The Mitochondrial ATP-Sensitive K+ Channel in Heart

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DESCRIPTION (provided by applicant): Cardiac ischemia and its sequelae are major causes of mortality. Opening KATP channels exerts a powerful anti-infarct effect, offering the possibilities of a therapeutic target and of insights into the mechanisms of endogenous cardioprotection. The mitochondrial ATP-sensitive K+ channel (mitoKATP) plays a pivotal role in cardioprotection. The long-term goal of this project is to uncover the mechanisms of this action with respect to its role in cell signaling and its role as an end effector of protection. The rationale for our approach is based on the widely accepted conclusion that signaling pathways involving mitoKATP must occur, at least in part, inside the mitochondrion. The specific aims are [1] To determine the topology and localization of kinases within the mitochondrial signaling pathway. [2] To determine how mitoKATP is opened by ischemic preconditioning and how mitoKATP opening is regulated under pathophysiological conditions. [3] To determine the mechanisms by which mitoKATP opening causes the signal to be transmitted to the end effectors of cardioprotection. [4] To determine the downstream targets of mitoKATP opening and mitoKATP-induced signaling by reactive oxygen species (ROS). [5] To determine the biochemistry of the signaling sequence extending from the cytosol to mitoKATP. [6] To determine the molecular identity of mitoKATP. To address these aims, experiments will focus on the perfused heart, isolated mitochondria, fractionation of mitochondrial membranes, and fractionation/purification of membrane proteins. Mechanisms of K+ transport will be studied using light scattering and ion-specific fluorescent probes. The bioenergetics of mitochondrial ROS production will be studied with fluorescent probes. Effects of treatment of the perfused heart will be evaluated in isolated mitochondria using immunodetection and K+ flux measurement techniques. Identification of signaling intermediates will utilize 32P-labeling and functional assays of phosphorylation.