Reconstituted Human Cardiac K ATP Channels

Abstract

Abstract —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 K ATP channels in human ventricular cells. The single-channel conductance of ≈80 pS measured at −40 mV in quasi-symmetrical ≈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 Mg 2+ (≈0.7 mmol/L) or in the absence of Mg 2+ , with an apparent IC 50 of ≈20 μmol/L and a pseudo-Hill coefficient of ≈1. They were “refreshed” by MgATP and stimulated by ADP in the presence of Mg 2+ when inhibited by ATP. The huSUR2A/huK IR 6.2 channels were stimulated by cromakalim and pinacidil in the presence of ATP and Mg 2+ 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.