MOLECULAR REGULATION--CARDIAC K+ATP CHANNELS IN ISCHEMIA

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The ATP-sensitive potassium channel (KATP) plays important physiological and pathophysiological roles in heart, pancreas, brain, vascular smooth muscle. Particularly in heart, KATP participates in ischemic arrhythmias and myocardial preservation. Regulation of KATP is complex. This proposal focuses on two aspects of KATP regulation: effects of cytoplasmic acidosis and effects of anionic phospholipids, both of which may be important in ischemia. This focus is motivated by previous work of the Principal Investigators on native cardiac KATP, by the recent availability of KATP clones (co- expressed BIR/SUR1 and KATP/SUR2), and by a structural hypothesis of phospholipid effects involving the cytoplasmic C-tail of the KATP. In the first aim, detailed characterization of clones KATPs will be performed and compared with native KATP. In the second aim, the effects of acidosis, in combination with other conditions found in ischemia, will be investigated in native KATP from acutely isolated cardiac cells. In the third aim, the effects of anionic phospholipids on native and cloned KATP and other related inward rectifier channel function will be investigated. In the fourth aim, regulation of KATP by native phospholipids and their regulatory enzymes (phosphatases and kinases) will be investigated using a novel phospholipid kinase. In the fifth aim, the 'C-terminus tethering' hypothesis for phospholipid effects will be tested using recombinant DNA technology including site directed mutagenesis, deletions, and chimera. Although the motivation for the studies is to account at the molecular level for effects of ischemic conditions on KATP, the expected results are likely to have wide implications for the structure/function of the superfamily of inward rectifier potassium channels, and will also have implications for the physiology of many tissues in addition to heart.