Reconstituted human cardiac KATP channels -: Functional identity with the native channels from the sarcolemma of human ventricular cells

ATP-sensitive potassium (K-ATP) channels in striated myocytes are heteromultimers of K(IR)6.2, a weak potassium inward rectifier, plus SUR2A, a low-affinity sulfonylurea receptor. We have cloned human K(IR)6.2 (huK(IR)6.2) and a huSUR2A that corresponds to the major, full-length splice variant identified by polymerase chain reaction analysis of human cardiac poly A(+) mRNA. ATP- and glibenclamide-sensitive K+ channels were produced when both subunits were coexpressed in COSm6 and Chinese hamster ovary cells lacking endogenous K-ATP channels, but not when huSUR2A or huK(IR)6.2 were transfected alone. Recombinant channels activated by metabolic inhibition in cell-attached configuration or in inside-out patches with ATP-free internal solution were compared with sarcolemmal KATP channels in human ventricular cells. The single-channel conductance of approximate to 80 pS measured at -40 mV in quasi-symmetrical approximate to 150 mmol/L K+ solutions, the intraburst kinetics that were dependent on K+ driving force, and the weak inward rectification were indistinguishable for both channels. Similar to the native channels, huSUR2A/huK(IR)6.2 recombinant channels were inhibited by ATP at quasi-physiological free Mg2+ (approximate to 0.7 mmol/L) or in the absence of Mg2+, with an apparent IC50 of approximate to 20 mu mol/L and a pseudo-Hill coefficient of approximate to 1. They were "refreshed" by MgATP and stimulated by ADP in the presence of Mg2+ when inhibited by ATP. The huSUR2A/huK(IR)6.2 channels were stimulated by cromakalim and pinacidil in the presence of ATP and Mg2+ but were insensitive to diazoxide. The results suggest that reconstituted huSUR2A/huK(IR)6.2 channels represent K-ATP channels in sarcolemma of human cardiomyocytes and are an adequate experimental model with which to examine structure-function relationships, molecular physiology, and pharmacology of these channels from human heart.