This is an interdisciplinary Program based on the collaboration of physicists, structural and molecular biologists, biophysicists and cardiovascular physiologists, aimed at applying structural approaches to the understanding of the mechanisms of cardiovascular ion transport by ion channels and enzymes. The structure of the cardiac Ca-ATPase and its association with phospholamban will be determined through cryoelectron crystallography and atomic force microscopy. Supported cross-linked phospholipid bilayers and other special preparatory methods and instrumentation will be developed for atomic force microscopy, for imaging at molecular resolution and nicotinic acetylcholine receptor, Ca-ATPase and Na,K-ATPase, with the ultimate aim of visualizing molecular conformational changes mediating ion transport. Intracellular (ryanodine- and InsP3- receptor) and plasmalemmal (dihydropyridine-binding protein) Ca2+-channels will be localized by laser scanning confocal fluorescence microscopy. The cardiac t-tubular network, its association with L-type Ca-channels and with gap junctions will be determined, and the molecular topology of the smooth muscle InsP3 receptor will be mapped. Chimeric constructs and site- directed mutants of Ca-ATPase and Na,K-ATPase will be used to determine the targeting mechanisms of these enzymes. A new, 200kV field emission gun- equipped electron microscope combined with electron energy loss spectroscopic methods, to be developed in the Program, will be used for imaging Ca-storage organelles and Ca bound to cardiac membranes, to determine the effects of Ca-binding on cardiac conduction and arrhythmias. New, generally applicable techniques of atomic force microscopy and scanning transmission energy filtered electron microscopy will be developed.
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