Coexpression of the type 2 diabetes susceptibility gene variants KCNJ11 E23K and ABCC8 S1369A alter the ATP and sulfonylurea sensitivities of the ATP-sensitive K(+) channel

Abstract

Objective: In the pancreatic beta-cell, ATP-sensitive K(+) (K(ATP)) channels couple metabolism with excitability and consist of Kir6.2 and SUR1 subunits encoded by KCNJ11 and ABCC8, respectively. Sulfonylureas, which inhibit the K(ATP) channel, are used to treat type 2 diabetes. Rare activating mutations cause neonatal diabetes, whereas the common variants, E23K in KCNJ11 and S1369A in ABCC8, are in strong linkage disequilibrium, constituting a haplotype that predisposes to type 2 diabetes. To date it has not been possible to establish which of these represents the etiological variant, and functional studies are inconsistent. Furthermore, there have been no studies of the S1369A variant or the combined effect of the two on K(ATP) channel function.

Research design and methods: The patch-clamp technique was used to study the nucleotide sensitivity and sulfonylurea inhibition of recombinant human K(ATP) channels containing either the K23/A1369 or E23/S1369 variants.

Results: ATP sensitivity of the K(ATP) channel was decreased in the K23/A1369 variant (half-maximal inhibitory concentration [IC(50)] = 8.0 vs. 2.5 mumol/l for the E23/S1369 variant), although there was no difference in ADP sensitivity. The K23/A1369 variant also displayed increased inhibition by gliclazide, an A-site sulfonylurea drug (IC(50) = 52.7 vs. 188.7 nmol/l for the E23/S1369 variant), but not by glibenclamide (AB site) or repaglinide (B site).

Conclusions: Our findings indicate that the common K23/A1369 variant K(ATP) channel displays decreased ATP inhibition that may contribute to the observed increased risk for type 2 diabetes. Moreover, the increased sensitivity of the K23/A1369 variant to the A-site sulfonylurea drug gliclazide may provide a pharmacogenomic therapeutic approach for patients with type 2 diabetes who are homozygous for both risk alleles.

FIG. 1.

The K23/A1369 variant K_ATP channel exhibits decreased sensitivity to MgATP. A: Representative macroscopic current recordings of recombinant human K_ATP channel activity at different MgATP concentrations. B: MgATP inhibition response curves illustrating that the K23/A1369 variant is less sensitive to MgATP inhibition than the E23/S1369 variant. n = 3–11 patches per concentration. Extrapolation of the curves to millimolar physiological MgATP levels (inset). C and D: Representative single-channel recordings of E23/S1369 and K23/A1369 variant K_ATP channels at 0 and 1 mmol/l MgATP (o, open state; c, closed state). Single-channel unitary current amplitude was not different between the variants. E and F: Grouped open probability (P_o) data from 3–6 patches (containing 1–4 K_ATP channels per patch) showing no difference in open probability at 0 mmol/l MgATP but a significantly increased open probability in the K23/A1369 variant K_ATP channels at 1 mmol/l MgATP. G: MgATP inhibition curves from quasi-heterologous K_ATP channels containing either the K23/S1369 or E23/A1369 variant combinations (n = 3–8 patches per MgATP concentration in each group). Dashed line, MgATP inhibition curve for the wild-type E23/S1369 variant replotted from panel B. *P < 0.05.

FIG. 2.

A: Representative macroscopic current recordings of the MgADP stimulatory effects of 0.1 mmol/l MgADP in the presence of 0.1 mmol/l MgATP. B: Concentration response curves for the stimulatory effects of increasing MgADP concentrations in the presence of 0.1 mmol/l MgATP. Results show no significant differences in MgADP stimulation between the E23/S1369 and K23/A1369 haplotypes across a range of MgADP concentrations (P > 0.05). n = 3–10 patches per group.

FIG. 3.

K23/A1369 variant K_ATP channels exhibit a greater sensitivity to gliclazide. A: Schematic representation of the SUR1 and Kir6.2 protein transmembrane topologies. Amino acids discussed in the text are labeled. The nucleotide-binding folds (NBF1 and 2) and the A and B ligand binding sites are indicated. B: The structure and binding-site classification of sulfonylureas and glinides used in this study: repaglinide (B site), glibenclamide (AB site), and gliclazide (A site). C and D: Representative macroscopic current recordings showing the effect of the A-site sulfonylurea, gliclazide (300 nmol/l), on the E23/S1369 and K23/A1369 variant K_ATP channels. E: Concentration response curves illustrating that the K23/A1369 variant K_ATP channel is significantly more sensitive to gliclazide inhibition (IC₅₀ = 52.7 ± 11.1 vs. 188.7 ± 32.6 nmol/l for K23/A1369 vs. E23/S1369, respectively). n = 3–12 patches per gliclazide concentration. F: Grouped data demonstrating that the K23/A1369 variant is significantly more sensitive to inhibition by gliclazide but not glibenclamide (means ± SE 0.47 ± 0.07 vs. 0.42 ± 0.05 for E23/S1369 vs. K23/A1369, respectively; P > 0.05, n = 11 patches) or repaglinide (0.40 ± 0.06 vs. 0.52 ± 0.05 for E23/S1369 vs. K23/A1369, respectively; P > 0.05, n = 11 patches). *P < 0.05. Glic, gliclazide; Glib, glibenclamide; Rep, repaglinide.

FIG. 4.

The increased gliclazide sensitivity of K23/A1369 variant K_ATP channels is maintained in the presence of MgADP and is conferred upon the K_ATP channel complex by the ABCC8 A1369 risk allele. A and B: Representative macroscopic current recordings showing the inhibitory effect of gliclazide (300 nmol/l) on the two variants in the presence of MgADP. C: Grouped data demonstrating that the K23/A1369 variant K_ATP channels are significantly more sensitive to gliclazide in the presence of MgADP than the E23/S1369 variant K_ATP channels. n = 10–12 patches per group. D–F: Representative current recordings and grouped data showing the increased gliclazide inhibitory effect is dependent on the presence of the ABCC8 A1369 variant and not the KCNJ11 K23 variant. n = 15 patches per group. *P < 0.05. Glic, gliclazide.