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 (KATP channel) to the murine sulphonylurea receptor, SUR2B, was investigated. SUR2B, a proposed subunit of the vascular KATP channel, was expressed in HEK 293 cells and binding assays were performed in membranes at 37 degrees C using the tritiated KATP 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 microM at free Mg2+ concentrations of 3 microM and 1 mM, respectively. At 1 mM Mg2+, binding was lower than at 3 microM Mg2+. 3. [3H]-P1075 saturation binding experiments, performed at 3 mM ATP and free Mg2+ concentrations of 3 microM and 1 mM, gave KD values of 1.8 and 3.4 nM and BMAX 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 Ki values of 0.35 and 2.4 microM at 3 Mm 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 microM affinity must be occupied to allow opener binding whereas Mg2+ concentrations > or = 10 microM 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.