Bi-directional Calcium Signaling in Striated Muscles

描述（由申请人提供）：在横纹肌细胞中，质膜的横管（TT）内陷接触肌浆网（S.R）的末端池，形成限制性三联体连接结构，从而为细胞内Ca释放的顺向调节和细胞外Ca进入的逆向调节提供结构框架。虽然已经详细研究了直接负责电压感应机制和Ca释放途径的分子机制，但对骨骼肌和心肌细胞中介导这种双向Ca信号传导过程的中间组分知之甚少。我们最近发现了一种新的肌肉特异性蛋白MG 53，其氨基端含有TRIM基序，羧基端含有SPRY结构域。与其他TRIM家族蛋白不同，MG 53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确定MG 53主要在肌膜周围区域，除了细胞内囊泡。活细胞成像显示，过表达MG 53的细胞表现出升高的膜运输和融合事件。生物化学分析表明，MG 53可以与位于TT膜上的二氢吡啶受体以及位于SR膜上的ryanodine受体相互作用。基于这些观察结果，本项目将测试中心假设，即“MG 53的TRIM和SPRY基序可以通过与Ca调节蛋白直接相互作用和/或调节骨骼肌中的膜运输和三联体连接结构参与肌细胞中的双向Ca信号传导过程”。具体而言，我们的实验将解决两个问题：1）MG 53的TRIM和SPRY基序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发挥什么作用（目的1）？2)MG 53是否通过与SOCE大分子复合物直接相互作用或通过改变膜运输来影响SOCE功能来调节钙库操纵的钙内流（SOCE）（目的2）？对上述问题的回答应该提供对在健康和患病状态下控制肌肉功能中的Ca信号传导的细胞和分子机制的新见解，以及在涉及受损的膜完整性或Ca信号传导事件的肌肉功能障碍中靶向MG 53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.