εPKC phosphorylates the mitochondrial K+ATP channel during induction of ischemic preconditioning in the rat hippocampus

Neuroprotection against cerebral ischemia conferred by ischemic preconditioning (IPC) requires translocation of epsilon protein kinase C (epsilon PKC). A major goal in our laboratory is to define the cellular targets by which ePKC confers protection. We tested the hypothesis that epsilon PKC targets the mitochondrial K-ATP(+) channel (mtK(ATP)(+)) after IPC. Our results demonstrated a rapid translocation of epsilon PKC to rat hippocampal mitochondria after IPC. Because in other tissues epsilon PKC targets mtK(ATP)(+) channels, but its presence in brain mitochondria is controversial, we determined the presence of the K-ATP(+) channel-specific subunits (Kir6.1 and Kir6.2) in mitochondria isolated from rat hippocampus. Next, we determined whether mtK(ATP)(+) channels play a role in the IPC induction. In hippocampal organotypic slice cultures, IPC and lethal ischemia were induced by oxygen-glucose deprivation. Subsequent cell death in the CA1 region was quantified using propidium iodide staining. Treatment with the K-ATP(+) channel openers diazoxide or pinacidil 48 h prior to lethal ischemia protected hippocampal CA1 neurons, mimicking the induction of neuroprotection conferred by either IPC or epsilon PKC agonist-induced preconditioning. Blockade of mtK(ATP)(+) channels using 5-hydroxydecanoic acid abolished the neuroprotection due to either IPC or epsilon PKC preconditioning. Both ischemic and epsilon PKC agonist-mediated preconditioning resulted in phosphorylation of the mtK(ATP)(+) channel subunit Kir6.2. After IPC, selective inhibition of epsilon PKC activation prevented Kir6.2 phosphorylation, consistent with Kir6.2 as a phosphorylation target of epsilon PKC or its downstream effectors. Our results support the hypothesis that the brain mtK(ATP)(+) channel is an important target of IPC and the signal transduction pathways initiated by epsilon PKC. (c) 2007 Elsevier B.V. All rights reserved.