STRUCTURAL BIOLOGY OF CARDIOVASCULAR ION TRANSPORT

这是一个跨学科的项目，基于 物理学家、结构和分子生物学家、生物物理学家和 心血管生理学家，旨在将结构性方法应用于 对离子转运心血管离子机制的认识 通道和酶。心肌细胞钙-三磷酸腺苷酶的结构及其功能 与磷蛋白的结合将通过冷电子来确定 结晶学和原子力显微镜。支持的交叉链接 磷脂双层及其他特殊制备方法和 将开发用于原子力显微镜、成像的仪器 在分子分辨率和烟碱型乙酰胆碱受体、Ca-ATPase和 Na，K-ATPase，最终目的是可视化分子构象 改变了调节离子传输的方式。细胞内(Ryanodine-和InsP3- 受体)和质膜(二氢吡啶结合蛋白)钙通道 将通过激光扫描共聚焦荧光显微镜进行定位。这个 心脏T管网络及其与L型钙通道的关系 缝隙结将被确定，并使分子的拓扑结构光滑 肌肉InsP3受体将被定位。嵌合结构和地点- 将使用Ca-ATPase和Na，K-ATPase的定向突变体来确定 这些酶的靶向机制。一种新型的200千伏场致发射枪- 结合电子能量损失的装备电子显微镜 将在该计划中开发的光谱方法将用于 成像钙储存细胞器和结合到心肌膜上的钙，以 测定钙结合对心脏传导和心律失常的影响。 新的、普遍适用的原子力显微镜技术和 扫描透射式能量过滤电子显微镜将 发展起来的。

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.