SUPPRESSION OF K-ATP CURRENTS BY GENE-TRANSFER OF A DOMINANT-NEGATIVEKIR6.2 CONSTRUCT

Cardiac ATP-sensitive K+ (K-ATP) channels (SUR2A plus Kir6.2) couple t he metabolic state of the myocyte to its electrical activity via a mec hanism that is not well understood. Recent pharmacological evidence su ggests that K-ATP channels may mediate ischemic preconditioning. Howev er, there is no potent pharmaceutical agent that specifically blocks t he sarcolemmal K-ATP channel without significant effects on other cell ular proteins. As a molecular tool, the GFG sequence in the H5 loop of the murine Kir6.2 channel was mutated to AFA. This mutated channel su bunit (6.2AFA) suppressed wildtype Kir6.2 (6.2WT) channel current in a dominant-negative manner: when co-expressed with SUR2A and 6.2WT, who le-cell K-ATP current recorded from HEK cells was greatly attenuated. The 6.2AFA subunit also co-assembled with endogenous subunits in both smooth-muscle-derived A10 cells and rat neonatal ventricular myocytes, resulting in a significant reduction of current compared with that re corded from non-transfected or mock-transfected cells (<15% of control for both cell types). This study shows that mutation of GFG-->AFA in the putative pore-forming region of Kir6.2 acts in a dominant-negative manner to suppress current in heterologous systems and in native cell s.