Pharmacology of human sulphonylurea receptor SUR1 and inward rectifier K+ channel Kir6.2 combination expressed in HEK-293 cells

1 The pharmacological properties of K-ATP channels generated by stable co-e xpression of the sulphonylurea receptor SUR1 and the inwardly rectifying K channel Kir6.2 were characterized in HEK-293 cells. 2 [H-3]-Glyburide (glibenclamide) bound to transfected cells with a B-max v alue of 18.5 pmol mg(-1) protein and with a KD Value of 0.7 nM. Specific bi nding was displaced by a series of sulphonylurea analogues with rank order potencies consistent with those observed in pancreatic RINm5F insulinoma an d in the brain. 3 Functional activity of K-ATP channels was assessed by whole cell patch cl amp, cation efflux and membrane potential measurements. Whole cell currents were detected in transfected cells upon depletion of internal ATP or by ex posure to 500 mu M diazoxide. The currents showed weak inward rectification and were sensitive to inhibition by glyburide (IC50 = 0.92 nM). 4 Metabolic inhibition by 2-deoxyglucose and oligomycin treatment triggered Rb-86(+) efflux from transfected cells that was sensitive to inhibition by glyburide (IC50 = 3.6 nM). 5 Diazoxide, but not levcromakalim, evoked concentration-dependent decrease s in DiBAC(4)(3) fluorescence responses with an EC50 value of 14.1 mu M whi ch were attenuated by the addition of glyburide. Diazoxide-evoked responses were inhibited by various sulphonylurea analogues with rank order potencie s that correlated well with their binding affinities. 6 In summary, results from ligand binding and functional assays demonstrate that the pharmacological properties of SUR1 and Kir6.2 channels co-express ed in HEK-293 cells resemble those typical of native KATP channels describe d in pancreatic and neuronal tissues.