Protein kinase C isoform-dependent modulation of ATP-sensitive K⁺ channels in mitochondrial inner membrane

Vivek Garg, and Keli Hu

Division of Pharmacology, College of Pharmacy, The Ohio State University, Columbus, Ohio

Abstract: The ATP-sensitive K⁺ (K_ATP) channels in both sarcolemmal (sarcK_ATP) and mitochondrial inner membrane (mitoK_ATP) are the critical mediators in cellular protection of ischemic preconditioning (IPC). Whereas cardiac sarcK_ATP contains Kir6.2 and sulfonylurea receptor (SUR)2A, the molecular identity of mitoK_ATP remains elusive. In the present study, we tested the hypothesis that protein kinase C (PKC) may promote import of Kir6.2-containing K_ATP into mitochondria. Fluorescence imaging of isolated mitochondria from both rat adult cardiomyocytes and COS-7 cells expressing recombinant Kir6.2/SUR2A showed that Kir6.2-containing K_ATP channels were localized in mitochondria and this mitochondrial localization was significantly increased by PKC activation with phorbol 12-myristate 13-acetate (PMA). Fluorescence resonance energy transfer microscopy further revealed that a significant number of Kir6.2-containing K_ATP channels were localized in mitochondrial inner membrane after PKC activation. These results were supported by Western blotting showing that the Kir6.2 protein level in mitochondria from COS-7 cells transfected with Kir6.2/SUR2A was enhanced after PMA treatment and this increase was inhibited by the selective PKC inhibitor chelerythrine. Furthermore, functional analysis indicated that the number of functional K_ATP channels in mitochondria was significantly increased by PMA, as shown by K_ATP-dependent decrease in mitochondrial membrane potential in COS-7 cells transfected with Kir6.2/SUR2A but not empty vector. Importantly, PKC-mediated increase in mitochondrial Kir6.2-containing K_ATP channels was blocked by a selective PKC inhibitor peptide in both COS-7 cells and cardiomyocytes. We conclude that the K_ATP channel pore-forming subunit Kir6.2 is indeed localized in mitochondria and that the Kir6.2 content in mitochondria is increased by activation of PKC. PKC isoform-regulated mitochondrial import of K_ATP channels may have significant implication in cardioprotection of IPC.

Key Words: ischemic preconditioning • mitochondria • Kir6.2

Address for reprint requests and other correspondence: K. Hu, 530 Parks Hall, Div. of Pharmacology, Coll. of Pharmacy, 500 W. 12th Ave., Ohio State Univ., Columbus, OH 43210 (e-mail: hu.175{at}osu.edu)

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