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Mitochondria are key mediators of the cardioprotective signal and the mitochondrial ATP-sensitive K+ channel (mitoK(ATP)) plays a crucial role in originating and transmitting that signal. Recently, protein kinase C epsilon (PKC epsilon) has been identified as a component of the mitoK(ATP) signaling cascade. We hypothesized that PKC epsilon and mitoK(ATP) 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 mitoK(ATP). 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 epsilon in the reconstitutively active fraction. Addition of the PKC activators 12-phorbol 13-myristate acetate, hydrogen peroxide, and the specific PKC epsilon peptide agonist, psi epsilonRACK, each activated mitoK(ATP)-dependent K+ flux in the reconstituted system. This effect of PKC epsilon was prevented by chelerythrine, by the specific PKC epsilon peptide antagonist, epsilonV(1-2), and by the specific mitoK(ATP) 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 epsilon and mitoK(ATP).