Mg2+ and ATP dependence of K(ATP) channel modulator binding to the recombinant sulphonylurea receptor, SUR2B

Abstract

1. The binding of modulators of the ATP-sensitive K+ channel (K(ATP) channel) to the murine sulphonylurea receptor, SUR2B, was investigated. SUR2B, a proposed subunit of the vascular K(ATP) channel, was expressed in HEK 293 cells and binding assays were performed in membranes at 37°C using the tritiated K(ATP) channel opener, [3H]-P1075. 2. Binding of [3H]-P1075 required the presence of Mg2+ and ATP. MgATP activated binding with EC50 values of 10 and 3 μM at free Mg2+ concentrations of 3 μM and 1 mM, respectively. At 1 mM Mg2+, binding was lower than at 3 μM Mg2+. 3. [3H]-P1075 saturation binding experiments, performed at 3 mM ATP and free Mg2+ concentrations of 3 μM and 1 mM, gave K(D) values of 1.8 and 3.4 nM and B(MAX) values of 876 and 698 fmol mg-1 respectively. 4. In competition experiments, openers inhibited [3H]-P1075 binding with potencies similar to those determined in rings of rat aorta. 5. Glibenclamide inhibited [3H]-P1075 binding with K(i) values of 0.35 and 2.4 μM at 3 μM and 1 mM free Mg2+, respectively. Glibenclamide enhanced the dissociation of the [3H]-P1075-SUR2B complex suggesting a negative allosteric coupling between the binding sites for P1075 and the sulphonylureas. 6. It is concluded that an MgATP site on SUR2B with μM affinity must be occupied to allow opener binding whereas Mg2+ concentrations ≤ 10 μM decrease the affinities for openers and glibenclamide. The properties of the [3H]-P1075 site strongly suggest that SUR2B represents the drug receptor of the openers in vascular smooth muscle.