Published online before print September 7, 2006, doi: 10.1161/01.RES.0000245106.80628.d3
© 2006 American Heart Association, Inc.
Cellular Biology |
Mitochondrial PKC
and Mitochondrial ATP-Sensitive K+ Channel Copurify and Coreconstitute to Form a Functioning Signaling Module in Proteoliposomes
rekFrom 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.
Correspondence to Keith D. Garlid, Department of Biology, Portland State University, PO Box 751, Portland, Oregon 97207. 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, V1-2, 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
Related Article:
and MitoKATP in the Inner Membrane of Mitochondria
Circ. Res. 2006 99: 798-800.
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