ABCMdb

PMID: 19095654

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., [PubMed]

Sentences

No. Mutations Sentence Comment

9 ABCC8 p.Met1289Thr ABCC8 p.Arg1379Cys ABCC8 p.Arg1379Leu 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 ABCC8 p.Met1289Thr ABCC8 p.Arg1379Cys ABCC8 p.Arg1379Leu 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 44 ABCC8 p.Met1289Thr ABCC8 p.Arg1379Cys ABCC8 p.Arg1379Leu 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 ABCC8 p.Met1289Thr ABCC8 p.Arg1379Cys ABCC8 p.Arg1379Leu 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 47 ABCC8 p.Met1289Thr ABCC8 p.Arg1379Cys ABCC8 p.Arg1379Leu 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 ABCC8 p.Met1289Thr 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 104 ABCC8 p.Met1289Thr ABCC8 p.Arg1379Cys ABCC8 p.Arg1379Leu 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 105 ABCC8 p.Arg1379Cys ABCC8 p.Arg1379Leu By contrast, apoptosis in cells expressing mutants SUR1(R1379C) or SUR1(R1379L) was potentiated to a large extent (Figs. Login to comment 106 ABCC8 p.Arg1379Cys ABCC8 p.Arg1379Leu By contrast, apoptosis in cells expressing mutants SUR1(R1379C) or SUR1(R1379L) was potentiated to a large extent (Figs. Login to comment 118 ABCC8 p.Met1289Thr ABCC8 p.Arg1379Cys ABCC8 p.Arg1379Leu 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 120 ABCC8 p.Met1289Thr ABCC8 p.Arg1379Cys ABCC8 p.Arg1379Leu 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 121 ABCC8 p.Met1289Thr ABCC8 p.Arg1379Cys ABCC8 p.Arg1379Leu 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 ABCC8 p.Met1289Thr ABCC8 p.Arg1379Cys 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 145 ABCC8 p.Met1289Thr ABCC8 p.Arg1379Cys 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 147 ABCC8 p.Met1289Thr ABCC8 p.Arg1379Cys 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 ABCC8 p.Met1289Thr 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 154 ABCC8 p.Met1289Thr 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 156 ABCC8 p.Met1289Thr 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 ABCC8 p.Met1289Thr ABCC8 p.Arg1379Cys ABCC8 p.Arg1379Leu 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 184 ABCC8 p.Met1289Thr ABCC8 p.Arg1379Cys ABCC8 p.Arg1379Leu 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 186 ABCC8 p.Met1289Thr ABCC8 p.Arg1379Cys ABCC8 p.Arg1379Leu 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 ABCC8 p.Met1289Thr 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 204 ABCC8 p.Met1289Thr 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 206 ABCC8 p.Met1289Thr 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 ABCC8 p.Met1289Thr The mutation M1289T, located in TM17 of SUR1, influences binding (19) and action (18) of several KATP channel openers. Login to comment 221 ABCC8 p.Met1289Thr In the mutant SUR1(M1289T), a single amino acid in TM17 is exchanged by the corresponding amino acid of SUR2. Login to comment 222 ABCC8 p.Met1289Thr The mutation M1289T, located in TM17 of SUR1, influences binding (19) and action (18) of several KATP channel openers. Login to comment 223 ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr In the mutant SUR1(M1289T), a single amino acid in TM17 is exchanged by the corresponding amino acid of SUR2. Login to comment 224 ABCC8 p.Met1289Thr The mutation M1289T, located in TM17 of SUR1, influences binding (19) and action (18) of several KATP channel openers. Login to comment 225 ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr ABCC8 p.Arg1379Cys ABCC8 p.Arg1379Leu 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 ABCC8 p.Met1289Thr ABCC8 p.Arg1379Cys ABCC8 p.Arg1379Leu 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 229 ABCC8 p.Arg1379Cys ABCC8 p.Arg1379Leu 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 235 ABCC8 p.Met1289Thr 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 ABCC8 p.Met1289Thr 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 237 ABCC8 p.Met1289Thr 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 ABCC8 p.Met1289Thr 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 239 ABCC8 p.Met1289Thr 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 ABCC8 p.Met1289Thr 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