Mitochondrial PKC and Mitochondrial ATP-Sensitive K Channel Copurify and Coreconstitute to Form a Functioning Signaling Module in Proteoliposomes

Mitochondria are key mediators of the cardioprotective signal and the mitochondrial ATP-sensitive K channel (mitoKATP) plays a crucial role in originating and transmitting that signal. Recently, protein kinase C (PKC ) has been identified as a component of the mitoKATP signaling cascade. We hypothesized that PKC and mitoKATP interact directly to form functional signaling modules in the inner mitochondria membrane. To examine this possibility, we studied K flux in liposomes containing partially purified mitoKATP. The reconstituted proteins were obtained after detergent extraction of isolated mitochondria, 200-fold purification by ion exchange chromatography, and reconstitution into lipid vesicles. Immunoblot analysis revealed the presence of PKC in the reconstitutively active fraction. Addition of the PKC activators 12-phorbol 13-myristate acetate, hydrogen peroxide, and the specific PKC peptide agonist, RACK, each activated mitoKATP-dependent K flux in the reconstituted system. This effect of PKC was prevented by chelerythrine, by the specific PKC peptide antagonist, V1-2, and by the specific mitoKATP inhibitor 5-hydroxydecanoate. In addition, the activating effect of PKC agonists was reversed by exogenous protein phosphatase 2A. These results demonstrate persistent, functional association of mitochondrial PKC and mitoKATP. (Circ Res. 2006;99:878-883.)