Calcium release units in skeletal and cardiac muscle

描述（由申请人提供）：本项目探索蛋白质的排列和膜的生物发生，这些膜组成负责骨骼肌和心肌中钙释放调节的大分子复合物。基本方法是建立分子-结构和结构-功能的相关性，前提是结构可以通过观察特定分子扰动后的变化来最好地理解，并且如果不了解潜在结构，功能就不能完全建模。关于蛋白质的排列，要检验的关键假设是：1。钙螯合蛋白的处置取决于在二价阳离子存在下与SR膜的连接和分子间键合的组合; 2.嗜连接蛋白1和2对于SR与表面膜的对接是必需的。在缺乏junctophilin的永久协会之间的表面和内部膜系统可能是不可能的; 3。骨骼型兴奋-收缩偶联是在低等脊索动物和脊椎动物之间的过渡时期进化的，它基于两种不同膜系统的蛋白质之间的分子连接，需要进化出一种新型的ryanodine受体; 4.小窝蛋白-3对于T小管的发育是必需的。实验策略涉及扰动的分子组成和发展的事件，通过选择添加，去除和取代的关键组件和扰动的功能状态的变化，离子组成。将使用的结构方法的中心技术的透射和冷冻断裂电子显微镜，与免疫荧光和共聚焦光学显微镜。

DESCRIPTION (provided by applicant): This project probes the arrangement of proteins and the biogenesis of membranes composing the macromolecular complexes responsible for regulated calcium release in skeletal and cardiac muscle. The basic approach is to establish molecular-structural and structural- functional correlations, under the premise that structure can be best understood by observing its changes following specific molecular perturbations, and that function cannot be fully modeled without knowledge of the underlying structure. Regarding the arrangement of proteins, the key hypotheses to be tested are: 1. calsequestrin's disposition depends on a combination of links to the SR membrane and intermolecular bonding in the presence of bivalent cations; 2. junctophilin 1 and 2 are essential for the docking of SR to surface membranes. In the absence of junctophilin the permanent association between surface and internal membrane systems may not be possible; 3. skeletal type excitation-contraction coupling, based on a molecular link between proteins of two different membrane systems, evolved at the transition between low chordates and vertebrates and it required the evolution of a new type of ryanodine receptor; 4. caveolin-3 is necessary for the development of T tubules. The experimental strategies involve perturbation of the molecular composition and developmental events by selected addition, removal and substitution of key components and perturbation of the functional state by changes in ionic composition. The structural approaches to be used center on techniques of transmission and freeze-fracture electron microscopy, correlated with immunofluorescence and confocal light microscopy.