Bi-directional Calcium Signaling in Striated Muscles

描述（由申请人提供）：在横面肌细胞中，质膜的横管（TT）内陷与肌浆网（S.R）的末端池池接触，形成受限的三联结结构，从而为正向调节细胞内钙释放和逆行调节细胞外钙进入提供结构框架。虽然对电压感应机制和钙释放途径直接负责的分子机制已经进行了详细的研究，但对骨骼肌和心肌细胞中介导这种双向钙信号传导过程的中间成分知之甚少。我们最近发现了一种名为MG53的新型肌肉特异性蛋白，它在氨基端含有TRIM基元，在羧基端含有SPRY结构域。与其他TRIM家族蛋白不同，MG53只在心脏和骨骼肌纤维中表达。免疫荧光染色和电镜定位发现，除了细胞内囊泡外，MG53主要位于肌膜周围区域。活细胞成像显示过表达MG53的细胞表现出升高的膜运输和融合事件。生化实验表明MG53能与TT膜上的二氢吡啶受体和SR膜上的ryanodine受体相互作用。基于这些观察结果，本项目将测试“MG53的TRIM和SPRY基元可以通过与钙调节蛋白的直接相互作用和/或调节骨骼肌中的膜运输和三联结结构参与肌肉细胞中的双向钙信号传导过程”的中心假设。具体来说，我们的实验将解决两个问题：1)MG53的TRIM和SPRY基元在整合骨骼肌中电压诱导的Ca释放和Ca诱导的Ca释放的各种动态过程中起什么作用（Aim 1）？2) MG53是否通过与SOCE大分子复合物的直接相互作用，或通过改变膜运输来影响SOCE功能来调节储存性钙离子进入（SOCE）（目的2）？上述问题的答案将为健康和患病状态下肌肉功能中控制Ca信号的细胞和分子机制提供新的见解，以及靶向MG53治疗涉及膜完整性受损或Ca信号事件的肌肉功能障碍的潜在治疗目的。

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.