The nucleotide regulatory sites on the mitochondrial K-ATP channel face the cytosol

The mitochondrial K-ATP channel (mitoK(ATP)) is richly endowed with regulatory sites for metabolites and drugs, but the topological location of these sites is unknown. Thus, it is not known whether ATP, GTP and acyl CoA esters regulate mitoK(ATP) from the matrix or cytosolic side of the inner membrane, nor whether they all act from the same side. The experiments reported in this paper provide an unambiguous answer to these questions. Electrophysiological experiments in bilayer membranes containing purified mitoK(ATP) showed that current is blocked asymmetrically by ATP. K+ flux experiments using proteoliposomes containing purified mitoK(ATP) showed that mitoK(ATP) is unipolar with respect to regulation by Mg2+, ATP, GTP, and palmitoyl CoA and that all of these ligands react on the same pole of the protein. This demonstration was made possible by the new finding that mitoK(ATP) is 85-90% oriented inward or outward in liposomes, depending on the presence or absence of Mg2+ in the reconstitution buffer. K+ flux experiments in respiring rat liver mitochondria showed that mitoK(ATP) was inhibited by palmitoyl CoA and activated by GTP when these agents were added to the external medium. Given that the inner membrane is impermeant to these ligands and that mitoK(ATP) is unipolar with respect to nucleotide regulation, it follows that the regulatory sites on mitoK(ATP) face the cytosol. (C) 1997 Elsevier Science B.V.