Pharmacological and histochemical distinctions between molecularly definedsarcolemmal K-ATP channels and native cardiac mitochondrial K-ATP channels

A variety of direct and indirect techniques have revealed the existence of ATP-sensitive potassium (K-ATP) channels in the inner membranes of mitochon dria. The molecular identity of these mitochondrial K-ATP, (mitoK(ATP)) cha nnels remains unclear. We used a pharmacological approach to distinguish mi toK(ATP) channels from classical, molecularly defined cardiac sarcolemmal K -ATP (surfaceK(ATP)) channels encoded by the sulfonylurea receptor SUR2A an d the pore-forming subunit K(lr)6.2. SUR2A and K(ir)6.2 were expressed in h uman embryonic kidney (HEK)293 cells, and their activities were measured by patch-clamp recordings of membrane current. SurfaceK(ATP) channels are act ivated potently by 100 mu M pinacidil but only weakly by 100 mu M diazoxide ; in addition, they are blocked by 10 mu M glibenclamide, but are insensiti ve to 500 mu M 5-hydroxydecanoate. This pharmacology, which was confirmed w ith patch-clamp recordings in intact rabbit: ventricular myocytes, contrast s with that of mitoK(ATP) channels as indexed by flavoprotein oxidation. Mi toK(ATP) channels in myocytes are activated equally by 100 mu M diazoxide a nd 100 mu M pinacidil. In contrast to its lack of effect on surfaceK(ATP) c hannels, 5-hydroxydecanoate is an effective blocker of mitoK(ATP) channels. Glibenclamide's effects on mitoK(ATP) channels are difficult to assess, be cause it independently activates flavoprotein fluorescence, consistent with a previously described primary uncoupling effect. Confocal imaging of the subcellular distribution of expressed fluorescent K(ir)6.2 in HEK cells and in myocytes revealed no targeting of mitochondrial membranes. The differen ces in drug sensitivity and subcellular localization indicate that mitoK(AT P) channels are distinct from surface K-ATP channels at a molecular level.