ATP binding without hydrolysis switches sulfonylurea receptor 1 (SUR1) to outward-facing conformations that activate KATP channels

Abstract

Neuroendocrine-type ATP-sensitive K⁺ (K_ATP) channels are metabolite sensors coupling membrane potential with metabolism, thereby linking insulin secretion to plasma glucose levels. They are octameric complexes, (SUR1/Kir6.2)₄, comprising sulfonylurea receptor 1 (SUR1 or ABCC8) and a K⁺-selective inward rectifier (Kir6.2 or KCNJ11). Interactions between nucleotide-, agonist-, and antagonist-binding sites affect channel activity allosterically. Although it is hypothesized that opening these channels requires SUR1-mediated MgATP hydrolysis, we show here that ATP binding to SUR1, without hydrolysis, opens channels when nucleotide antagonism on Kir6.2 is minimized and SUR1 mutants with increased ATP affinities are used. We found that ATP binding is sufficient to switch SUR1 alone between inward- or outward-facing conformations with low or high dissociation constant, K_D , values for the conformation-sensitive channel antagonist [³H]glibenclamide ([³H]GBM), indicating that ATP can act as a pure agonist. Assembly with Kir6.2 reduced SUR1's K_D for [³H]GBM. This reduction required the Kir N terminus (KNtp), consistent with KNtp occupying a "transport cavity," thus positioning it to link ATP-induced SUR1 conformational changes to channel gating. Moreover, ATP/GBM site coupling was constrained in WT SUR1/WT Kir6.2 channels; ATP-bound channels had a lower K_D for [³H]GBM than ATP-bound SUR1. This constraint was largely eliminated by the Q1179R neonatal diabetes-associated mutation in helix 15, suggesting that a "swapped" helix pair, 15 and 16, is part of a structural pathway connecting the ATP/GBM sites. Our results suggest that ATP binding to SUR1 biases K_ATP channels toward open states, consistent with SUR1 variants with lower K_D values causing neonatal diabetes, whereas increased K_D values cause congenital hyperinsulinism.

Keywords: ABC transporter; ATP-binding cassette transporter subfamily C member 8 (ABCC8); ATP-sensitive potassium channel; KATP channel; KCNJ11 (Kir6.2); SUR1; allosteric regulation; congenital hyperinsulinism; diabetes; glibenclamide; hyperinsulinism; ion channel; metabolic sensor; neonatal diabetes.

Figure 1.

Assay for the action of nucleotides on K_ATP channels. ATP⁴⁻ (10 mm), without Mg²⁺ (A), or MgATP (1 mm) (B) inhibits WT SUR1/WT Kir6.2 channels. C, WT SUR1/Kir6.2_G334D channels, where the G334D mutant Kir6.2 subunit has a low affinity for adenine nucleotides, are activated by MgATP (1 mm) but not significantly affected by ATP⁴⁻ (10 mm). In B and C, the NP_o values (Current = Number of open channels × Channel open probability) were estimated over 20–45-s intervals before, during, or after the application of nucleotides.

Figure 2.

ATP⁴⁻ (10 mm) and GTP⁴⁻ (10 mm) activate K_ATP channels with ND mutations in SUR1 that have a lower K_D for ATP. A and B, activation of SUR1_Q1179R/Kir6.2_G334D channels by ATP⁴⁻ or GTP⁴⁻. D and E, summary of the results from 12 experiments on SUR1_Q1179R/Kir6.2_G334D channels. The activation of SUR1_E1507Q/Kir6.2_G334D channels by ATP⁴⁻ (C) and a summary of results from seven experiments on SUR1_E1507Q/Kir6.2_G334D channels (F) are shown. Red and blue error bars are the means ± S.D. Significance was determined using the nonparametric Wilcoxon signed-rank test.

Figure 3.

Activation of SUR1_E1507K/WT Kir6.2 channels associated with congenital hyperinsulinism by the agonist diazoxide. At the arrows, patches were pulled into nucleotide-free medium, which activates a large number of channels as inhibitory nucleotides leave the pore. Application of ATP⁴⁻ (10 mm) (A) or MgATP (1 mm) (B) rapidly inhibits channel activity. Concurrent application of diazoxide (340 μm) increases channel activity in both cases. Activation was observed in five of five trials for ATP⁴⁻ and for MgATP.

Figure 4.

Assembly with Kir6.2 increases the affinity of SUR1 for [³H]GBM. A–C, dissociation constants, K_G values, for [³H]GBM were determined by displacement of 0.3 (blue) or 1 (red) nm [³H]GBM by unlabeled GBM. Plots are for WT SUR1 alone, WT SUR1/WT Kir6.2, and WT SUR1/Kir6.2_G334D, respectively. The data are presented as the mean values (error bars are ±S.E., n = 9) of the total bound radioactivity. The radioactivity remaining at the foot of the binding curves shows nonspecific binding, which increases with higher [³H]GBM concentrations. The curves are global best fits to a homologous displacement model with radioligand depletion (74). Table 1 gives a summary of K_G values for WT and mutant SUR1s and corresponding channels. D, the thermodynamic cycle used to estimate GBM stabilization of K_ATP channels. Binding assays were done in the absence of ATP and Mg²⁺ with 1 mm EDTA added.

Figure 5.

Association with Kir6.2 affects the allosteric properties of WT SUR1. Increasing MgATP reduces the binding of [³H]GBM (1 nm). The data are given as specific bound [³H]GBM defined as [³H]GBM bound in the presence of MgATP divided by the radioactivity bound without nucleotide. The inset shows the four-state ternary complex model. R is defined as SUR1 with ATP bound to NBD1, the noncanonical nucleotide-binding site. ATP can induce NBD dimerization by binding to NBD2 with or without bound GBM. The K_G values from Fig. 4, estimated in the absence of ATP, were used to estimate β and K_T. The curves were generated using the global-fit parameters in Table 2; data are means with error bars indicating ±S.E., n = 6.

Figure 6.

Effect of the SUR1_Q1179R ND mutation on allosteric coupling in K_ATP channels. Increasing MgATP reduces bound [³H]GBM (1 nm). Curves are the best fits of the ternary complex model (Fig. 5, inset) to the data. The K_G values from Table 1 were used to estimate β and K_T. The best-fit parameters are given in the text. Points are means with error bars indicating ±S.E., n = 6. The WT SUR1_Q1179/Kir6.2 channel values include those shown in Fig. 5.

Figure 7.

Effect of MgADP on allosteric coupling in SUR1 alone versus SUR1 in K_ATP channels. Increasing MgADP reduces bound [³H]GBM (1 nm). The K_G values from Fig. 4 were used to estimate β and K_ADP. The SUR1 curve was drawn using best-fit parameters (mean (lower confidence interval–upper confidence interval): K_G = 0.25 (0.19–0.32) nm, K_ADP = 55 (28–107) μm, and β = 1.8 (1.6–1.9)). Points are means ± S.E., n = 6.

Figure 8.

Positioning of amino acid Gln-1179 in helix 15 relative to the GBM-binding site. A shows one Kir6.2 subunit (green) interacting with SUR1 core (gray) through TMD0 (tan) and L0 (pink). Helices 15 and 16 are orange and cyan, respectively. Residues Gln-1179 (red), Lys-1180 (light blue), Arg-1182 and Arg-1187 (dark blue) and GBM (magenta) are shown. NBD1 is shown in yellow. B is an enlarged view of the GBM site with the helices in the forefront hidden. The dotted surfaces show the amino acids identified in Martin et al. (41) as part of the GBM-binding site.