Published online before print September 7, 2006, doi: 10.1161/01.RES.0000245106.80628.d3
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Submitted on August 18, 2005
Revised on August 7, 2006
Accepted on August 30, 2006
Mitochondrial PKC
and Mitochondrial ATP-Sensitive K+ Channel Copurify and Coreconstitute to Form a Functioning Signaling Module in Proteoliposomes
Martin Jaburek ;
From the Department of Biology (M.J., A.D.T.C., J.R.B., C.L.C., K.D.G.), Portland State University, Portland, Ore.; and the Department of Membrane Transport Biophysics (M.J.), Institute of Physiology, Academy of Sciences of the Czech Republic, Prague.
* To whom correspondence should be addressed. E-mail: garlid{at}pdx.edu.
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 to 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.
Key words: ATP-sensitive K+ channel • protein kinase C
•
protein phosphatase •
reactive oxygen species •
reconstitution •
cardioprotection •
mitochondria
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