ABCMdb

PMID: 15561900

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., [PubMed]

Sentences

No. Mutations Sentence Comment

6 ABCC8 p.Met1289Thr ABCC8 p.Thr1285Leu 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 ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr ABCC8 p.Thr1285Leu 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 ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr ABCC8 p.Thr1285Leu ABCC8 p.Thr1285Leu 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 ABCC8 p.Met1289Thr ABCC8 p.Thr1285Leu 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 ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr ABCC8 p.Thr1285Leu 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 96 ABCC8 p.Thr1285Leu ABCC8 p.Met1289Leu ABCC8 p.Met1289Leu As we intended to compare binding properties of SUR1 with the mutants SUR1(T1285L, M1289L) and SUR1(M1289L), [3 H]glibenclamide was chosen as the radioligand due to its high affinity to SUR1. Login to comment 100 ABCC8 p.Thr1285Leu ABCC8 p.Met1289Leu ABCC8 p.Met1289Leu Displacing [3 H]glibenclamide by increasing concentrations of unlabeled glibenclamide (Fig. 2A) resulted in similar monophasic inhibition curves for SUR1, SUR1 (M1289L), and SUR1(T1285L, M1289L) with KD values of thioformamide: aprikalim [F]; and benzothiadiazine: diazoxide [G]. Login to comment 103 ABCC8 p.Met1289Thr ABCC8 p.Thr1285Leu 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 111 ABCC8 p.Thr1285Leu ABCC8 p.Met1289Leu ABCC8 p.Met1289Leu Glibenclamide binding was not significantly changed by the mutations [pKi SUR1 (M1289L) vs. SUR1: P ϭ 0.0868; pKi SUR1(T1285L, M1289L) vs. SUR1: P ϭ 0.0825]. Login to comment 112 ABCC8 p.Thr1285Leu ABCC8 p.Met1289Leu ABCC8 p.Met1289Leu In the following experiments, binding of openers representative of different chemical classes was systematically compared between SUR1 and SUR1(T1285L, M1289L); for P1075, levcromakalim, and diazoxide, the mutant SUR1 (M1289L) was also investigated. Login to comment 115 ABCC8 p.Met1289Thr ABCC8 p.Thr1285Leu 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 ABCC8 p.Met1289Thr ABCC8 p.Thr1285Leu In any case, for SUR1(T1285L, M1289T), A (55%) was not larger than for SUR1. Login to comment 118 ABCC8 p.Met1289Thr ABCC8 p.Thr1285Leu ABCC8 p.Met1289Leu Binding of P1075 to SUR1(T1285L, M1289T) did not differ much from binding to SUR1(M1289L) (Table 1). Login to comment 120 ABCC8 p.Met1289Thr ABCC8 p.Thr1285Leu 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 ABCC8 p.Met1289Thr ABCC8 p.Thr1285Leu 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 ABCC8 p.Met1289Thr ABCC8 p.Thr1285Leu 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 ABCC8 p.Met1289Thr Binding of levcromakalim was also tested with SUR1(M1289T). Login to comment 124 ABCC8 p.Met1289Thr ABCC8 p.Thr1285Leu Here, inhibition curves were very similar to those obtained for SUR1(T1285L, M1289T) (Table 1). Login to comment 128 ABCC8 p.Met1289Thr ABCC8 p.Thr1285Leu 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 ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr ABCC8 p.Thr1285Leu 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 ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr ABCC8 p.Thr1285Leu ABCC8 p.Thr1285Leu ABCC8 p.Thr1285Leu 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 ABCC8 p.Met1289Thr ABCC8 p.Thr1285Leu 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 ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr ABCC8 p.Thr1285Leu 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 ABCC8 p.Met1289Thr ABCC8 p.Thr1285Leu 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 ABCC8 p.Met1289Thr ABCC8 p.Thr1285Leu 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 ABCC8 p.Met1289Thr ABCC8 p.Thr1285Leu 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 ABCC8 p.Met1289Thr ABCC8 p.Thr1285Leu 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 ABCC8 p.Met1289Thr ABCC8 p.Met1289Thr ABCC8 p.Thr1285Leu 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 ABCC8 p.Met1289Thr ABCC8 p.Thr1285Leu 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