ABCMdb

ABCC8 p.Thr1285Leu

Predicted by SNAP2:	A: N (53%), C: D (71%), D: D (75%), E: D (80%), F: D (75%), G: D (71%), H: D (75%), I: D (75%), K: D (85%), L: D (53%), M: D (59%), N: D (66%), P: D (80%), Q: D (66%), R: D (85%), S: N (97%), V: D (71%), W: D (80%), Y: D (80%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: N, V: D, W: D, Y: D,

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[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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No. Sentence Comment
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
96 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 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 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 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 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 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
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