Molecular Mechanism and Functional Role of SOCE in Skeletal Muscle

 描述（由申请人提供）：钙池操纵的钙内流（SOCE）于2001年首次报道，是骨骼肌中一种相对较新的现象。SOCE通过两种蛋白质之间的偶联来协调：肌浆网（SR）中的STIM 1钙传感器和横小管（TT）膜中的Ca 2+渗透性Orai 1通道。SOCE增强肌肉生长/发育，限制疲劳，并促进抗疲劳I型纤维规格。另一方面，SOCE功能障碍导致衰老中的肌无力/疲劳，加剧肌营养不良，STIM 1和Orai 1基因的突变导致衰弱性肌病。出现的情况是，STIM 1/Orai 1依赖性SOCE活性的严格调节对于最佳肌肉性能至关重要，因此SOCE活性的增加或减少可能导致肌肉疲劳，肌肉减少症和肌病。虽然SOCE活性明显影响肌肉性能，但肌肉中STIM 1-Orai 1偶联的位点仍不清楚。对于这种更新，我们开发了可诱导的肌肉特异性Orai 1和STIM 1 KO小鼠，以确定STIM 1-Orai 1偶联限制疲劳的机制。我们提供了令人兴奋的证据，即疲劳运动驱动TT和SR之间迄今未描述的连接的形成，其中STIM 1-Orai 1偶联发生，我们称之为“Ca 2+进入单位”（CEU）。CEU与三联体或Ca 2+释放单位相连，但与三联体或Ca 2+释放单位不同。我们提供的初步数据表明，与雄性小鼠相比，Orai 1对雌性小鼠肌纤维收缩功能的影响更大。我们还提供了初步的协同免疫沉淀和质谱可行性数据，用于表征CEU形成前后的STIM 1蛋白质组。我们将使用这些研究工具，方法，发现和合作，以推进对SOCE在骨骼肌中的分子决定因素，亚细胞位置和功能作用的理解。基于我们已发表的和初步的数据，我们假设疲劳运动触发了含有激活的STIM 1-Orai 1复合物的三联体的连接延伸的形成，该复合物协调SOCE以增强SR钙再填充，限制肌肉疲劳，并在长期内促进NFATc 1核定位和I型纤维特化。我们还假设疲劳诱导的CEU在肌肉中的形成涉及多种蛋白质组分（包括Bin 1、STIM 1和Orai 1）的复杂协调。我们建议根据以下两个具体目标来检验这些假设。目的1将描述Orai 1在肌肉疲劳和I型纤维规格中的作用。目的2将确定新发现的CEU在成人骨骼肌中的亚细胞位置、分子组成和稳定性，并确定CEU的形成和分解对疲劳的发展和恢复的依赖性。该项目将：1）为SOCE在肌肉中的生理作用和亚细胞位置提供新的机制见解，2）使用有针对性和无偏见的发现方法来识别和验证参与CEU形成的蛋白质，3）确定性别对Orai 1依赖性SOCE功能，疲劳，纤维类型规格和CEU形成的影响。

 DESCRIPTION (provided by applicant): Being first reported in 2001, store-operated Ca2+ entry (SOCE) is a relatively new phenomenon in skeletal muscle. SOCE is coordinated by coupling between two proteins: STIM1 calcium sensors in the sarcoplasmic reticulum (SR) and Ca2+-permeable Orai1 channels in the transverse tubule (TT) membrane. SOCE enhances muscle growth/development, limits fatigue, and promotes fatigue-resistant type I fiber specification. On the other hand, SOCE dysfunction contributes to muscle weakness/fatigue in aging, exacerbates muscular dystrophy, and mutations in STIM1 and Orai1 genes result in debilitating myopathies. The picture that emerges is that tight regulation of STIM1/Orai1-dependent SOCE activity is critical for optimal muscle performance such that increases or decreases in SOCE activity can lead to muscle fatigue, sarcopenia, and myopathy. While SOCE activity clearly impacts muscle performance, sites of STIM1-Orai1 coupling in muscle remain unclear. For this renewal, we developed inducible, muscle-specific Orai1 and STIM1 KO mice to determine the mechanism by which STIM1-Orai1 coupling limits fatigue. We provide exciting evidence that fatiguing exercise drives the formation of heretofore undescribed junctions between the TT and SR where STIM1-Orai1 coupling occurs, which we refer to as "Ca2+ entry units" (CEUs). CEUs are connected to, but distinct from, the triad or Ca2+ release unit. We provide preliminary data that Orai1 has a stronger impact on muscle fiber contractile function in female compared to male mice. We also provide preliminary collaborative immunoprecipitation and mass spectroscopy feasibility data for characterizing the STIM1 proteome before and after CEU formation. We will use these research tools, approaches, discoveries, and collaborations to advance understanding of the molecular determinants, subcellular location, and functional role of SOCE in skeletal muscle. Based on our published and preliminary data, we hypothesize that fatiguing exercise triggers formation of junctional extensions of the triad containing activated STIM1-Orai1 complexes that coordinate SOCE to enhance SR calcium refilling, limit muscle fatigue, and over the long-term, promote NFATc1 nuclear localization and type I fiber specification. We also hypothesize that fatigue-induced CEU formation in muscle involves a complex coordination of multiple protein components (including Bin1, STIM1, and Orai1). We propose to test these hypotheses according to the following two Specific Aims. Aim 1 will characterize the role of Orai1 in muscle fatigue and Type I fiber specification. Aim 2 will identif the subcellular location, molecular components, and stability of newly identified CEUs in adult skeletal muscle and determine the dependence of CEU formation and disassembly on the development of and recovery from fatigue. This project will: 1) provide novel mechanistic insights into the physiological role and subcellular location of SOCE in muscle, 2) use targeted and non-biased discovery approaches to identify and validate proteins involved CEU formation, and 3) determine the impact of gender on Orai1-dependent SOCE function, fatigue, fiber type specification, and CEU formation.