Suppression of KATP currents by gene transfer of a dominant negative Kir6.2 construct

Cardiac ATP-sensitive K+ (K-ATP) channels (SUR2A plus Kir6.2) couple the metabolic state of the myocyte to its electrical activity via a mechanism that is not well understood. Recent pharmacological evidence suggests that K-ATP channels may mediate ischemic preconditioning. However, there is no potent pharmaceutical agent that specifically blocks the sarcolemmal K-ATP channel without significant effects on other cellular 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 subunit (6.2AFA) suppressed wildtype Kir6.2 (6.2WT) channel current in a dominant-negative manner: when co-expressed with SUR2A and 6.2WT, whole-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 recorded 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 cells.