ABCMdb

PMID: 24849284

Zhou Q, Chen PC, Devaraneni PK, Martin GM, Olson EM, Shyng SL
Carbamazepine inhibits ATP-sensitive potassium channel activity by disrupting channel response to MgADP.
Channels (Austin). 2014;8(4):376-82., [PubMed]

Sentences

No. Mutations Sentence Comment

14 ABCC8 p.Phe27Ser Mutations that render channels less sensitive to ATP inhibition, or more sensitive to MgADP stimulation, cause neonatal diabetes and in some cases also DEND syndrome.3,12-14 By contrast, mutations which render channels unresponsive to the stimulatory effect of MgADP result in loss of channel function and are frequently found in patients with congenital hyperinsulinism, which is characterized by persistent insulin secretion despite life-threatening hypoglycemia.15,16 Aside from gating defects, channel biogenesis and trafficking defects which prevent expression of functional channels in the b2;-cell plasma membrane are also major causes of congenital hyperinsulinism.17 Sulfonylureas such as tolbutamide and glibenclamide inhibit KATP channel activity to stimulate insulin secretion and are thus effective in treating type II diabetes as well as some cases of neonatal diabetes/ DEND syndrome caused by hyperactive KATP channels.3 The inhibitory effect of sulfonylureas on KATP channel activity is largely attributed to an inhibition of channel response to MgADP.18 In addition to inhibiting channel activity, sulfonylureas have been shown to act as KATP channel pharmacological chaperones and correct trafficking defects caused by a subset of mutations in SUR1, specifically those in the first transmembrane domain TMD0.19,20 We have previously shown that mutant channels rescued to the cell surface by the high-affinity sulfonylurea glibenclamide are unable to open in response to metabolic inhibition in intact cells as assessed by 86 Rb+ efflux assays.19 Closer examination by inside-out patch-clamp recording revealed that the rescued channels failed to respond to MgADP, likely because the drug remained bound to the channel even after extensive washout.19 However, a lower affinity sulfonylurea tolbutamide could be washed out from rescued surface channels to recover channel response to MgADP and to allow channels to open upon metabolic inhibition in 86 Rb+ efflux assays.19,20 In a recent study, we identified carbamazepine as a novel KATP channel 'corrector` able to rescue trafficking defects caused by the same set of TMD0 mutations rescued by sulfonylureas.21 Interestingly, functional analysis of a SUR1-TMD0 trafficking mutant, F27S, showed that channels rescued to the cell surface by carbamazepine also failed to open upon metabolic inhibition.21 However, mutant channel activity gradually recovered as carbamazepine was washed out, with activity near the level observed in wild-type (WT) channels after 90 min washout.21 In this study, we investigated how carbamazepine affects KATP channel gating to prevent rescued channels from opening in metabolically stressed cells. Login to comment 17 ABCC8 p.Phe27Ser We therefore determined the response of carbamazepine-rescued F27S mutant channels to ATP and ADP by inside-out patch-clamp recording. Login to comment 19 ABCC8 p.Phe27Ser Note although the F27S mutation severely hinders channel trafficking to the cell surface, sufficient currents (~10% of averaged WT channel current amplitude) can be detected in a small number of cells to allow analysis of ATP and MgADP sensitivity. Login to comment 20 ABCC8 p.Phe27Ser ABCC8 p.Phe27Ser Compared with WT channels, mutant F27S channels exhibited comparable sensitivity to ATP inhibition and MgADP stimulation (Fig. 1), indicating that the F27S mutation per se does not have a significant effect on channel response to intracellular nucleotides. Login to comment 21 ABCC8 p.Phe27Ser Next, we recorded F27S mutant channels from cells treated overnight with Figure 1. Login to comment 22 ABCC8 p.Phe27Ser The F27S mutation in SUR1 does not alter KATP channel gating property. Login to comment 23 ABCC8 p.Phe27Ser (A) Representative recordings testing MgADP responses of WT and F27S channels expressed in COSm6 cells without overnight carbamazepine treatment. Login to comment 25 ABCC8 p.Phe27Ser There is no statistical significance in MgADP response between F27S and WT channels. Login to comment 29 ABCC8 p.Phe27Ser As the MgADP stimulatory effect is crucial for KATP channels to open upon glucose deprivation,15,16 this explains why carbamazepine-rescued F27S mutant channels were unable to open upon metabolic inhibition, as observed in our previous study.21 Interestingly, washout of carbamazepine by incubating cells in fresh media without the drug for 2 h prior to recording recovered the channel`s response to MgADP, suggesting carbamazepine impairs MgADP response. Login to comment 31 ABCC8 p.Phe27Ser As observed in the F27S mutant, WT channels recorded from cells treated overnight with carbamazepine also had little MgADP response; upon washout for more than 2 h, the MgADP response recovered to nearly the extent seen in channels not previously exposed to carbamazepine (Fig. 2). Login to comment 34 ABCC8 p.Phe27Ser We found that both WT and F27S mutant channels from cells treated overnight with carbamazepine also exhibited greatly reduced response to diazoxide; and again, after washout, diazoxide response recovered to the level comparable to that reported previously for WT channels (Fig. 3). Login to comment 43 ABCC8 p.Phe27Ser (A) Representative inside-out patch-clamp recordings testing MgADP responses of WT and F27S channels expressed in COSm6 cells treated overnight with 10 &#b5;M carbamazepine with (right) or without (left) 2 h of washout prior to recording. Login to comment 61 ABCC8 p.Phe27Ser (A) Representative inside-out patch-clamp recordings testing diazoxide responses of WT and F27S channels expressed in cells treated overnight with 10 &#b5;M carbamazepine with (right) or without (left) 2 h of washout prior to recording. Login to comment