Carbamazepine as a novel small molecule corrector of trafficking-impaired ATP-sensitive potassium channels identified in congenital hyperinsulinism

Abstract

ATP-sensitive potassium (KATP) channels consisting of sulfonylurea receptor 1 (SUR1) and the potassium channel Kir6.2 play a key role in insulin secretion by coupling metabolic signals to β-cell membrane potential. Mutations in SUR1 and Kir6.2 that impair channel trafficking to the cell surface lead to loss of channel function and congenital hyperinsulinism. We report that carbamazepine, an anticonvulsant, corrects the trafficking defects of mutant KATP channels previously identified in congenital hyperinsulinism. Strikingly, of the 19 SUR1 mutations examined, only those located in the first transmembrane domain of SUR1 responded to the drug. We show that unlike that reported for several other protein misfolding diseases, carbamazepine did not correct KATP channel trafficking defects by activating autophagy; rather, it directly improved the biogenesis efficiency of mutant channels along the secretory pathway. In addition to its effect on channel trafficking, carbamazepine also inhibited KATP channel activity. Upon subsequent removal of carbamazepine, however, the function of rescued channels was recovered. Importantly, combination of the KATP channel opener diazoxide and carbamazepine led to enhanced mutant channel function without carbamazepine washout. The corrector effect of carbamazepine on mutant KATP channels was also demonstrated in rat and human β-cells with an accompanying increase in channel activity. Our findings identify carbamazepine as a novel small molecule corrector that may be used to restore KATP channel expression and function in a subset of congenital hyperinsulinism patients.

Keywords: ABC Transporter; CFTR; Carbamazepine; Hyperinsulinism; Intracellular Trafficking; KATP Channel; Molecular Chaperone; Potassium Channels; Sulfonylurea Receptor 1; β-Cells.

FIGURE 1.

Carbamazepine corrects SUR1 processing defects caused by a subset of mutations previously identified in congenital hyperinsulinism. A, location of SUR1 mutations included in this study. SUR1 topology is based on Conti et al. (70). B, chemical structure of carbamazepine. C, Western blots of SUR1 from COSm6 cells co-transfected with WT Kir6.2 and WT or mutant SUR1 cDNAs and treated with 0.1% DMSO (Veh), 5 μm glibenclamide (Glib), or 10 μm carbamazepine (CBZ) for 16 h. Untransfected cells (Unt) and cells expressing WT channels were included for comparison. Only mutations that exhibit a significant response to carbamazepine are shown (all in TMD0). The thin lines separate different parts of the same blot, and thick vertical lines separate different blots. The empty arrow points to the core-glycosylated immature SUR1, and the solid arrow points to the complex-glycosylated mature SUR1. Molecular mass markers shown on the right side of the blots are in kDa in this and all subsequent figures. D, dose response of SUR1 mutants to the rescue effect of carbamazepine.

FIGURE 2.

Time course and duration of the rescue effect of carbamazepine. A, COSm6 cells were transiently transfected with WT Kir6.2 and WT or F27S mutant SUR1 cDNAs and treated with 10 or 50 μm carbamazepine (C) or 5 μm glibenclamide (G) for 0, 1, 2, 4, 6, 10, 12, or 16 h. Panel i, blots showing cells treated with glibenclamide or carbamazepine for 1 h (left) when an effect on the upper band signal was first detected and for 6 h (right) when the effect began to plateau. WT channels and mutant receiving various treatments for 16 h are included for comparison. Panel ii, time course of the effect of glibenclamide and carbamazepine. Upper and lower band signals (left and right plots, respectively) from blots similar to those shown in panel i were quantified by densitometry and expressed as percentage of the total signal (upper + lower) of each sample. Each data point represents the mean ± S.E. of three to five independent experiments. WT without any treatment and F27S mutant treated with DMSO (0.1%) for 16 h are shown for comparison. B, COSm6 cells transfected with WT Kir6.2 and F27S mutant SUR1 cDNAs were treated with 0.1% DMSO, 10 or 50 μm carbamazepine (CBZ), or 5 μm glibenclamide (Glib) for 16 h. The drugs were then removed from the culture medium, and cells were cultured for an additional 2, 4, or 6 h in fresh medium. Panel i, blots showing that the rescue effect of carbamazepine remains for at least 6 h after drug removal. Panel ii, quantification of blots shown in panel i by densitometry. Upper and lower band signals (left and right plots, respectively) were expressed as percentage of the total signal of each sample. F27S treated with DMSO for 16 h is shown for comparison. Each data point represents the mean ± S.E. of three independent experiments. No statistical difference was found between the 0 time point and 2, 4, or 6 h of washout in all three treatment groups (p > 0.05 by Student's t test). Error bars represent S.E.

FIGURE 3.

Carbamazepine restores surface expression of trafficking-impaired SUR1 mutants. A, surface SUR1 detected using surface protein biotinylation followed by immunoprecipitation and immunoblotting. COSm6 cells were transiently transfected with WT Kir6.2 and WT or mutant SUR1 cDNAs; treated with 0.1% DMSO (Veh), 10 or 50 μm carbamazepine (CBZ), or 5 μm glibenclamide (Glib) for 12 h; and then subjected to surface biotinylation. Only the upper complex-glycosylated band was pulled down by the NeutrAvidin beads (top). Total SUR1 detected in whole-cell lysate in the corresponding samples is also shown with tubulin as a loading control (middle and lower). B, surface expression of mutant SUR1 upon rescue by carbamazepine and tolbutamide (Tolb) as detected by immunostaining of the extracellular FLAG epitope tag of f-SUR1 in non-permeabilized cells. Scale bar, 5 μm. C, quantification of surface expression of K_ATP channels by chemiluminescence assays. Each bar represents mean ± S.E. of three to four experiments. *, p < 0.001 comparing F27S vehicle with various treatment groups by one-way analysis of variance with Bonferroni post hoc test. Error bars represent S.E. Unt, untransfected cells.

FIGURE 4.

Carbamazepine increases F27S mutant channel surface expression by improving processing and maturation of the channel complex during biogenesis. A, COSm6 cells transfected with F27S SUR1 and WT Kir6.2 were pulse-labeled with Tran³⁵S-Label for an hour and chased for 0–4 h in regular medium. SUR1 was immunoprecipitated using anti-FLAG-agarose beads and analyzed by phosphorimaging. Shown are representative gels from cells treated with 10 μm carbamazepine (CBZ) or DMSO vehicle control during the chase. B, the upper and lower band signals from gels shown in A were quantified using a Bio-Rad phosphorimaging system and expressed as percentage of total signal at time 0 of DMSO-treated group. Data points represent the mean ± S.E. of three to four experiments. The upper band signals in the carbamazepine-treated group are significantly higher than those in the DMSO-treated group at 2 and 4 h of chase. *, p < 0.001 by Student's t test. Error bars represent S.E.

FIGURE 5.

The rescue effect of carbamazepine is not due to activation of autophagy. A, Western blot of SUR1 from COSm6 cells transfected with F27S SUR1 and WT Kir6.2 and treated with carbamazepine (CBZ), chloroquine (Chloroq), or both for 16 h (upper panel) or carbamazepine, Ly294002, or both for 16 h (lower panel) as indicated. WT SUR1 is shown as a control. B, Western blot of SUR1 from COSm6 cells transfected with F27S SUR1 and WT Kir6.2 and treated with carbamazepine or rapamycin (Rapa) or Li⁺, both of which are autophagy inducers, for 16 h as indicated.

FIGURE 6.

Effects of carbamazepine on K_ATP channel function. A, carbamazepine inhibits WT channel activity in isolated membrane patches. Carbamazepine (CBZ) (10 μm) was applied acutely to the cytoplasmic face of isolated membrane patches containing WT channels. A representative current trace is shown on the left, and the averaged data (mean ± S.E.) are shown on the right (n = 3). Cont, control. *, p < 0.001 by Student's t test. B, K_ATP channel activity in intact cells in response to metabolic inhibition as assessed by the ⁸⁶Rb⁺ efflux assay. Cells were treated with metabolic inhibitors for 30 min prior to efflux measurements. Efflux over a 40-min period is expressed as percentage of total counts. F27S cells were treated overnight (O.N.) with 10 μm carbamazepine, and carbamazepine was removed 15, 30, or 60 min (washout) prior to incubation with metabolic inhibitors. Untreated untransfected cells (Unt) and cells transfected with WT channels are included as controls. C, same as B except that carbamazepine was not washed out prior to incubation with metabolic inhibitors, and the efflux was measured with or without the addition of 200 μm diazoxide (Diaz). In B and C, each bar represents the mean ± S.E. (n = 3–10). *, p < 0.001 comparing F27S vehicle (Veh) with various treatment groups by one-way analysis of variance with Bonferroni post hoc test. Error bars represent S.E. D, Western blots show the effects of different drug combinations on the processing efficiency of F27S SUR1 co-expressed with WT Kir6.2 in COSm6 cells. The concentrations of the drugs used in the overnight (16-h) combination treatment are 5 μm glibenclamide (Glib), 300 μm tolbutamide (Tolb), 10 μm carbamazepine, and 200 μm diazoxide. Note that although diazoxide nearly abolished the rescue effect of glibenclamide or tolbutamide it had no effect on the ability of carbamazepine to correct the processing defect of F27S SUR1. The lines separate different blots.

FIGURE 7.

Carbamazepine restores surface expression and function of trafficking-impaired SUR1 mutants in β-cells. A, panel i, representative SUR1 blots from uninfected human islets (probed with anti-SUR1 antibody) and human islets infected with adenoviruses carrying WT Kir6.2 and WT or F27S or A116P mutant f-SUR1 cDNAs (probed with anti-FLAG antibody) and treated with DMSO, 5 μm glibenclamide (Glib), or 10 μm carbamazepine (CBZ) for 16 h. Panel ii, representative whole-cell patch clamp recordings measuring K_ATP current density in control and drug-treated human β-cells infected with the F27S mutant viruses (recordings are from two cells with similar membrane capacitance of ∼10 picofarads (pF)). Dissociated human islet β-cells were identified by staining with 0.01% dithizone briefly followed by washout before recording. K_ATP currents were activated upon perfusion with Tyrode's solution containing 200 μm diazoxide. Tolbutamide (Tolb) (300 μm) was used to verify that the currents observed were from K_ATP channels. Panel iii, the averaged current density measured by whole-cell recordings. Each bar represents the mean ± S.E. of the number of patches shown in the bar. B, same as in A except INS-1 cells were used for the experiments. #, p < 0.05; *, p < 0.01; **, p < 0.001 by one-way analysis of variance with Bonferroni post hoc test. Error bars represent S.E. uninf, uninfected; Veh, vehicle.