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

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 degrees C using the tritiated K-ATP channel opener, [H-3]-P1075. 2 Binding of [H-3]-P1075 required the presence of Mg2+ and ATP. MgATP activated binding with EC50 values of 10 and 3 mu M at free Mg2+ concentrations of 3 mu M and 1 mM, respectively. At 1 mM Mg2+, binding was lower than at 3 mu M Mg2+. 3 [H-3]-P1075 saturation binding experiments, performed at 3 mM ATP and free Mg2+ concentrations gave K-D values of 1.8 and 3.4 nM and B-MAX values of 876 and 698 fmol mg(-1) of 3 mu M and 1 mM, respectively. 4 In competition experiments, openers inhibited [H-3]-P1075 binding with potencies similar to those determined in rings of rat aorta. 5 Glibenclamide inhibited [H-3]-P1075 binding with K-i values of 0.35 and 2.4 mu M at 3 mu M and 1 mM free Mg2+, respectively. Glibenclamide enhanced the dissociation of the [H-3]-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 I mu M affinity must be occupied to allow opener binding whereas Mg2+ concentrations greater than or equal to 10 mu M decrease the affinities for openers and glibenclamide. The properties of the [H-3]-P1075 site strongly suggest that SUR2B represents the drug receptor of the openers in vascular smooth muscle.