Conserved functional consequences of disease-associated mutations in the slide helix of Kir6.1 and Kir6.2 subunits of the ATP-sensitive potassium channel

Abstract

Cantu syndrome (CS) is a condition characterized by a range of anatomical defects, including cardiomegaly, hyperflexibility of the joints, hypertrichosis, and craniofacial dysmorphology. CS is associated with multiple missense mutations in the genes encoding the regulatory sulfonylurea receptor 2 (SUR2) subunits of the ATP-sensitive K⁺ (K_ATP) channel as well as two mutations (V65M and C176S) in the Kir6.1 (KCNJ8) subunit. Previous analysis of leucine and alanine substitutions at the Val-65-equivalent site (Val-64) in Kir6.2 indicated no major effects on channel function. In this study, we characterized the effects of both valine-to-methionine and valine-to-leucine substitutions at this position in both Kir6.1 and Kir6.2 using ion flux and patch clamp techniques. We report that methionine substitution, but not leucine substitution, results in increased open state stability and hence significantly reduced ATP sensitivity and a marked increase of channel activity in the intact cell irrespective of the identity of the coassembled SUR subunit. Sulfonylurea inhibitors, such as glibenclamide, are potential therapies for CS. However, as a consequence of the increased open state stability, both Kir6.1(V65M) and Kir6.2(V64M) mutations essentially abolish high-affinity sensitivity to the K_ATP blocker glibenclamide in both intact cells and excised patches. This raises the possibility that, at least for some CS mutations, sulfonylurea therapy may not prove to be successful and highlights the need for detailed pharmacogenomic analyses of CS mutations.

Keywords: ATP; cardiovascular; cardiovascular disease; drug resistance; mutagenesis; nucleotide; potassium channel.

Figure 1.

CS-associated mutations in Kir6.1. To date, two mutations in Kir6.1 (KCNJ8) have been identified in CS patients, Kir6.1(C176S) and Kir6.1(V65M), at residues that are conserved in Kir6.2. A, a Kir6.1 homology model based upon the recent cryo-EM structures for Kir6.2/SUR1 (18, 19) shows that Cys-176 and Val-65 (equivalent to Cys-166 and Val-64 in Kir6.2) lie in close proximity. Inset, Val-65 (Val-64) faces TM2 on the slide helix, whereas Cys-176 (Cys-166) lies nearby in TM2. B, Kir6.x and SUR subunits coassemble as obligate hetero-octamers in a 1:1 stoichiometry. Here two SUR subunits are omitted for display. C, the Kir6.1(V65M) mutation lies in a highly conserved N-terminal sequence within the slide helix. D, average root mean square deviation (RMSD) from three independent 100-ns MD simulations of the modeled Kir6.1 subunits show that Kir6.1 WT, Kir6.1(V65L), and Kir6.1(V65M) subunits are stable.

Figure 2.

Kir6.1(V65M), but not Kir6.1(V65L), increases SUR1-dependent K_ATP channel activity in intact cells. Cumulative ⁸⁶Rb⁺ efflux as a function of time was measured in GFP-transfected control COSm6 cells and in cells transiently expressing WT or mutant Kir6.1 with SUR1. Experiments were performed in basal conditions in Ringer's solution (A) or in the presence of the K⁺ channel opener diazoxide (B) or the metabolic inhibitors (MI) oligomycin and 2-deoxy-d-glucose (C). Data points and error bars represent mean and S.E. of eight to 18 experiments. Summary representations of the mean cumulative flux at 25 min are shown on the right (* denotes statistical significance as determined by unpaired Student's t tests; p < 0.05).

Figure 3.

Kir6.1(V65M), but not Kir6.1(V65L), increases SUR2A-dependent K_ATP channel activity in intact cells. Cumulative ⁸⁶Rb⁺ efflux as a function of time was measured in GFP-transfected control COSm6 cells and in cells transiently expressing WT or mutant Kir6.1 with SUR2A. Experiments were performed in basal conditions in Ringer's solution (A) or in the presence of the K⁺ channel opener pinacidil (B) or the metabolic inhibitors (MI) oligomycin and 2-deoxy-d-glucose (C). Data points and error bars represent mean and S.E. of three experiments. Summary representations of the mean cumulative flux at 25 min are shown on the right (* denotes statistical significance as determined by Mann-Whitney U test; p < 0.05).

Figure 4.

Kir6.2(V64M), but not Kir6.2(V64L), increases SUR1-dependent K_ATP channel activity in intact cells. Cumulative ⁸⁶Rb⁺ efflux as a function of time was measured in GFP-transfected control COSm6 cells and in cells transiently expressing WT or mutant Kir6.2 with SUR1. Experiments were performed in basal conditions in Ringer's solution (A) or in the presence of the K⁺ channel opener diazoxide (B) or the metabolic inhibitors (MI) oligomycin and 2-deoxy-d-glucose (C). Data points and error bars represent mean and S.E. of three to five experiments. Summary representations of the mean cumulative flux at 25 min are shown on the right (* denotes statistical significance as determined by Mann-Whitney U test; p < 0.05).

Figure 5.

Kir6.2(V64M), but not Kir6.2(V64L), increases SUR2A-dependent K_ATP channel activity in intact cells. Cumulative ⁸⁶Rb⁺ efflux as a function of time was measured in GFP-transfected control COSm6 cells and in cells transiently expressing WT or mutant Kir6.2 with SUR2A. Experiments were performed in basal conditions in Ringer's solution (A) or in the presence of the K⁺ channel opener pinacidil (B) or the metabolic inhibitors (MI) oligomycin and 2-deoxy-d-glucose (C). Data points and error bars represent mean and S.E. of three to six experiments. Summary representations of the mean cumulative flux at 25 min are shown on the right (* denotes statistical significance as determined by Mann-Whitney U test; p < 0.05).

Figure 6.

Gain of function in Kir6.2(V64M) results from decreased ATP sensitivity. Representative excised patch clamp recordings from COSm6 cells coexpressing WT or mutant Kir6.2 subunits with SUR2A are shown. A, membrane potential was held at −50 mV, and currents were recorded continuously in inside-out excised patches exposed to KINT in the absence or presence of 0.01, 0.1, or 5 mm Mg²⁺-free ATP. B, summary dose-response data (data points and error bars represent mean and S.E.; 10 patches each) was fit using a four-parameter Hill equation to estimate the ATP concentration for half-maximal inhibition. IC₅₀ values were 32.2 ± 6.6 μm (Hill coefficient, 2.2 ± 0.2) for WT, 198 ± 31.1 μm (Hill coefficient, 2.9 ± 0.3) for Kir6.2(V64M), and 31.0 ± 2.2 μm (Hill coefficient, 2.2 ± 0.1) for Kir6.2(V64L) (* denotes statistical significance as determined by unpaired Student's t tests; p < 0.05). In this and in representative current recordings in subsequent figures, the dashed lines represent zero channel current.

Figure 7.

Kir6.2(V64M) increases channel open state stability. Representative K_ATP currents recorded from patches expressing WT (A) or V64M mutant Kir6.2 (B) with SUR2A following excision at the arrow and in the presence of 10 mm ATP, 1 mm ATP, or 5 μg/ml PIP₂ as indicated are shown. C, relative P_o determined as a ratio of steady-state current in the patch upon excision in the absence of nucleotides to the maximum current measured following PIP₂. Individual patch data are represented by symbols (n = 8–14); bars and error bars are the means and S.E. Relative P_o = 0.59 ± 0.07 (WT) and 1.05 ± 0.07 (Kir6.2(V64M)) (* denotes statistical significance as determined by unpaired Student's t tests; p < 0.05).

Figure 8.

Kir6.2(V64M) decreases glibenclamide sensitivity in intact cells and excised patches. Cumulative ⁸⁶Rb⁺ efflux as a function of time was measured from COSm6 cells incubated in metabolic inhibitors oligomycin and 2-deoxy-d-glucose in the presence or absence of 10 μm glibenclamide (Glib). A, top left, cells were transfected either with GFP alone or with WT Kir6.2 and SUR2A. Top right, cells were transfected either with GFP alone or with Kir6.2(V64M) and SUR2A. Bottom left, cells were transfected either with GFP alone or with a 1:1 mixture of Kir6.2 WT and Kir6.2(V64M) plus SUR2A. Data points and error bars represent mean and S.E. of three experiments. B, percent inhibition of K_ATP flux at the 25-min time point from three independent experiments. C, representative traces from inside-out patch clamp recordings from cells transfected with Kir6.2 WT and SUR2A (top trace) or Kir6.2(V64M) and SUR2A (bottom trace) at −50 mV in the presence and absence of 3 mm ATP or increasing concentrations of glibenclamide as indicated. D, summary glibenclamide dose response from inside-out patch recordings (data points and error bars represent mean and S.E. from three to five patches). Asterisks (*) denote statistical significance as determined by Mann-Whitney U test (p < 0.05).

Figure 9.

The Kir6.1(V65M) mutation decreases glibenclamide sensitivity in heterotetrameric channels expressed in intact cells and excised patches. A, representative inside-out patch current recordings from COSm6 cells transfected with a 1:1 mixture of Kir6.2 WT and either Kir6.1 WT (top) or Kir6.1(V65M) (bottom) and SUR2A. B, summary dose-response data (mean ± S.E. from three patches) was fit using a four-parameter Hill equation to estimate the ATP concentration for half-maximal inhibition.The IC₅₀ for Kir6.2 WT/Kir6.1 WT-containing channels was 3.2 ± 1.2 μm (Hill coefficient 0.9 ± 0.1; n = 3) compared with 15.1 ± 2.6 μm (Hill coefficient 1.2 ± 0.1; n = 3) for Kir6.2 WT/Kir6.1(V65M)-containing channels (* denotes statistical significance as determined by Mann-Whitney U tests; p < 0.05). C, cumulative ⁸⁶Rb⁺ efflux as a function of time was measured from COSm6 cells incubated in metabolic inhibitors oligomycin and 2-deoxy-d-glucose in the presence or absence of 10 μm glibenclamide (Glib). Left, cells were transfected either with GFP alone or with a 1:1 ratio of Kir6.2 WT with Kir6.1 WT and SUR2A. Right, cells were transfected either with GFP alone or with a 1:1 ratio of Kir6.2 WT with Kir6.1(V65M) and SUR2A. Data points and error bars represent mean and S.E. of three experiments. D, the inhibition of K_ATP flux by 10 μm glibenclamide (calculated as in Fig. 8). Kir6.2 WT/Kir6.1 WT-containing channels were inhibited by 46.1 ± 2.7% compared with 31.4 ± 4.0% for Kir6.2 WT/Kir6.1(V65M)-containing channels. E, representative traces from inside-out patch clamp recordings from cells transfected with Kir6.2 WT/Kir6.1 WT and SUR2A (top trace) or Kir6.2 WT/Kir6.1(V65M) and SUR2A (bottom trace). Currents were recorded at −50 mV in the presence and absence of 3 mm ATP or increasing concentrations of glibenclamide as indicated. F, summary glibenclamide dose response from inside-out patch clamp recordings (data points and error bars represent mean and S.E. from three patches). Asterisks (*) denote statistical significance as determined by Mann-Whitney U test (p < 0.05).