ABCMdb

PMID: 12196472

Hansen AM, Christensen IT, Hansen JB, Carr RD, Ashcroft FM, Wahl P
Differential interactions of nateglinide and repaglinide on the human beta-cell sulphonylurea receptor 1.
Diabetes. 2002 Sep;51(9):2789-95., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC8 p.Ser1237Tyr Mutation of serine 1237 in SUR1 to tyrosine (S1237Y) abolished tolbutamide and nateglinide block, suggesting that these drugs share a common point of interaction on the SUR1 subunit of the ATP-sensitive K؉ channel. Login to comment 4 ABCC8 p.Ser1237Tyr In contrast, repaglinide inhibition was unaffected by the S1237Y mutation (IC50 ‫؍‬ 23 nmol/l). Login to comment 7 ABCC8 p.Ser1237Tyr This is consistent with the idea that binding of nateglinide and tolbutamide, but not repaglinide, is abolished by the SUR1[S1237Y] mutation and that the binding site for repaglinide is not identical to that of nateglinde/tolbutamide. Login to comment 34 ABCC8 p.Ser1237Tyr SUR1 currents by the nonsulphonylurea mitiglinide, as with tolbutamide, is abolished by the S1237Y mutation (16). Login to comment 35 ABCC8 p.Ser1237Tyr SUR1 currents by the nonsulphonylurea mitiglinide, as with tolbutamide, is abolished by the S1237Y mutation (16). Login to comment 45 ABCC8 p.Ser1237Tyr The point mutation SUR1[S1237Y] was constructed by standard molecular biology techniques and confirmed by DNA sequencing. Login to comment 46 ABCC8 p.Ser1237Tyr The point mutation SUR1[S1237Y] was constructed by standard molecular biology techniques and confirmed by DNA sequencing. Login to comment 48 ABCC8 p.Ser1237Tyr Cells were seeded at 50% confluency and transfected with Kir6.2 and SUR1[S1237Y] at a plasmid ratio of 1:3 on the next day. Login to comment 49 ABCC8 p.Ser1237Tyr Cells were seeded at 50% confluency and transfected with Kir6.2 and SUR1[S1237Y] at a plasmid ratio of 1:3 on the next day. Cells to be used for electrophysiological experiments were also cotransfected with green fluorescent protein (GFP) to enable visual identification of transfected cells. Login to comment 63 ABCC8 p.Ser1237Tyr Binding experiments were performed in triplicate (Kir6.2/SUR1) or duplicate (Kir6.2/SUR1[S1237Y]). Login to comment 64 ABCC8 p.Ser1237Tyr Binding experiments were performed in triplicate (Kir6.2/SUR1) or duplicate (Kir6.2/SUR1[S1237Y]). Login to comment 92 ABCC8 p.Ser1237Tyr RESULTS Electrophysiology. Whole-cell currents were recorded from HEK293 cells coexpressing Kir6.2 and either SUR1 or SUR1[S1237Y]. Login to comment 93 ABCC8 p.Ser1237Tyr ABCC8 p.Ser1237Tyr After establishment of the whole-cell configuration and dialysis with intracellular solution, there was a gradual increase in both Kir6.2/SUR1 and Kir6.2/ SUR1[S1237Y] currents due to opening of KATP channels. Login to comment 94 ABCC8 p.Ser1237Tyr After establishment of the whole-cell configuration and dialysis with intracellular solution, there was a gradual increase in both Kir6.2/SUR1 and Kir6.2/ SUR1[S1237Y] currents due to opening of KATP channels. Login to comment 95 ABCC8 p.Ser1237Tyr In contrast, tolbutamide had very little effect on Kir6.2/SUR1[S1237Y] currents (Fig. 1B). Login to comment 96 ABCC8 p.Ser1237Tyr In contrast, tolbutamide had very little effect on Kir6.2/SUR1[S1237Y] currents (Fig. 1B). Login to comment 97 ABCC8 p.Ser1237Tyr Although glibenclamide also inhibited Kir6.2/SUR1[S1237Y] channels (by 67 Ϯ 6%; n ϭ 3), in this case, inhibition was largely reversed on wash-out of the drug (Fig. 1B). Login to comment 98 ABCC8 p.Ser1237Tyr Although glibenclamide also inhibited Kir6.2/SUR1[S1237Y] channels (by 67 afe; 6%; n afd; 3), in this case, inhibition was largely reversed on wash-out of the drug (Fig. 1B). Login to comment 101 ABCC8 p.Ser1237Tyr A similar extent of block was observed for Kir6.2/ SUR1[S1237Y] (94 Ϯ 1%, n ϭ 4) (Fig. 2B), suggesting that S1237 is not required for repaglinide inhibition. Login to comment 102 ABCC8 p.Ser1237Tyr A similar extent of block was observed for Kir6.2/ SUR1[S1237Y] (94 afe; 1%, n afd; 4) (Fig. 2B), suggesting that S1237 is not required for repaglinide inhibition. Login to comment 103 ABCC8 p.Ser1237Tyr In contrast, nateglinide (100 ␮mol/l) produced reversible inhibition of Kir6.2/SUR1 currents (96 Ϯ 2%, n ϭ 4) (Fig. 2A) but was without significant effect on Kir6.2/SUR1[S1237Y] channels (Fig. 2B). Login to comment 104 ABCC8 p.Ser1237Tyr In contrast, nateglinide (100 òe;mol/l) produced reversible inhibition of Kir6.2/SUR1 currents (96 afe; 2%, n afd; 4) (Fig. 2A) but was without significant effect on Kir6.2/SUR1[S1237Y] channels (Fig. 2B). Login to comment 105 ABCC8 p.Ser1237Tyr Repaglinide blocked Kir6.2/SUR1 and Kir6.2/SUR1[S1237Y] currents with similar potency: IC50 ϭ 21 nmol/l (95% CI 17-26) and IC50 ϭ 23 nmol/l (18-28), respectively. Login to comment 106 ABCC8 p.Ser1237Tyr Repaglinide blocked Kir6.2/SUR1 and Kir6.2/SUR1[S1237Y] currents with similar potency: IC50 afd; 21 nmol/l (95% CI 17-26) and IC50 afd; 23 nmol/l (18-28), respectively. Login to comment 114 ABCC8 p.Ser1237Tyr Similarly, binding of [3 H]repaglinide to Kir6.2/SUR1[S1237Y] revealed a single binding site (Fig. 4B) with a KD of 0.31 Ϯ 0.02 nmol/l and a Bmax of 1.6 Ϯ 0.2 pmol/mg protein (n ϭ 3). Login to comment 115 ABCC8 p.Ser1237Tyr Similarly, binding of [3 H]repaglinide to Kir6.2/SUR1[S1237Y] revealed a single binding site (Fig. 4B) with a KD of 0.31 afe; 0.02 nmol/l and a Bmax of 1.6 afe; 0.2 pmol/mg protein (n afd; 3). Login to comment 118 ABCC8 p.Ser1237Tyr HEK 293 cells expressing Kir6.2/SUR1 (A) or Kir6.2/SUR1[S1237Y] (B) channels were clamped at -70 mV. Login to comment 119 ABCC8 p.Ser1237Tyr HEK 293 cells expressing Kir6.2/SUR1 (A) or Kir6.2/SUR1[S1237Y] (B) channels were clamped at d1a;70 mV. Login to comment 122 ABCC8 p.Ser1237Tyr HEK 293 cells expressing Kir6.2/SUR1 (A) or Kir6.2/ SUR1[S1237Y] (B) channels were clamped at -70 mV. Login to comment 123 ABCC8 p.Ser1237Tyr HEK 293 cells expressing Kir6.2/SUR1 (A) or Kir6.2/ SUR1[S1237Y] (B) channels were clamped at d1a;70 mV. Login to comment 125 ABCC8 p.Ser1237Tyr Concentration-response curves for inhibition of Kir6.2/SUR1 (f/F) and Kir6.2/SUR1[S1237Y] (Ⅺ/E) channels by repaglinide (f/Ⅺ) or nateglinide (F/E). Login to comment 126 ABCC8 p.Ser1237Tyr Concentration-response curves for inhibition of Kir6.2/SUR1 (f/F) and Kir6.2/SUR1[S1237Y] (ǧa;/E) channels by repaglinide (f/ǧa;) or nateglinide (F/E). Login to comment 130 ABCC8 p.Ser1237Tyr We therefore examined the ability of unlabelled nateglinide to displace [3 H]repaglinide binding to membranes isolated from HEK293 cells expressing Kir6.2/SUR1 or Kir6.2/SUR1[S1237Y]. Login to comment 131 ABCC8 p.Ser1237Tyr We therefore examined the ability of unlabelled nateglinide to displace [3 H]repaglinide binding to membranes isolated from HEK293 cells expressing Kir6.2/SUR1 or Kir6.2/SUR1[S1237Y]. Login to comment 137 ABCC8 p.Ser1237Tyr In the case of Kir6.2/SUR1[S1237Y], repaglinide displaced [3 H]repaglinide binding with a Ki of 0.4 Ϯ 0.2 nmol/l (n ϭ 3), which is similar to that found for the wild-type channel. Login to comment 138 ABCC8 p.Ser1237Tyr In the case of Kir6.2/SUR1[S1237Y], repaglinide displaced [3 H]repaglinide binding with a Ki of 0.4 afe; 0.2 nmol/l (n afd; 3), which is similar to that found for the wild-type channel. Login to comment 141 ABCC8 p.Ser1237Tyr The data are consistent with the idea that the nateglinide binding, as with that of tolbutamide, is abolished by the S1237Y mutation in SUR1. Login to comment 142 ABCC8 p.Ser1237Tyr The data are consistent with the idea that the nateglinide binding, as with that of tolbutamide, is abolished by the S1237Y mutation in SUR1. Login to comment 147 ABCC8 p.Ser1237Tyr Saturation binding of [3 H]repaglinide to membranes prepared from HEK 293 cells expressing Kir6.2/SUR1 (A) or Kir6.2/ SUR1[S1237Y] (B). Login to comment 148 ABCC8 p.Ser1237Tyr ABCC8 p.Ser1237Tyr Data are from a single representative experiment in which data points were collected in triplicate (Kir6.2/SUR1) or duplicate (Kir6.2/SUR1[S1237Y]). Login to comment 149 ABCC8 p.Ser1237Tyr Data are from a single representative experiment in which data points were collected in triplicate (Kir6.2/SUR1) or duplicate (Kir6.2/SUR1[S1237Y]). Login to comment 150 ABCC8 p.Ser1237Tyr Competition binding to membranes from HEK 293 cells expressing Kir6.2/SUR1 (A) or Kir6.2/SUR1[S1237Y] (B). Login to comment 151 ABCC8 p.Ser1237Tyr Competition binding to membranes from HEK 293 cells expressing Kir6.2/SUR1 (A) or Kir6.2/SUR1[S1237Y] (B). Login to comment 153 ABCC8 p.Ser1237Tyr Data are from a single representative experiment in which data points were collected in triplicate (Kir6.2/SUR1) or duplicate (Kir62/SUR1[S1237Y]). Login to comment 154 ABCC8 p.Ser1237Tyr Data are from a single representative experiment in which data points were collected in triplicate (Kir6.2/SUR1) or duplicate (Kir62/SUR1[S1237Y]). Login to comment 179 ABCC8 p.Ser1237Tyr ABCC8 p.Ser1237Tyr TABLE 1 Comparison of [3 H]repaglinide binding data on Kir6.2/SUR1 and Kir6.2/SUR1[S1237Y] Compound Kir6.2/SUR1 Kir6.2/SUR1[S1237Y] IC50 (nmol/l) nH Ki (nmol/l) IC50 (nmol/l) nH Ki (nmol/l) Repaglinide 1.9 Ϯ 0.8 -1.19 Ϯ 0.10 0.6 Ϯ 0.3 1.2 Ϯ 0.7 -1.05 Ϯ 0.14 0.4 Ϯ 0.2 Glibenclamide 0.7 Ϯ 0.2 -1.14 Ϯ 0.13 0.2 Ϯ 0.1 105 Ϯ 17 -0.90 Ϯ 0.25 36 Ϯ 6 Nateglinide 679 Ϯ 121 -0.98 Ϯ 0.05 235 Ϯ 42 Ͼ30,000 N/A N/A Tolbutamide 26,000 Ϯ 9,300 -0.91 Ϯ 0.12 9,000 Ϯ 3,220 Ͼ300,000 N/A N/A Data are means Ϯ SD (n ϭ 3). Login to comment 180 ABCC8 p.Ser1237Tyr ABCC8 p.Ser1237Tyr TABLE 1 Comparison of [3 H]repaglinide binding data on Kir6.2/SUR1 and Kir6.2/SUR1[S1237Y] Compound Kir6.2/SUR1 Kir6.2/SUR1[S1237Y] IC50 (nmol/l) nH Ki (nmol/l) IC50 (nmol/l) nH Ki (nmol/l) Repaglinide 1.9 afe; 0.8 afa;1.19 afe; 0.10 0.6 afe; 0.3 1.2 afe; 0.7 afa;1.05 afe; 0.14 0.4 afe; 0.2 Glibenclamide 0.7 afe; 0.2 afa;1.14 afe; 0.13 0.2 afe; 0.1 105 afe; 17 afa;0.90 afe; 0.25 36 afe; 6 Nateglinide 679 afe; 121 afa;0.98 afe; 0.05 235 afe; 42 b0e;30,000 N/A N/A Tolbutamide 26,000 afe; 9,300 afa;0.91 afe; 0.12 9,000 afe; 3,220 b0e;300,000 N/A N/A Data are means afe; SD (n afd; 3). Login to comment 181 ABCC8 p.Ser1237Tyr Glibenclamide produced a reversible block of Kir6.2/ SUR1[S1237Y]. Login to comment 182 ABCC8 p.Ser1237Tyr Glibenclamide produced a reversible block of Kir6.2/ SUR1[S1237Y]. Login to comment 184 ABCC8 p.Ser1237Tyr Thus, glibenclamide displaced [3 H]repaglinide binding to the mutant channel with a much lower potency than for the wild-type channel, consistent with a larger dissociation rate constant for glibenclamide binding to Kir6.2/SUR1[S1237Y]. Login to comment 185 ABCC8 p.Ser1237Tyr Thus, glibenclamide displaced [3 H]repaglinide binding to the mutant channel with a much lower potency than for the wild-type channel, consistent with a larger dissociation rate constant for glibenclamide binding to Kir6.2/SUR1[S1237Y]. Login to comment 188 ABCC8 p.Ser1237Tyr This suggests that the binding affinity of repaglinide is enhanced by interaction with additional residues in SUR1 and that this interaction is not disrupted by the S1237Y mutation. Login to comment 189 ABCC8 p.Ser1237Tyr ABCC8 p.Ser1237Tyr The effect of the S1237Y mutation on nateglinide-induced KATP channel inhibition could be due to either reduced drug binding or an impaired ability of SUR1 to transduce drug binding into channel closure. Login to comment 190 ABCC8 p.Ser1237Tyr ABCC8 p.Ser1237Tyr However, because Kir6.2/SUR1[S1237Y] retains the ability to be blocked fully by repaglinide and glibenclamide, the transduction mechanism does not appear to be compromised by the mutation. Login to comment 191 ABCC8 p.Ser1237Tyr However, because Kir6.2/SUR1[S1237Y] retains the ability to be blocked fully by repaglinide and glibenclamide, the transduction mechanism does not appear to be compromised by the mutation. Login to comment