The mutation Y1206S increases the affinity of the sulphonylurea receptor SUR2A for glibenclamide and enhances the effects of coexpression with Kir6.2

Abstract

1. ATP-sensitive K(+) channels (K(ATP) channels) are tetradimeric complexes of inwardly rectifying K(+) channels (Kir6.x) and sulphonylurea receptors (SURs). The SURs SUR2A (cardiac) and SUR2B (smooth muscle) differ only in the last 42 amino acids. In SUR2B, the mutation Y1206S, located at intracellular loop 8, increases the affinity for glibenclamide (GBC) about 10-fold. Here, we examined whether the mutation Y1206S in SUR2A had effects similar to those in SUR2B.2. GBC bound to SUR2A with K(D)=20 nM; the mutation increased affinity approximately 5 x. 3. In cells, coexpression of SUR2A with Kir6.2 increased the affinity for GBC approximately 3 x; with the mutant, the increase was 9 x. 4. The mutation did not affect the affinity of SUR2A for openers; coexpression with Kir6.2 reduced opener affinity of wild-type and mutant SUR2A by about 2 x. 5. The negative allosteric interaction between the opener, P1075, and GBC at wild-type and mutant SUR2A was markedly affected by the presence of MgATP and by coexpression with Kir6.2. 6. In inside-out patches, GBC inhibited the wild-type Kir6.2/SUR2A and 2B channels with IC(50) values of 27 nM; the mutation shifted the IC(50) values to approximately 1 nM. 7. The data show that the mutation Y1206S increased the affinity of SUR2A for GBC and modulated the effects of coexpression. Overall, the changes were similar to those observed with SUR2B(Y1206S), suggesting that the differences in the last 42 carboxy-terminal amino acids of SUR2A and 2B are of limited influence on the binding of GBC and P1075 to the SUR2 isoforms.

Figure 1

Inhibition of [³H]GBC binding to wild-type and mutant SUR2A by GBC in membranes at 37°C in the absence of MgATP (1 mM EDTA, 0 ATP). Data are expressed as % of total binding (B_TOT) to show the contributions of GBC binding to SUR2A and to endogenous GBC sites in HEK cell membranes. Individual inhibition curves for GBC (n=4–6) were analysed according to the two-component model with n_H=1 and the resulting parameters were averaged as described in Methods. The parameters of the high-affinity components are listed in Table 1; the parameters of the low-affinity component were (wild-type/mutant SUR2A) A₂=44±1/15±1% B_TOT and K_D,2=900 (780, 1100)/130 (90, 200) nM. The broken curves represent the high-affinity component (i.e. binding to SUR2A and SUR2A(YS), respectively), and the dotted horizontal lines the amount of [³H]GBC displaced by 100 μM P1075. Experiments with wild-type were conducted at 3.5 to 3.8 nM [³H]GBC and B_TOT was 200±16 fmol mg⁻¹ (n=16); for SUR2A(YS), [³H]GBC was 2.3 nM and B_TOT 310±38 fmol mg⁻¹ (n=6).

Figure 2

Saturation of [³H]GBC binding to SUR2A(YS) in membranes (a, b) and cells (c). Data represent normalised specific binding pooled from three to four experiments. Binding is to SUR2A(YS) expressed alone (a) and to Kir6.2/SUR2A(YS) (b, c). Experiments in membranes were performed in the presence of MgATP (1 mM). Individual experiments were evaluated according to equations (4) and (5) in Methods section; mean parameters of specific binding are listed in Table 2. Nonspecific binding was given by equation (5) with a=39±2, 32±2 and 67±2 fmol mg⁻¹ nM⁻¹ for SUR2A(AS) and Kir6.2/SUR2A(YS) in membranes and cells, respectively.

Figure 3

Inhibition of [³H]GBC binding to SUR2A(YS) by P1075: effect of MgATP (1 mM) and of coexpression with Kir6.2. Data are means±s.e.m. from four to five independent experiments and are expressed as % specific binding (%B_S). Mean fitting parameters obtained from the analysis of the individual inhibition curves are listed in Table 1. B_TOT values, determined at [³H]GBC concentrations of 2.5–1.5 nM, ranged from 440 to 200 fmol mg⁻¹ and nonspecific binding from 50 to 20% of B_TOT.

Figure 4

Inhibition of [³H]P1075 binding to wild-type and mutant SUR2A by GBC: effect of coexpression with Kir6.2. (a) SUR2A and (b) SUR2A(YS). Data are means from three to six experiments; fitting parameters are listed in Table 3. Experiments were performed in membrane in the presence of MgATP (1 mM).

Figure 5

Recording from an inside-out patch showing inhibition of the Kir6.2/SUR2A(YS) channel by GBC. After excision of the patch into nucleotide-free solution, a current was present, which was abolished by superfusion with MgATP (1 mM) and showed some run-down. MgATP was applied repeatedly to induce refreshment of channels from run-down and GBC intermittently as indicated by the hatched bars. Holding potential was −50 mV; experiments were performed in symmetrical high K⁺ buffer at 23°C. Data were corrected manually for run-down and for weakening of the MgATP block. Note the slow washout of GBC (1000 nM).

Figure 6

Concentration-dependent inhibition of wild-type and mutant Kir6.2/SUR2A channels by GBC. Data were obtained as shown in Figure 5 and normalised with respect to the ATP-sensitive current () prior to the application of GBC. Concentration dependencies were analysed using the Hill equation with n_H=1; the parameters are listed in Table 4. n=4–10 per data point; data for wild type are from Russ et al. (2001).