STRUCTURAL BIOLOGY OF CARDIOVASCULAR ION TRANSPORT

这是一个基于合作的跨学科计划， 物理学家、结构和分子生物学家、生物药理学家和 心血管生理学家，旨在应用结构方法， 离子对心血管离子转运机制的认识 通道和酶。 心肌Ca-ATP酶的结构及其与心肌细胞的关系 与受磷蛋白的结合将通过冷冻电子 晶体学和原子力显微镜。 支持交联 磷脂双层和其他特殊制备方法， 将开发用于原子力显微镜的仪器，用于成像， 在分子分辨率和烟碱乙酰胆碱受体，钙ATP酶和 Na，K-ATPase，最终目的是可视化分子构象 介导离子运输的变化。 细胞内（ryanodine-和InsP 3- 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定位。 的 心脏t-管网络，其与L-型钙通道和 间隙连接将被确定，和光滑的分子拓扑结构 将绘制肌肉InsP 3受体。 嵌合构建体和位点- Ca-ATP酶和Na，K-ATP酶的定向突变体将用于确定 这些酶的靶向机制。 一种新型的200 kV场发射电子枪- 电子能量损失电子显微镜 光谱方法，将在该计划中开发，将用于 成像钙储存细胞器和钙结合到心脏膜， 确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， 扫描透射能量过滤电子显微镜将是 开发

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.