Pore loop-mutated rat KIR6.1 and KIR6.2 suppress KATP current in rat cardiomyocytes

Cardiomyocytes express mRNA for all major subunits of ATP-sensitive potassium (K(ATP)) channels: KIR6.1, KIR6.2, SUR1A, SUR2A, and SUR2B. It has remained controversial as to whether KIR6.1 may associate with KIR6.2 to form the tetrameric pore of K(ATP) channels in cardiomyocytes. To explore this possibility, cultured rat cardiomyocytes were examined for an inhibition of K(ATP) current by overexpression of pore loop-mutated (inactive) KIR6.x. Bicistronic plasmids were constructed encoding loop-mutated (AFA or SFG for GFG) rat KIR6.x followed by EGFP. In ventricular myocytes, the overexpression of KIR6.1SFG-pIRES(2)-EGFP or KIR6.2AFA-pIRES(2)-EGFP DNA caused, after 72 h, a major decrease of K(ATP) current density of 85.8% and 82.7%, respectively (P < 0.01), relative to EGFP controls (59 +/- 9 pA/pF). In atrial myocytes, overexpression of these pore-mutated KIR6.x by 6.0-fold and 10.6-fold, as assessed by quantitative immunohistochemistry, caused a decrease of K(ATP) current density of 73.7% and 58.5%, respectively (P < 0.01). Expression of wild-type rat KIR6.2 increased the ventricular and atrial K(ATP) current density by 58.3% and 42.9%, respectively (P < 0.01), relative to corresponding EGFP controls, indicating a reserve of SUR to accommodate increased KIR6.x trafficking to the sarcolemma. The results favor the view that KIR6.1 may associate with KIR6.2 to form heterotetrameric pores of native K(ATP) channels in cardiomyocytes.