Molecular Mechanism and Functional Role of SOCE in Skeletal Muscle

 描述(申请人提供)：2001年首次报道，在骨骼肌中，存储操作的钙离子进入(SOCE)是一个相对较新的现象。SOCE是通过两种蛋白质之间的偶联来协调的：位于肌浆网(SR)的STIM1钙感受器和位于横管(TT)膜的钙通透性Orai1通道。SOCE可促进肌肉生长/发育，限制疲劳，并促进抗疲劳的I型纤维规格。另一方面，SOCE功能障碍导致衰老时肌肉无力/疲劳，加重肌肉营养不良，STIM1和Orai1基因突变导致衰弱的肌肉疾病。目前的情况是，严格调节依赖STIM1/Orai1的SOCE活性对于优化肌肉性能至关重要，因为SOCE活性的增加或减少可能导致肌肉疲劳、肌萎缩症和肌病。虽然SOCE活动明显影响肌肉表现，但STIM1-Orai1偶联在肌肉中的位置仍然不清楚。为了这种更新，我们建立了可诱导的、肌肉特异性的Orai1和STIM1KO小鼠，以确定STIM1-Orai1偶联限制疲劳的机制。我们提供了令人兴奋的证据，即疲劳运动促使TT和SR之间形成迄今未描述的连接，在那里发生STIM1-Orai1偶联，我们称之为“钙进入单位”(CEU)。CEU与三联体或钙释放单位相连，但与之不同。我们提供的初步数据表明，与雄性小鼠相比，Orai1对雌性小鼠的肌肉纤维收缩功能有更强的影响。我们还提供了初步的协作性免疫沉淀和质谱学的可行性数据，用于表征CEU形成前后的STIM1蛋白质组。我们将使用这些研究工具、方法、发现和合作来促进对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。根据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STIM1-Orai1复合体的三联体的连接延伸的形成，这些复合体协调SOCE来增强SR钙再充盈，限制肌肉疲劳，并在长期内促进NFATc1核定位和I型纤维规范。我们还假设，疲劳诱导的肌肉CEU的形成涉及多个蛋白质组分(包括BIN1、STIM1和OraI1)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。目标1将描述Orai1在肌肉疲劳和I型纤维规格中的作用。目的2鉴定新发现的成人骨骼肌中CEU的亚细胞位置、分子组成和稳定性，并确定CEU的形成和拆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定位提供新的机制见解；2)使用定向和无偏见的发现方法来识别和验证与CEU形成相关的蛋白质；以及3)确定性别对Orai1依赖的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.