EDTA. 2 Binding of openers to SUR2B was measured using a mutant with high affinity for [(3)H]glibenclamide ([(3)H]GBC). In the absence of Mg(2+), 'typical' openers (benzopyrans, cyanoguanidines and aprikalim) inhibited [(3)H]GBC binding with K(i) values approximately 200 x higher than in the presence of MgATP. Minoxidil sulphate and nicorandil were inactive, whereas binding of diazoxide was unaffected by MgATP. 3 In the absence/presence of MgATP, N-cyano-N'-(1,1-dimethylpropyl)-N"-3-pyridylguanidine (P1075) activated the Kir6.2/SUR2B channel in inside-out patches with EC(50)=2000/67nM and E(max)=32/134%. In the absence of Mg(2+), responses were variable with only a small part of the variability being explained by a decrease in channel responsiveness with time after patch excision and to differences in the ATP sensitivity between patches. 4 The rank order of efficacy of the openers was P1075>rilmakalim approximately nicorandil>diazoxide>minoxidil sulphate. 5 The data show that structurally diverse openers are able to bind to, and to activate the Kir6.2/SUR2B channel by a pathway independent of ATP hydrolysis. These effects are observed at concentrations used to define the biochemical mechanism of the openers in the presence of MgATP and allow the openers to be classified into 'typical' and 'atypical' KCOs with diazoxide standing apart.

beta3-Adrenergic regulation of an ion channel in the heart-inhibition of the slow delayed rectifier potassium current I(Ks) in guinea pig ventricular myocytes.

OBJECTIVES: I(Ks), the slow component of the delayed rectifier potassium current, underlies a strong beta-adrenergic regulation in the heart. Catecholamines, like isoproterenol, induce a strong increase in I(Ks). Recent work has pointed to an opposing biological effect of beta(1)- and beta(3)-adrenoceptors in the heart. However the role of these subtypes in the regulation of cardiac ion channel function is unknown. METHODS: We investigated the effects of beta(1)- and beta(3)-adrenoceptor modulation on I(Ks) in guinea-pig ventricular myocytes, using patch-clamp techniques. RESULTS: Superfusion with 100 nmol/l isoproterenol increased the step current amplitude by 81.3+/-8.0%. In contrast, after block of beta(1)- (1 micromol/l atenolol) and beta(2)-receptors (1 micromol/l ICI118,551), isoproterenol induced a reduction of the step current amplitude by 34.3+/-3.5%. The beta(3)-selective agonist BRL37344 significantly reduced the I(Ks) step current at +70 mV in a concentration-dependent manner (IC(50): 5.01 nmol/l). In the presence of bupranolol (beta(1)-, beta(2)- and beta(3)-adrenoceptor antagonist), the effect of BRL37344 was markedly attenuated, from 27.3+/-5.6% (100 nmol/l BRL37344 alone) to 4.0+/-1.3% (100 nmol/l BRL37344+1 micromol/l bupranolol). BRL37344 (100 micromol/) did not alter current amplitudes of KvLQT1/minK expressed in CHO cells or in Xenopus oocytes, excluding a direct effect of BRL37344 on the channel. 1 micromol/l BRL37344 mildly prolonged action potentials in guinea pig ventricle (APD(90):+7.8%) CONCLUSIONS: 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.

Mg2+ sensitizes KATP channels to inhibition by DIDS: dependence on the sulphonylurea receptor subunit.

1. ATP-sensitive potassium channels (K(ATP) channels) consist of pore-forming Kir6.x subunits and of sulphonylurea receptors (SURs). In the absence of Mg(2+), the stilbene disulphonate, DIDS, irreversibly inhibits K(ATP) channels by binding to the Kir subunit. Here, the effects of Mg(2+) on the interaction of DIDS with recombinant K(ATP) channels were studied in electrophysiological and [(3)H]-glibenclamide binding experiments. 2. In inside-out macropatches, Mg(2+) (0.7 mM) increased the sensitivity of K(ATP) channels towards DIDS up to 70 fold (IC(50)=2.7 micro M for Kir6.2/SUR2B). Inhibition of current at DIDS concentrations > or =10 micro M was irreversible. 3. Mg(2+) sensitized the truncated Kir6.2Delta26 channel towards inhibition by DIDS only upon coexpression with a SUR subunit (SUR2B). The effect of Mg(2+) did not require the presence of nucleotides. 4. [(3)H]-glibenclamide binding to SUR2B(Y1206S), a mutant with improved affinity for glibenclamide, was inhibited by DIDS. The potency of inhibition was increased by Mg(2+) and by coexpression with Kir6.2. 5. In the presence of Mg(2+), DIDS inhibited binding of [(3)H]-glibenclamide to Kir6.2/SUR2B(Y1206S) with IC(50)=7.9 micro M by a non-competitive mechanism. Inhibition was fully reversible. 6. It is concluded that the binding site of DIDS on SUR that is sensed by glibenclamide does not mediate channel inhibition. Instead, Mg(2+) binding to SUR may allosterically increase the accessibility and/or reactivity of the DIDS site on Kir6.2. The fact that the Mg(2+) effect does not require the presence of nucleotides underlines the importance of this ion in modulating the properties of the K(ATP) channel.

The stereoenantiomers of a pinacidil analog open or close cloned ATP-sensitive K+ channels.

ATP-dependent K(+) channels (K(ATP) channels) are composed of pore-forming subunits Kir6.x and sulfonylurea receptors (SURs). Cyanoguanidines such as pinacidil and P1075 bind to SUR and enhance MgATP binding to and hydrolysis by SUR, thereby opening K(ATP) channels. In the vasculature, openers of K(ATP) channels produce vasorelaxation. Some novel cyanoguanidines, however, selectively reverse opener-induced vasorelaxation, suggesting that they might be K(ATP) channel blockers. Here we have analyzed the interaction of the enantiomers of a racemic cyanoguanidine blocker, PNU-94750, with Kir6.2/SUR channels. In patch clamp experiments, the R-enantiomer (PNU-96293) inhibited Kir6.2/SUR2 channels (IC(50) approximately 50 nm in the whole cell configuration), whereas the S-enantiomer (PNU-96179) was a weak opener. Radioligand binding studies showed that the R-enantiomer was more potent and that it was negatively allosterically coupled to MgATP binding, whereas the S-enantiomer was weaker and positively coupled. Binding experiments also suggested that both enantiomers bound to the P1075 site of SUR. This is the first report to show that the enantiomers of a K(ATP) channel modulator affect channel activity and coupling to MgATP binding in opposite directions and that these opposite effects are apparently mediated by binding to the same (opener) site of SUR.

Glibenclamide binding to sulphonylurea receptor subtypes: dependence on adenine nucleotides.

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 affinity, 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 approximately 60%, to SUR2A(Y1206S) by 21%). Inhibition curves for the SUR2(Y1206S) isoforms were monophasic with IC(50) values of 5-10 microM; the curve for SUR1 was biphasic (IC(50) values 4.7 and 1300 microM). 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 efficacy of ATP in inhibiting glibenclamide binding; this was particularly marked for Kir6.2/SUR1. 6: The data show (a) that the inhibitory effects of ATP and ADP on glibenclamide binding differ from one another, (b) that they depend on the SUR subtype, and (c) that they are weakened by coexpression with Kir6.2.

Four novel splice variants of sulfonylurea receptor 1.

ATP-sensitive K(+) (K(ATP)) channels are composed of pore-forming Kir6.x subunits and regulatory sulfonylurea receptor (SUR) subunits. SURs are ATP-binding cassette proteins with two nucleotide-binding folds (NBFs) and binding sites for sulfonylureas, like glibenclamide, and for channel openers. Here we report the identification and functional characterization of four novel splice forms of guinea pig SUR1. Three splice forms originate from alternative splicing of the region coding for NBF1 and lack exons 17 (SUR1Delta17), 19 (SUR1Delta19), or both (SUR1Delta17Delta19). The fourth (SUR1C) is a COOH-terminal SUR1-fragment formed by exons 31-39 containing the last two transmembrane segments and the COOH terminus of SUR1. RT-PCR analysis showed that these splice forms are expressed in several tissues with strong expression of SUR1C in cardiomyocytes. Confocal microscopy using enhanced green fluorescent protein-tagged SUR or Kir6.x did not provide any evidence for involvement of these splice forms in the mitochondrial K(ATP) channel. Only SUR1 and SUR1Delta17 showed high-affinity binding of glibenclamide (K(d) approximately 2 nM in the presence of 1 mM ATP) and formed functional K(ATP) channels upon coexpression with Kir6.2.

Interaction of K(ATP) channel modulators with sulfonylurea receptor SUR2B: implication for tetramer formation and allosteric coupling of subunits.

Sulfonylurea receptors (SURs) are subunits of ATP-sensitive K(+) channels (K(ATP) channels); they mediate the channel-closing effect of sulfonylureas such as glibenclamide and the channel-activating 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 )= 4 versus 22 nM, respectively) but otherwise exhibiting similar pharmacological properties. In the absence of MgATP, [(3)H]glibenclamide binding to both SURs was homogenous. MgATP inhibited [(3)H]glibenclamide binding to both SURs to 25% by reducing the apparent number of glibenclamide binding sites, leaving the affinity unchanged. In the absence of MgATP, P1075 inhibited [(3)H]glibenclamide binding in a monophasic manner with K(i) approximately 1 microM. In the presence of MgATP (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 (approximately 1 microM). 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.