ABCMdb

ABCC8 p.Met1289Thr

Predicted by SNAP2:	A: D (59%), C: D (75%), D: D (75%), E: D (63%), F: D (71%), G: D (71%), H: D (80%), I: D (66%), K: D (66%), L: D (66%), N: D (59%), P: D (85%), Q: N (66%), R: D (63%), S: N (53%), T: D (53%), V: D (63%), W: D (85%), Y: D (75%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, L: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: N, Y: N,

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[hide] Resveratrol binds to the sulfonylurea receptor (SU... J Biol Chem. 2007 Feb 2;282(5):3347-56. Epub 2006 Nov 30. Hambrock A, de Oliveira Franz CB, Hiller S, Grenz A, Ackermann S, Schulze DU, Drews G, Osswald H
Resveratrol binds to the sulfonylurea receptor (SUR) and induces apoptosis in a SUR subtype-specific manner.
J Biol Chem. 2007 Feb 2;282(5):3347-56. Epub 2006 Nov 30., [PMID:17138562]

Abstract [show]
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.

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8 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). Login to comment
30 This effect was not observed in cells expressing the vascular smooth muscle isoform SUR2B or the mutant SUR1(M1289T), in which a single amino acid in the last C-terminal transmembrane helix 17 was exchanged with the corresponding amino acid of SUR2. Login to comment
51 The apoptotic potency of resveratrol was compared in cells expressing SUR1, SUR2A (cardiac and skeletal muscle isoform), SUR2B, or SUR1(M1289T). Login to comment
57 EXPERIMENTAL PROCEDURES Cell Culture-HEK293 cell lines were stably transfected with the pcDNA3.1 expression vector (Invitrogen, Karlsruhe, Germany) containing the coding sequence of SUR1 (GenBankTM accession number X97279), SUR1(S1238Y), SUR1(M1289T), SUR2A (accession number D86037), SUR2B (accession number D86038), or SUR2B(Y1206S), or they were transfected with the empty pcDNA3.1 expression vector (Invitrogen). Login to comment
86 Radioligand Binding Assays-Binding of resveratrol to different SUR forms was determined in heterologous competition experiments using [3 H]glibenclamide as the radioligand (SUR1, SUR1(M1289T), and pcDNA controls, 1 nM; SUR1(S1238Y), SUR2B, and pcDNA controls, 4-5 nM; and SUR2B(Y1206S), 3 nM). Login to comment
88 Membranes prepared from the respective cell lines (SUR1, SUR1(M1289T), and pcDNA controls, 0.05-0.10 mg/ml membrane protein; SUR2B, SUR2B(S1238Y), and pcDNA controls, 0.5-0.7 mg/ml membrane protein; and SUR2B(Y1206S), 0.1-0.2 mg/ml membrane protein) were incubated in the presence of 1 mM MgATP for 15 min at 37 °C, and binding properties were analyzed as described by Hambrock et al. (36). Login to comment
112 Resveratrol-induced cell detachment was enhanced by ϳ2-fold in cells expressing SUR1 (but not SUR2B or the mutant SUR1(M1289T)) compared with pcDNA control cells. Login to comment
113 Detachment of cells expressing SUR2B or SUR1(M1289T) tended to be even slightly but not significantly lower than that of pcDNA control cells. Login to comment
120 These typical signs of apoptosis were very frequent and pronounced in HEK cells expressing SUR1, but occurred only to a small extent in pcDNA control cells or SUR1(M1289T)- and SUR2B-expressing cells (SUR1 ϩ resveratrol, 22.33 Ϯ 1.86% apoptotic nuclei/total number of nuclei; SUR1 ϩ solvent, 1.90 Ϯ 0.67%; SUR1(M1289T) ϩ resveratrol, 6.02 Ϯ 0.90%; SUR1(M1289T) ϩ solvent, 3.18 Ϯ 0.77%; SUR2B ϩ resveratrol, 4.46 Ϯ 0.79%; SUR2B ϩ solvent, 2.53 Ϯ 0.62%; pcDNA ϩ resveratrol, 5.89 Ϯ 1.02%; pcDNA ϩ solvent, 2.10 Ϯ 0.53%; SUR1 ϩ resveratrol versus pcDNA/SUR1(M1289T)/SUR2B ϩ resveratrol, p Ͻ 0.001; SUR1 ϩ resveratrol versus SUR1 ϩ solvent, p Ͻ 0.001; pcDNA/SUR1(M1289T) ϩ resveratrol versus pcDNA/ SUR1(M1289T) ϩ solvent, p Ͻ 0.05). Login to comment
124 If resveratrol-induced changes in caspase-3 activity were calculated in the form of "induction factors," i.e. the ratio of the activity after substance treatment to the activity after solvent treatment, values of 2.14 Ϯ 0.30 (pcDNA), 2.49 Ϯ 0.30 (SUR1), and 1.81 Ϯ 0.20 (SUR1(M1289T)) were obtained (Fig. 4A). Login to comment
126 With induction factors of ϳ1, caspase-8 activity (IETD-caspase) was obviously not induced (induction factors, 1.02 Ϯ 0.05 (pcDNA), 1.23 Ϯ 0.11 (SUR1), and 0.87 Ϯ 0.08 (SUR1(M1289T)) (Fig. 4B). Login to comment
127 The activity of caspase-9 (LEHD-caspase) was clearly enhanced, however, after resveratrol treatment, with an additional significant increase in SUR1-expressing cells (induction factors, 1.73 Ϯ 0.15 (pcDNA), 3.12 Ϯ 0.26 (SUR1), and 1.76 Ϯ 0.18 (SUR1(M1289T)) (Fig. 4C). Login to comment
128 The activity of caspase-12 (ATAD-caspase) was generally elevated after resveratrol treatment, but exhibited no significant differences between the cell lines (induction factors, 1.87 Ϯ 0.27 (pcDNA), 1.72 Ϯ 0.24 (SUR1), and 1.63 Ϯ 0.19 (SUR1(M1289T)) (Fig. 4D). Login to comment
129 The activities of caspase-3 and caspase-9 were also determined for etoposide-treated cells, and no significant differences were detected between the SUR1-expressing cells and the other HEK cell lines (induction factors: caspase-3, 2.35 Ϯ 0.14 (pcDNA), 2.89 Ϯ 0.60 (SUR1), and 2.25 Ϯ 0.18 (SUR1(M1289T); and caspase-9, 1.17 Ϯ 0.09 (pcDNA), 1.36 Ϯ 0.06 (SUR1), and 1.54 Ϯ 0.10 (SUR1(M1289T) (n ϭ 5-6)). Login to comment
132 SUR1-, SUR1(M1289T)-, and SUR2B-expressing HEK cells and pcDNA control cells were treated with 100 ␮M resveratrol (RSV) or 100 ␮M etoposide (ETO) for 24 h. Login to comment
156 After 24 h of treatment with 100 ␮M resveratrol (RSV) or Me2SO solvent (solv.), pcDNA con- trolcellsandSUR1-orSUR1(M1289T)-expressingHEK293cellswerestainedwith Hoechst 33258. Login to comment
159 Nuclei from (former) cells in the supernatant were apoptotic to 100%(notshown).TheresultsforSUR2B-expressingHEKcells(notshown)arethe same as those for pcDNA control and SUR1(M1289T)-expressing cells. Login to comment
163 The resveratrol binding properties of SUR1 were not significantly different from those determined for the mutant SUR1(M1289T) (Ki ϭ 99 Ϯ 21 ␮M; extent of inhibition, 91 Ϯ 9%). Login to comment
176 Changes in the activities of different caspase enzymes in pcDNA control cells and SUR1- or SUR1(M1289T)-expressing HEK cells after resveratrol treatment. Login to comment
223 In addition to demonstrating the SUR-specific apoptotic effect ofresveratrol,wehaveconfirmedinourradioligandbindingassays that resveratrol specifically binds to SUR (SUR1, SUR2B, and SUR1(M1289T)) and is able to displace specific binding of glibenclamide.Byshowingthat,likeglibenclamide,resveratrolcanactas a blocker of the SUR1/KIR6.2 channel, we have provided further evidence for analogies between both substances. Login to comment
227 Binding of resveratrol to SUR1 and SUR1(M1289T). Login to comment
228 Binding of resveratrol to membranes from HEK cells expressing SUR1 or SUR1(M1289T) was analyzed in heterologous competition experiments using [3 H]glibenclamide as the radioligand. Login to comment
243 This is underlined by the fact that no significant differences in the resveratrol or glibenclamide (36) binding properties of SUR1 and the mutant SUR1(M1289T) could be detected, although the expression of these different SURs is linked to a significantly different susceptibility to resveratrol- or glibenclamide-induced apoptosis. Login to comment
29 This effect was not observed in cells expressing the vascular smooth muscle isoform SUR2B or the mutant SUR1(M1289T), in which a single amino acid in the last C-terminal transmembrane helix 17 was exchanged with the corresponding amino acid of SUR2. Login to comment
50 The apoptotic potency of resveratrol was compared in cells expressing SUR1, SUR2A (cardiac and skeletal muscle isoform), SUR2B, or SUR1(M1289T). Login to comment
56 EXPERIMENTAL PROCEDURES Cell Culture-HEK293 cell lines were stably transfected with the pcDNA3.1 expression vector (Invitrogen, Karlsruhe, Germany) containing the coding sequence of SUR1 (GenBankTM accession number X97279), SUR1(S1238Y), SUR1(M1289T), SUR2A (accession number D86037), SUR2B (accession number D86038), or SUR2B(Y1206S), or they were transfected with the empty pcDNA3.1 expression vector (Invitrogen). Login to comment
85 Radioligand Binding Assays-Binding of resveratrol to different SUR forms was determined in heterologous competition experiments using [3 H]glibenclamide as the radioligand (SUR1, SUR1(M1289T), and pcDNA controls, 1 nM; SUR1(S1238Y), SUR2B, and pcDNA controls, 4-5 nM; and SUR2B(Y1206S), 3 nM). Login to comment
87 Membranes prepared from the respective cell lines (SUR1, SUR1(M1289T), and pcDNA controls, 0.05-0.10 mg/ml membrane protein; SUR2B, SUR2B(S1238Y), and pcDNA controls, 0.5-0.7 mg/ml membrane protein; and SUR2B(Y1206S), 0.1-0.2 mg/ml membrane protein) were incubated in the presence of 1 mM MgATP for 15 min at 37 &#b0;C, and binding properties were analyzed as described by Hambrock et al. (36). Login to comment
111 Resveratrol-induced cell detachment was enhanced by b03;2-fold in cells expressing SUR1 (but not SUR2B or the mutant SUR1(M1289T)) compared with pcDNA control cells. Login to comment
119 These typical signs of apoptosis were very frequent and pronounced in HEK cells expressing SUR1, but occurred only to a small extent in pcDNA control cells or SUR1(M1289T)- and SUR2B-expressing cells (SUR1 af9; resveratrol, 22.33 afe; 1.86% apoptotic nuclei/total number of nuclei; SUR1 af9; solvent, 1.90 afe; 0.67%; SUR1(M1289T) af9; resveratrol, 6.02 afe; 0.90%; SUR1(M1289T) af9; solvent, 3.18 afe; 0.77%; SUR2B af9; resveratrol, 4.46 afe; 0.79%; SUR2B af9; solvent, 2.53 afe; 0.62%; pcDNA af9; resveratrol, 5.89 afe; 1.02%; pcDNA af9; solvent, 2.10 afe; 0.53%; SUR1 af9; resveratrol versus pcDNA/SUR1(M1289T)/SUR2B af9; resveratrol, p b0d; 0.001; SUR1 af9; resveratrol versus SUR1 af9; solvent, p b0d; 0.001; pcDNA/SUR1(M1289T) af9; resveratrol versus pcDNA/ SUR1(M1289T) af9; solvent, p b0d; 0.05). Login to comment
123 If resveratrol-induced changes in caspase-3 activity were calculated in the form of "induction factors," i.e. the ratio of the activity after substance treatment to the activity after solvent treatment, values of 2.14 afe; 0.30 (pcDNA), 2.49 afe; 0.30 (SUR1), and 1.81 afe; 0.20 (SUR1(M1289T)) were obtained (Fig. 4A). Login to comment
125 With induction factors of b03;1, caspase-8 activity (IETD-caspase) was obviously not induced (induction factors, 1.02 afe; 0.05 (pcDNA), 1.23 afe; 0.11 (SUR1), and 0.87 afe; 0.08 (SUR1(M1289T)) (Fig. 4B). Login to comment
131 SUR1-, SUR1(M1289T)-, and SUR2B-expressing HEK cells and pcDNA control cells were treated with 100 òe;M resveratrol (RSV) or 100 òe;M etoposide (ETO) for 24 h. Login to comment
155 After 24 h of treatment with 100 òe;M resveratrol (RSV) or Me2SO solvent (solv.), pcDNA con- trolcellsandSUR1-orSUR1(M1289T)-expressingHEK293cellswerestainedwith Hoechst 33258. Login to comment
158 Nuclei from (former) cells in the supernatant were apoptotic to 100%(notshown).TheresultsforSUR2B-expressingHEKcells(notshown)arethe same as those for pcDNA control and SUR1(M1289T)-expressing cells. Login to comment
162 The resveratrol binding properties of SUR1 were not significantly different from those determined for the mutant SUR1(M1289T) (Ki afd; 99 afe; 21 òe;M; extent of inhibition, 91 afe; 9%). Login to comment
175 Changes in the activities of different caspase enzymes in pcDNA control cells and SUR1- or SUR1(M1289T)-expressing HEK cells after resveratrol treatment. Login to comment
222 In addition to demonstrating the SUR-specific apoptotic effect ofresveratrol,wehaveconfirmedinourradioligandbindingassays that resveratrol specifically binds to SUR (SUR1, SUR2B, and SUR1(M1289T)) and is able to displace specific binding of glibenclamide.Byshowingthat,likeglibenclamide,resveratrolcanactas a blocker of the SUR1/KIR6.2 channel, we have provided further evidence for analogies between both substances. Login to comment
226 Binding of resveratrol to SUR1 and SUR1(M1289T). Login to comment
242 This is underlined by the fact that no significant differences in the resveratrol or glibenclamide (36) binding properties of SUR1 and the mutant SUR1(M1289T) could be detected, although the expression of these different SURs is linked to a significantly different susceptibility to resveratrol- or glibenclamide-induced apoptosis. Login to comment

[hide] 17beta-Estradiol modulates apoptosis in pancreatic... J Biol Chem. 2009 Feb 20;284(8):4905-13. Epub 2008 Dec 18. Ackermann S, Hiller S, Osswald H, Losle M, Grenz A, Hambrock A
17beta-Estradiol modulates apoptosis in pancreatic beta-cells by specific involvement of the sulfonylurea receptor (SUR) isoform SUR1.
J Biol Chem. 2009 Feb 20;284(8):4905-13. Epub 2008 Dec 18., [PMID:19095654]

Abstract [show]
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.

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9 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). Login to comment
43 In addition, we explored in which manner the action of 17beta-estradiol was influenced by mutations (M1289T, R1379C, R1379L) in the SUR1 gene (ABCC8) that are of special importance for SUR function (18-20). Login to comment
46 EXPERIMENTAL PROCEDURES Mutagenesis, Transfection, and Cell Culture-HEK293 cells (German Collection of Microorganisms and Cell Cultures, DSMZ, Braunschweig, Germany) were stably transfected with pcDNA3.1 expression vector (Invitrogen) containing the coding sequence of rat SUR1 (GenBankTM accession number X97279), SUR1(M1289T), SUR1(R1379C), SUR1(R1379L), murine SUR2A (GenBank D86037), SUR2A(Y1206S), murine SUR2B (GenBank D86038), or SUR2B(Y1206S), or they were transfected with empty pcDNA3.1 expression vector (Invitrogen). Login to comment
103 The apoptotic effect of 17beta-estradiol in SUR1 cells, visible in the form of enhanced cell detachment or intensive condensation and fragmentation of nuclei, was abolished in cells expressing the mutant SUR1(M1289T) (Figs. Login to comment
120 After treatment of SUR1-, SUR1(M1289T)-, SUR2A-, and SUR2B-expressing HEK293 cells (A and B) or of SUR1, SUR1(R1379C), and SUR1(R1379L) cells (C) with 17beta-estradiol (100 ␮mol/liter, 24 h), cell detachmentorchangesinnuclearmorphologyweredeterminedandcomparedwiththeresultsobtainedwith pcDNA control cells (please note the different scales in A and C). Login to comment
145 HEK293 cells stably expressing SUR1, SUR1(M1289T), SUR1(R1379C), or pcDNA control cells (A) were compared with cells from the clonal beta-cell lines HIT-T15 and RIN-m5F (B) or to isolated islets from SUR1-expressing wild type mice (SUR1wt) or SUR1KO mice (C) after treatment with 100 ␮mol/liter 17beta-estradiol (E2) or solvent (solv.) Login to comment
154 A comparable nucleotide dependence was also observed in the case of the mutant SUR1(M1289T) with binding curves being similar to those obtained for SUR1. Login to comment
184 This SUR1-dependent effect of 17beta-estradiol is either abolished by mutation M1289T or enhanced by mutations R1379C or R1379L in SUR1. Login to comment
204 Binding of 17beta-estradiol to membranes from HEK293 cells stably expressing SUR1 or mutants SUR1(M1289T), SUR2A(Y1206S), or SUR2B(Y1206S) was analyzed in heterologous competition experiments using [3 H]glibenclamide ([3 H]GBC) as the radioligand (final label concentration 1.0-3.0 nmol/liter) and 17beta-estradiol as the competitor. Login to comment
222 The mutation M1289T, located in TM17 of SUR1, influences binding (19) and action (18) of several KATP channel openers. Login to comment
223 In the mutant SUR1(M1289T), a single amino acid in TM17 is exchanged by the corresponding amino acid of SUR2. Login to comment
225 The mutation M1289T completely abolishes the SUR1-specific apoptotic effects of KATP channel blockers glibenclamide (3) or resveratrol (4), or 17beta-estradiol obviously without directly affecting binding of these substances to SUR1. Login to comment
227 To see whether SUR1-mediated apoptosis is linked with ATP hydrolysis, we explored the effects of mutations R1379C and R1379L in nucleotide binding fold 2 of SUR1 on 17beta-estradiol action. Login to comment
237 Several mutations in the genes encoding both KATP channel subunits, SUR or Kir6.x, are known to confer an increased TABLE 1 Binding parameters derived from the heterologous competition experiments using ͓3 H͔glibenclamide as the radioligand and 17beta-estradiol as the competitor Parametera SUR1 SUR1(M1289T) SUR2A(Y1206S) SUR2B(Y1206S) ؉MgATP -MgATPb ؉MgATP -MgATPb ؉MgATP -MgATPb ؉MgATP -MgATPb Ki (␮mol/liter) 81.4 Ϯ 26.3 - 13.1 Ϯ 5.8 - 57.1 Ϯ 23.4 201.1 83.3 Ϯ 22.7 70.2 A (% BS) 55.1 Ϯ 11.2 - 26.6 Ϯ 3.0 - 75.9 Ϯ 10.8 32.1 76.4 Ϯ 10.4 34.3 n 11 6 6 4 7 7 7 7 a Binding parameters (Ki, inhibition constant, A: amount of displaced specific radioligand binding, (Bs)) are means from the data determined in the individual ͓3 H͔glibenclamide inhibition experiments (n ϭ number of experiments) as described in the legend to Fig. 7. Login to comment
238 For correction of the inhibition curves according to the Cheng-Prusoff equation (see "Experimental Procedures") the following KD ϭ Ki values for glibenclamide were taken into account (SUR1 ϩMgATP/-MgATP: 3.2/1.4 nmol/liter, SUR1(M1289T) ϩMgATP/-MgATP: 3.5/1.2 nmol/l, SUR2A(Y1206S) ϩMgATP/-MgATP: 15.2/15.4 nmol/liter, SUR2B(Y1206S) ϩMgATP/-MgATP: 11.1/9.8 nmol/liter). Login to comment
44 In addition, we explored in which manner the action of 17beta-estradiol was influenced by mutations (M1289T, R1379C, R1379L) in the SUR1 gene (ABCC8) that are of special importance for SUR function (18-20). Login to comment
47 EXPERIMENTAL PROCEDURES Mutagenesis, Transfection, and Cell Culture-HEK293 cells (German Collection of Microorganisms and Cell Cultures, DSMZ, Braunschweig, Germany) were stably transfected with pcDNA3.1 expression vector (Invitrogen) containing the coding sequence of rat SUR1 (GenBankTM accession number X97279), SUR1(M1289T), SUR1(R1379C), SUR1(R1379L), murine SUR2A (GenBank D86037), SUR2A(Y1206S), murine SUR2B (GenBank D86038), or SUR2B(Y1206S), or they were transfected with empty pcDNA3.1 expression vector (Invitrogen). Login to comment
104 By contrast, apoptosis in cells expressing mutants SUR1(R1379C) or SUR1(R1379L) was potentiated to a large extent (Figs. 3C and 4A): compared with SUR1, cell detachment was increased by a factor of 3.0 or 2.7, respectively, and the rate of apoptotic nuclei was elevated b07;3-fold. Login to comment
118 After treatment of SUR1-, SUR1(M1289T)-, SUR2A-, and SUR2B-expressing HEK293 cells (A and B) or of SUR1, SUR1(R1379C), and SUR1(R1379L) cells (C) with 17beta-estradiol (100 òe;mol/liter, 24 h), cell detachmentorchangesinnuclearmorphologyweredeterminedandcomparedwiththeresultsobtainedwith pcDNA control cells (please note the different scales in A and C). Login to comment
143 HEK293 cells stably expressing SUR1, SUR1(M1289T), SUR1(R1379C), or pcDNA control cells (A) were compared with cells from the clonal beta-cell lines HIT-T15 and RIN-m5F (B) or to isolated islets from SUR1-expressing wild type mice (SUR1wt) or SUR1KO mice (C) after treatment with 100 òe;mol/liter 17beta-estradiol (E2) or solvent (solv.) Login to comment
152 A comparable nucleotide dependence was also observed in the case of the mutant SUR1(M1289T) with binding curves being similar to those obtained for SUR1. Login to comment
182 This SUR1-dependent effect of 17beta-estradiol is either abolished by mutation M1289T or enhanced by mutations R1379C or R1379L in SUR1. Login to comment
202 Binding of 17beta-estradiol to membranes from HEK293 cells stably expressing SUR1 or mutants SUR1(M1289T), SUR2A(Y1206S), or SUR2B(Y1206S) was analyzed in heterologous competition experiments using [3 H]glibenclamide ([3 H]GBC) as the radioligand (final label concentration 1.0-3.0 nmol/liter) and 17beta-estradiol as the competitor. Login to comment
220 The mutation M1289T, located in TM17 of SUR1, influences binding (19) and action (18) of several KATP channel openers. Login to comment
221 In the mutant SUR1(M1289T), a single amino acid in TM17 is exchanged by the corresponding amino acid of SUR2. Login to comment
235 Several mutations in the genes encoding both KATP channel subunits, SUR or Kir6.x, are known to confer an increased TABLE 1 Binding parameters derived from the heterologous competition experiments using ᐵ3 Hᐶglibenclamide as the radioligand and 17beta-estradiol as the competitor Parametera SUR1 SUR1(M1289T) SUR2A(Y1206S) SUR2B(Y1206S) d19;MgATP d1a;MgATPb d19;MgATP d1a;MgATPb d19;MgATP d1a;MgATPb d19;MgATP d1a;MgATPb Ki (òe;mol/liter) 81.4 afe; 26.3 - 13.1 afe; 5.8 - 57.1 afe; 23.4 201.1 83.3 afe; 22.7 70.2 A (% BS) 55.1 afe; 11.2 - 26.6 afe; 3.0 - 75.9 afe; 10.8 32.1 76.4 afe; 10.4 34.3 n 11 6 6 4 7 7 7 7 a Binding parameters (Ki, inhibition constant, A: amount of displaced specific radioligand binding, (Bs)) are means from the data determined in the individual ᐵ3 Hᐶglibenclamide inhibition experiments (n afd; number of experiments) as described in the legend to Fig. 7. Login to comment
236 For correction of the inhibition curves according to the Cheng-Prusoff equation (see "Experimental Procedures") the following KD afd; Ki values for glibenclamide were taken into account (SUR1 af9;MgATP/afa;MgATP: 3.2/1.4 nmol/liter, SUR1(M1289T) af9;MgATP/afa;MgATP: 3.5/1.2 nmol/l, SUR2A(Y1206S) af9;MgATP/afa;MgATP: 15.2/15.4 nmol/liter, SUR2B(Y1206S) af9;MgATP/afa;MgATP: 11.1/9.8 nmol/liter). Login to comment
121 After treatment of SUR1-, SUR1(M1289T)-, SUR2A-, and SUR2B-expressing HEK293 cells (A and B) or of SUR1, SUR1(R1379C), and SUR1(R1379L) cells (C) with 17beta-estradiol (100 òe;mol/liter, 24 h), cell detachmentorchangesinnuclearmorphologyweredeterminedandcomparedwiththeresultsobtainedwith pcDNA control cells (please note the different scales in A and C). Login to comment
147 HEK293 cells stably expressing SUR1, SUR1(M1289T), SUR1(R1379C), or pcDNA control cells (A) were compared with cells from the clonal beta-cell lines HIT-T15 and RIN-m5F (B) or to isolated islets from SUR1-expressing wild type mice (SUR1wt) or SUR1KO mice (C) after treatment with 100 òe;mol/liter 17beta-estradiol (E2) or solvent (solv.) Login to comment
156 A comparable nucleotide dependence was also observed in the case of the mutant SUR1(M1289T) with binding curves being similar to those obtained for SUR1. Login to comment
186 This SUR1-dependent effect of 17beta-estradiol is either abolished by mutation M1289T or enhanced by mutations R1379C or R1379L in SUR1. Login to comment
206 Binding of 17beta-estradiol to membranes from HEK293 cells stably expressing SUR1 or mutants SUR1(M1289T), SUR2A(Y1206S), or SUR2B(Y1206S) was analyzed in heterologous competition experiments using [3 H]glibenclamide ([3 H]GBC) as the radioligand (final label concentration 1.0-3.0 nmol/liter) and 17beta-estradiol as the competitor. Login to comment
224 The mutation M1289T, located in TM17 of SUR1, influences binding (19) and action (18) of several KATP channel openers. Login to comment
239 Several mutations in the genes encoding both KATP channel subunits, SUR or Kir6.x, are known to confer an increased TABLE 1 Binding parameters derived from the heterologous competition experiments using ᐵ3 Hᐶglibenclamide as the radioligand and 17beta-estradiol as the competitor Parametera SUR1 SUR1(M1289T) SUR2A(Y1206S) SUR2B(Y1206S) d19;MgATP d1a;MgATPb d19;MgATP d1a;MgATPb d19;MgATP d1a;MgATPb d19;MgATP d1a;MgATPb Ki (òe;mol/liter) 81.4 afe; 26.3 - 13.1 afe; 5.8 - 57.1 afe; 23.4 201.1 83.3 afe; 22.7 70.2 A (% BS) 55.1 afe; 11.2 - 26.6 afe; 3.0 - 75.9 afe; 10.8 32.1 76.4 afe; 10.4 34.3 n 11 6 6 4 7 7 7 7 a Binding parameters (Ki, inhibition constant, A: amount of displaced specific radioligand binding, (Bs)) are means from the data determined in the individual ᐵ3 Hᐶglibenclamide inhibition experiments (n afd; number of experiments) as described in the legend to Fig. 7. Login to comment
240 For correction of the inhibition curves according to the Cheng-Prusoff equation (see "Experimental Procedures") the following KD afd; Ki values for glibenclamide were taken into account (SUR1 af9;MgATP/afa;MgATP: 3.2/1.4 nmol/liter, SUR1(M1289T) af9;MgATP/afa;MgATP: 3.5/1.2 nmol/l, SUR2A(Y1206S) af9;MgATP/afa;MgATP: 15.2/15.4 nmol/liter, SUR2B(Y1206S) af9;MgATP/afa;MgATP: 11.1/9.8 nmol/liter). Login to comment

[hide] Glibenclamide-induced apoptosis is specifically en... J Pharmacol Exp Ther. 2006 Mar;316(3):1031-7. Epub 2005 Nov 23. Hambrock A, de Oliveira Franz CB, Hiller S, Osswald H
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).
J Pharmacol Exp Ther. 2006 Mar;316(3):1031-7. Epub 2005 Nov 23., [PMID:16306272]

Abstract [show]
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.

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0 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) Annette Hambrock, Claudia Bernardo de Oliveira Franz, Sabrina Hiller, and Hartmut Osswald Department of Pharmacology and Toxicology, Medical Faculty, University of Tu¨ bingen, Tu¨ bingen, Germany Received October 20, 2005; accepted November 21, 2005 ABSTRACT Sulfonylurea receptor 1 (SUR1) is the regulatory subunit of the pancreatic ATP-sensitive Kϩ channel (KATP channel), which is essential for triggering insulin secretion via membrane depolarization. Login to comment
3 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. Login to comment
4 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. Login to comment
6 In conclusion, expression of SUR1, but not of SUR2B or SUR1(M1289T), renders cells more susceptible to glibenclamide-induced apoptosis. Login to comment
37 Cells expressing the mutant SUR1(M1289T) at which a single amino acid in transmembrane helix 17 (TM17) was exchanged by the corresponding amino acid of SUR2 were also investigated. Login to comment
43 Cells were transfected with pcDNA3.1 expression vector (Invitrogen, Karlsruhe, Germany) containing the coding sequences of SUR1 (GenBank X97279), SUR1(M1289T), or SUR2B (GenBank D86038). Login to comment
44 The mutant SUR1(M1289T) had been constructed from SUR1 using the QuikChange Mutagenesis System (Stratagene, Amsterdam, The Netherlands). Login to comment
89 Results Cell Morphology after Glibenclamide Treatment of Different Cell Lines. HEK293 cells stably expressing either SUR1, SUR2B, or the mutant SUR1(M1289T) were incubated with glibenclamide concentrations (5-100 ␮M) over a 7-day culture period and compared with cells that were only treated with the same amount (0.1%) of solvent. Login to comment
95 Cells expressing the mutant SUR1(M1289T) resembled pcDNA cells after glibenclamide treatment. Login to comment
104 Effect of glibenclamide treatment on the morphology of SUR1, SUR1(M1289T), SUR2B, or pcDNA cells. Login to comment
111 B, pcDNA, SUR1, and SUR1(M1289T) cells were compared in parallel experiments with n ϭ 4. Login to comment
114 For cells expressing SUR1(M1289T), the number of detached cells was reduced compared with SUR1 cells but slightly higher than the amount of detached pcDNA cells (Fig. 2B). Login to comment
115 However, these differences between SUR1(M1289T) cells and SUR1 or pcDNA cells were not significant. Login to comment
120 Again, the degree of detached SUR1 cells after glibenclamide treatment (462 Ϯ 82% of solvent control) was considerably higher than that of the other cells (pcDNA, 320 Ϯ 66%; SUR1(M1289T), 262 Ϯ 71%; and SUR2B, 254 Ϯ 30%). Login to comment
121 The amount of glibenclamide-induced detachment of SUR1 cells was nearly the same as that of etoposide-induced cell detachment (pcDNA, 434 Ϯ 75%; SUR1, 475 Ϯ 127%; SUR1(M1289T), 488 ee; 78%; and SUR2B, 389 Ϯ 85%). Login to comment
132 In SUR1 cells, apoptotic nuclei occurred to a much larger extent and were significantly more condensed and fragmented than in SUR2B (data not shown), SUR1(M1289T) , or pcDNA cells (Fig. 4). Login to comment
150 After 48 h, caspase-3-like activity in SUR1 and pcDNA cells was significantly higher in the glibenclamide-treated groups than in the solvent-treated groups, but this difference was not observed in SUR1(M1289T) cells (Fig. 5). Login to comment
152 SUR1(M1289T) cells showed even slightly lower activity than pcDNA controls after glibenclamide treatment, but this difference was not significant. Login to comment
154 Caspase-3-like activity was a little higher in SUR1 cells than in pcDNA and SUR1(M1289T) cells in most experiments, and pcDNA cells for their part showed slightly but not significantly higher activity than SUR1(M1289T) cells. Login to comment
155 Yet, if total activity after glibenclamide treatment was referred to the respective solvent-dependent activity, the increase in caspase-3-like activity due to glibenclamide was still higher in SUR1 cells (323 Ϯ 40%) compared with pcDNA cells (269 Ϯ 37%) or SUR1(M1289T) cells (200 Ϯ 41%). Login to comment
158 A basal level of apoptosis was also present in sham-transfected control cells as well as in cells expressing the SUR2B isoform or the mutant SUR1(M1289T). Login to comment
172 Hoechst dye 33258 staining after 48 h of treatment with 100 ␮M glibenclamide (GBC) or ethanol/DMSO treatment of pcDNA, SUR1, and SUR1(M1289T) cells. Login to comment
174 Results for SUR2B cells (data not shown) were the same as for pcDNA or SUR1(M1289T) cells. Login to comment
176 Determination of caspase-3-like activity in pcDNA, SUR1, and SUR1(M1289T) cells after treatment with 100 ␮M glibenclamide (GBC) or solvent for 48 h. Login to comment
197 It still has to be noted that caspase-3-like activity in solvent-treated controls is slightly higher in SUR1 cells compared with pcDNA cells but lower in SUR1(M1289T) cells. Login to comment
199 Perhaps the expression of SUR1 per se [but not of SUR1(M1289T)] results in slightly higher caspase-3-like activity, which could be due to interaction of SUR1 with endogenous compounds or supplements in the culture medium, for example. Login to comment
202 SUR1(M1289T) is able to form functional plasmalemmal channels with Kir6.2 according to electrophysiological experiments (Moreau et al., 2000) and thus possesses essential properties of an intact SUR protein. Login to comment
204 However, the maximal amount of glibenclamide-binding sites is reduced at SUR1(M1289T) by approximately 30 to 50% at physiological MgATP concentrations (S. Hiller, unpublished results), which might explain the lower sensitivity of SUR1(M1289T) cells to glibenclamide-induced apoptosis. Login to comment
206 In contrast to this finding, glibenclamide-induced apoptosis in SUR1(M1289T) cells is at the same level as that of control cells (or is even lower for SUR1(M1289T) cells in case of caspase-3-like activity). Login to comment
208 In this respect, SUR1(M1289T) is probably similar to the SUR2B isoform, which is also less effective in triggering glibenclamide-induced apoptosis. Login to comment
209 It has been shown previously that binding and effect of several KATP channel openers are clearly enhanced in SUR1(M1289T) cells (Moreau et al., 2000; Hambrock et al., 2004). Login to comment
211 Therefore, it will be an interesting task to investigate whether openers exert different protective effects in cells expressing SUR1, SUR1(M1289T), or SUR2. Login to comment
212 In conclusion, glibenclamide induces apoptotic processes in HEK293 cells that are markedly enhanced by expression of SUR1 but not of SUR2B or the mutant SUR1(M1289T). Login to comment
90 Results Cell Morphology after Glibenclamide Treatment of Different Cell Lines. HEK293 cells stably expressing either SUR1, SUR2B, or the mutant SUR1(M1289T) were incubated with glibenclamide concentrations (5-100 òe;M) over a 7-day culture period and compared with cells that were only treated with the same amount (0.1%) of solvent. Login to comment
96 Cells expressing the mutant SUR1(M1289T) resembled pcDNA cells after glibenclamide treatment. Login to comment
105 Effect of glibenclamide treatment on the morphology of SUR1, SUR1(M1289T), SUR2B, or pcDNA cells. Login to comment
112 B, pcDNA, SUR1, and SUR1(M1289T) cells were compared in parallel experiments with n afd; 4. Login to comment
116 However, these differences between SUR1(M1289T) cells and SUR1 or pcDNA cells were not significant. Login to comment
122 The amount of glibenclamide-induced detachment of SUR1 cells was nearly the same as that of etoposide-induced cell detachment (pcDNA, 434 afe; 75%; SUR1, 475 afe; 127%; SUR1(M1289T), 488 afe; 78%; and SUR2B, 389 afe; 85%). Login to comment
133 In SUR1 cells, apoptotic nuclei occurred to a much larger extent and were significantly more condensed and fragmented than in SUR2B (data not shown), SUR1(M1289T) , or pcDNA cells (Fig. 4). Login to comment
151 After 48 h, caspase-3-like activity in SUR1 and pcDNA cells was significantly higher in the glibenclamide-treated groups than in the solvent-treated groups, but this difference was not observed in SUR1(M1289T) cells (Fig. 5). Login to comment
153 SUR1(M1289T) cells showed even slightly lower activity than pcDNA controls after glibenclamide treatment, but this difference was not significant. Login to comment
156 Yet, if total activity after glibenclamide treatment was referred to the respective solvent-dependent activity, the increase in caspase-3-like activity due to glibenclamide was still higher in SUR1 cells (323 afe; 40%) compared with pcDNA cells (269 afe; 37%) or SUR1(M1289T) cells (200 afe; 41%). Login to comment
159 A basal level of apoptosis was also present in sham-transfected control cells as well as in cells expressing the SUR2B isoform or the mutant SUR1(M1289T). Login to comment
173 Hoechst dye 33258 staining after 48 h of treatment with 100 òe;M glibenclamide (GBC) or ethanol/DMSO treatment of pcDNA, SUR1, and SUR1(M1289T) cells. Login to comment
175 Results for SUR2B cells (data not shown) were the same as for pcDNA or SUR1(M1289T) cells. Login to comment
177 Determination of caspase-3-like activity in pcDNA, SUR1, and SUR1(M1289T) cells after treatment with 100 òe;M glibenclamide (GBC) or solvent for 48 h. Login to comment
198 It still has to be noted that caspase-3-like activity in solvent-treated controls is slightly higher in SUR1 cells compared with pcDNA cells but lower in SUR1(M1289T) cells. Login to comment
200 Perhaps the expression of SUR1 per se [but not of SUR1(M1289T)] results in slightly higher caspase-3-like activity, which could be due to interaction of SUR1 with endogenous compounds or supplements in the culture medium, for example. Login to comment
203 SUR1(M1289T) is able to form functional plasmalemmal channels with Kir6.2 according to electrophysiological experiments (Moreau et al., 2000) and thus possesses essential properties of an intact SUR protein. Login to comment
205 However, the maximal amount of glibenclamide-binding sites is reduced at SUR1(M1289T) by approximately 30 to 50% at physiological MgATP concentrations (S. Hiller, unpublished results), which might explain the lower sensitivity of SUR1(M1289T) cells to glibenclamide-induced apoptosis. Login to comment
207 In contrast to this finding, glibenclamide-induced apoptosis in SUR1(M1289T) cells is at the same level as that of control cells (or is even lower for SUR1(M1289T) cells in case of caspase-3-like activity). Login to comment
210 It has been shown previously that binding and effect of several KATP channel openers are clearly enhanced in SUR1(M1289T) cells (Moreau et al., 2000; Hambrock et al., 2004). Login to comment
213 In conclusion, glibenclamide induces apoptotic processes in HEK293 cells that are markedly enhanced by expression of SUR1 but not of SUR2B or the mutant SUR1(M1289T). Login to comment

[hide] Effect of two amino acids in TM17 of Sulfonylurea ... Diabetes. 2004 Dec;53 Suppl 3:S128-34. Hambrock A, Kayar T, Stumpp D, Osswald H
Effect of two amino acids in TM17 of Sulfonylurea receptor SUR1 on the binding of ATP-sensitive K+ channel modulators.
Diabetes. 2004 Dec;53 Suppl 3:S128-34., [PMID:15561900]

Abstract [show]
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.

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6 In competition experiments using [3 H]glibenclamide as radioligand, SUR1(T1285L, M1289T) showed much higher affinity toward the cyanoguanidine openers pinacidil and P1075 than SUR1 wild type. Login to comment
50 A: Binding of glibenclamide to wild-type SUR1, SUR1(M1289T), and SUR1(T1285L, M1289T) was tested in homologous competition experiments that were performed with membranes from stably transfected HEK 293 cells. Login to comment
51 Binding studies were done at 37°C in the presence of 1 mmol/l MgATP using [3 H]glibenclamide as the radioliand [SUR1, SUR1(T1285L, M1289T): L0 ‫؍‬ 0.7 nmol/l; SUR1(M1289T): L0 ‫؍‬ 0.6 nmol/l]. Login to comment
53 B-G: Heterologous competition experiments were performed with SUR1 and SUR1(T1285L, M1289T) in the presence of 1 mmol/l MgATP using [3 H]glibenclamide as the radioligand (L0 ‫؍‬ 0.6-0.8 nmol/l) and openers from different chemical classes as competitors (cyanoguanidines: P1075 [B] and pinacidil [C]; benzopyrans: rilmakalim [D] and levcromakalim [E]; binding or efficacy of several KCOs (9,12-15). Login to comment
67 The mutants SUR1(M1289T) and SUR1(T1285L, M1289T) were constructed from rat SUR1 (GenBank X97279) using the QuikChange Site-Directed Mutagenesis System (Stratagene, Amsterdam, the Netherlands). Login to comment
103 The curves for binding of rilmakalim and levcromakalim to wild-type SUR1, for binding of aprikalim to SUR1(T1285L, M1289T), and for binding of diazoxide to wild type and mutant were fitted for two components, one considered the activatory component and one the inhibitory component. Login to comment
115 For SUR1 (T1285L, M1289T), these curves were shifted toward higher opener concentrations with a 27-fold (P1075) or 14-fold (pinacidil) decrease in Ki values. Login to comment
117 In any case, for SUR1(T1285L, M1289T), A (55%) was not larger than for SUR1. Login to comment
118 Binding of P1075 to SUR1(T1285L, M1289T) did not differ much from binding to SUR1(M1289L) (Table 1). Login to comment
120 In case of the benzopyran openers rilmakalim and levcromakalim, Ki values for SUR1(T1285L, M1289T) were not significantly altered compared with those for the wild type (ϫ1.6 and ϫ1.2, respectively) (Fig. 2D and E; Table 1). Login to comment
121 The amount of displaced specific [3 H]glibenclamide binding, however, was markedly increased at SUR1(T1285L, M1289T) (rilmakalim: from 40 to 68%; levcromakalim: from 27 to 52%). Login to comment
122 The slight increase in [3 H]glibenclamide binding observed for wild-type SUR1 in the presence of low rilmakalim or levcromakalim concentrations was not seen with SUR1 (T1285L, M1289T). Login to comment
123 Binding of levcromakalim was also tested with SUR1(M1289T). Login to comment
124 Here, inhibition curves were very similar to those obtained for SUR1(T1285L, M1289T) (Table 1). Login to comment
128 Although an increase was also seen with SUR1 (T1285L, M1289T) at lower aprikalim concentrations, an inhibition of [3 H]glibenclamide binding was seen at higher concentrations (Table 1). Login to comment
130 Diazoxide was the only opener tested for which no significant difference in binding properties was seen between SUR1, SUR1(T1285L, M1289T), and SUR1(M1289T) (Fig. 2G; Table 1). Login to comment
139 Our data show no significant difference in diazoxide binding to wild-type SUR1 and the mutants SUR1(T1285L, M1289T) and SUR1(M1289T), and are in agreement with other publications that postulate that one or more regions of SUR (probably TM6-11 in TABLE 1 Inhibition of ͓3 H͔glibenclamide binding by different KATP channel openers at SUR1, SUR1(T1285L, M1289T), and SUR1 (M1289T) Opener SUR1 SUR1(T1285L, M1289T) SUR1(M1289T) Ki (␮mol/l) A (%Bs) Ki (␮mol/l) A (%Bs) Ki (␮mol/l) A (%Bs) P1075 163 Ϯ 48 (71 Ϯ 5)* 6 Ϯ 1 55 Ϯ 3 5 Ϯ 1 57 Ϯ 2 Pinacidil 1,023 Ϯ 254 (86 Ϯ 8)* 73 Ϯ 20 50 Ϯ 6 ND ND Rilmakalim 16 Ϯ 3 40 Ϯ 4 10 Ϯ 3 68 Ϯ 2 ND ND Levcromakalim 30 Ϯ 20 27 Ϯ 3 26 Ϯ 4 52 Ϯ 1 25 Ϯ 9 51 Ϯ 3 Aprikalim - - 90 Ϯ 33 41 Ϯ 4 ND ND Diazoxide 54 Ϯ 11 39 Ϯ 2 34 Ϯ 6 46 Ϯ 11 45 Ϯ 19 47 Ϯ 2 Data are means Ϯ SEM. Login to comment
142 Comparison of pKi values gave a significant difference between SUR1(T1285L, M1289T) and wild type in case of P1075 and pinacidil (P Ͻ 0.005), but no significant difference in case of rilmakalim, levcromakalim, and diazoxide (P Ͼ 0.05). Login to comment
143 pKi values were not significantly different between SUR1(T1285L, M1289T) and SUR1 (M1289T) in case of all the openers tested at both mutants (P Ͼ 0.05). Login to comment
149 As the amount of displaced glibenclamide binding cannot be determined precisely for wild-type SUR1 due to the low affinity of these openers, it is either the same or maybe to some extent smaller at SUR1(T1285L, M1289T), where [3 H]glibenclamide is only displaced to 50-55%. Login to comment
150 For the thioformamide aprikalim, an increase in affinity is seen at SUR1(T1285L, M1289T): while aprikalim does not displace glibenclamide binding to wild-type SUR1 at concentrations up to 300 ␮mol/l, binding to mutant SUR1 is inhibited with Ki ϭ 90 ␮mol/l. Login to comment
151 Binding of the benzopyranes rilmakalim and levcromakalim is also markedly altered at SUR1(T1285L, M1289T), but in a different manner: Here, affinity is not significantly different between wild type and mutant. Login to comment
153 In addition, the increase in [3 H]glibenclamide binding seen with wild-type SUR1, indicating additional positive allosteric interactions at lower concentrations of these openers at SUR1, is missing at SUR1(T1285L, M1289T). Login to comment
161 In our study, we also tested binding of P1075, levcromakalim, diazoxide, and glibenclamide to a mutant that was only altered at position 1,289 and received nearly the same results with this mutant, SUR1(M1289T), as with SUR1(T1285L, M1289T). Login to comment
165 As the affinity for P1075 and pinacidil is clearly altered at SUR1(T1285L, M1289T) without affecting the amount of displaced specific [3 H]glibenclamide, this may suggest that the amino acids at positions 1,285 and especially 1,289 are located within the binding sites for cyanoguanidines. Login to comment