Calcium release units in skeletal and cardiac muscle

项目描述（由申请人提供）：本项目探讨蛋白质的排列和构成骨和心肌中钙释放调控的大分子复合物的膜的生物发生。基本方法是建立分子-结构和结构-功能的相关性，前提是通过观察特定分子扰动后的结构变化可以最好地理解结构，并且如果不了解底层结构，功能就不能完全建模。关于蛋白质的排列，需要验证的关键假设是：1.蛋白质的排列。calsequestrin的配置取决于与SR膜的连接和二价阳离子存在下的分子间键的组合；2. 亲结膜蛋白1和2是SR与表面膜对接所必需的。在没有亲结蛋白的情况下，表面和内部膜系统之间的永久联系可能是不可能的；3. 基于两种不同膜系统的蛋白质之间的分子连接的骨骼型兴奋-收缩耦合，在低脊索动物和脊椎动物之间的过渡中进化而来，它需要一种新型的ryanodine受体的进化；4. 小洞蛋白-3是T小管发育所必需的。实验策略包括通过选择性添加、去除和取代关键组分来扰动分子组成和发育事件，以及通过改变离子组成来扰动功能状态。采用的结构方法以透射电镜和冷冻断裂电镜技术为中心，与免疫荧光和共聚焦光显微镜相关。

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.