Abstract

A variety of direct and indirect techniques have revealed the existence of ATP-sensitive potassium (K_ATP) channels in the inner membranes of mitochondria. The molecular identity of these mitochondrial K_ATP (mitoK_ATP) channels remains unclear. We used a pharmacological approach to distinguish mitoK_ATP channels from classical, molecularly defined cardiac sarcolemmal K_ATP (surfaceK_ATP) channels encoded by the sulfonylurea receptor SUR2A and the pore-forming subunit K_ir6.2. SUR2A and K_ir6.2 were expressed in human embryonic kidney (HEK)293 cells, and their activities were measured by patch-clamp recordings of membrane current. SurfaceK_ATP channels are activated potently by 100 μM pinacidil but only weakly by 100 μM diazoxide; in addition, they are blocked by 10 μM glibenclamide, but are insensitive to 500 μM 5-hydroxydecanoate. This pharmacology, which was confirmed with patch-clamp recordings in intact rabbit ventricular myocytes, contrasts with that of mitoK_ATP channels as indexed by flavoprotein oxidation. MitoK_ATP channels in myocytes are activated equally by 100 μM diazoxide and 100 μM pinacidil. In contrast to its lack of effect on surfaceK_ATP channels, 5-hydroxydecanoate is an effective blocker of mitoK_ATPchannels. Glibenclamide’s effects on mitoK_ATP channels are difficult to assess, because it independently activates flavoprotein fluorescence, consistent with a previously described primary uncoupling effect. Confocal imaging of the subcellular distribution of expressed fluorescent K_ir6.2 in HEK cells and in myocytes revealed no targeting of mitochondrial membranes. The differences in drug sensitivity and subcellular localization indicate that mitoK_ATP channels are distinct from surface K_ATP channels at a molecular level.