Bi-directional Calcium Signaling in Striated Muscles

描述(申请人提供)：在横纹肌细胞中，质膜的横管(TT)内陷与肌浆网(S.R)的终池接触，形成受限的三元连接结构，从而为正交化调节细胞内钙释放和逆行调节细胞外钙内流提供结构框架。虽然直接负责电压敏感机制和钙释放途径的分子机制已经被详细研究，但对骨骼肌和心肌细胞中介导这一双向钙信号过程的中间成分知之甚少。我们最近发现了一种新的肌肉特异性蛋白MG53，它在氨基末端含有一个修剪基序，在羧基末端含有一个SPRY结构域。与其他TRIM家族蛋白不同，MG53仅在心肌和骨骼肌纤维中表达。免疫荧光染色和电子显微镜定位发现MG53主要位于肌膜周围区域，此外还有细胞内的小泡。活细胞成像显示，过度表达MG53的细胞表现出膜转运和融合事件的增加。生化分析表明，MG53能与TT膜上的二氢吡啶受体和SR膜上的兰尼定受体相互作用。在这些观察的基础上，本项目将检验中心假说，即MG53的TRIM和SPRY基序可以通过直接与钙调节蛋白相互作用和/或调节骨骼肌膜运输和三联体连接结构，参与肌肉细胞的双向钙信号传递过程。我们的实验将具体解决两个问题：1)MG53的TRIM和SPRY基序在整合电压诱导的骨骼肌钙释放和钙诱导的钙释放的各种动态过程中扮演什么角色(目标1)？2)MG53是通过直接与SOCE大分子复合体相互作用还是通过改变膜运输来影响SOCE功能(目标2)来调节储存操作的钙进入(SOCE)？对上述问题的回答将为在健康和疾病状态下控制肌肉功能中钙信号的细胞和分子机制提供新的见解，以及针对涉及膜完整性或钙信号事件的肌肉功能障碍的MG53的潜在治疗目的。

DESCRIPTION (provided by applicant): In striated muscle cells, the transverse tubular (TT) invagination of plasma membrane contacts the terminal cisternae of sarcoplasmic reticulum (S.R) to form a restricted triad junction structure, thereby providing the structural framework for orthograde regulation of intracellular Ca release and retrograde regulation of extracellular Ca entry. While the molecular machinery directly responsible for the voltage- sensing mechanism and the Ca release pathway has been studied in detail, little is known about the intermediate components that mediate this bi-directional Ca signaling process in skeletal and cardiac muscle cells. We have recently discovered a novel muscle-specific protein named MG53 that contains a TRIM motif at the amino-terminus and a SPRY domain at the carboxyl-terminus. Unlike other TRIM family proteins, MG53 is exclusively expressed in cardiac and skeletal muscle fibers. Immunofluorescent staining and electron microscopy localization identify MG53 predominately at the peri-sarcolemma region, in addition to intracellular vesicles. Live cell imaging reveals that cells overexpressing MG53 exhibit elevated membrane trafficking and fusion events. Biochemical assays demonstrate that MG53 can interact with the dihydropyridine receptor located on TT membrane, as well as with the ryanodine receptor located on SR membrane. Built on these observations, the present project will test the central hypothesis that "the TRIM and SPRY motifs of MG53 can participate in the bi-directional Ca signaling process in muscle cells, through direct interaction with Ca regulatory proteins and/or modulation of membrane trafficking and triad-junction architecture in skeletal muscle". Specifically, our experiments will address two questions: 1) What roles do the TRIM and SPRY motifs of MG53 play in integrating the various dynamic processes of voltage-induced Ca release and Ca-induced Ca release in skeletal muscle (Aim 1)? 2) Does MG53 regulate store-operated Ca entry (SOCE) by direct interaction with the SOCE macromolecular complex, or by altering membrane trafficking to affect SOCE function (Aim 2)? Answers to the above questions should provide new insights into the cellular and molecular mechanisms that control Ca signaling in muscle function in both healthy and diseased states, as well as potential therapeutic purpose for targeting MG53 in muscle dysfunction involving compromised membrane integrity or Ca signaling events.