Characterization of the Exercise-induced Orai1 Proteome in Skeletal Muscle

骨骼肌中钙池操纵的钙内流（SOCE）是由刺激1钙传感器之间的耦合介导的， 肌浆网（SR）和横小管（TT）中的Ca 2+渗透性Orai 1通道。SOCE 活性也受到钙螯合蛋白-1（Casq 1）的调节，钙螯合蛋白-1是肌肉中主要的SR Ca 2+缓冲剂。SOCE增强 肌肉发育，限制疲劳，并促进抗疲劳I型纤维规格。另一方面，在一项研究中， SOCE功能障碍导致衰老中的肌肉无力，加剧肌肉萎缩症和突变 在Stim 1、Orai 1和Casq 1中，所有这些都导致以管状聚集体的存在为特征的肌病。因此，在本发明中， Orai 1依赖的SOCE活性在正常肌肉功能和疾病中起着关键作用。 我们最近发现，急性运动驱动新的SR-TT连接（Ca 2+进入单位，CEU）的形成。 CEU促进Orai 1依赖的组成性和钙库操作的Ca 2+进入，增强SR钙库再填充， Ca 2+的释放和重复刺激过程中的力的产生。有趣的是，CEU本质上也是 缺乏Casq 1的久坐小鼠和野生型小鼠在1个月的自愿轮跑（VWR）后存在。 对于该项目，我们开发了在Orai 1 C末端末端具有V5-3xHA表位标签的敲入小鼠 （Orai 1V 5 HA/+小鼠）。使用这些小鼠，我们在骨骼肌中鉴定了两种不同的Orai 1亚型（短型和长型）， 肌肉（两者都是高度糖基化的），但脾脏中只有一种同种型。我们的初步数据表明， 使用Orai 1V 5 HA/+小鼠鉴定休息时和急性运动后肌肉中Orai 1相互作用蛋白的可行性 锻炼的将使用N端和C端Orai 1-TurboBioID串联进行平行研究 在诱导型肌肉特异性Orai 1敲除小鼠的肌肉中引入后的构建体。拟议 “高风险/高回报”研究将采用尖端的、无偏见的蛋白质组学方法（HA-IP、邻近 生物素化）以鉴定在缺氧条件下骨骼肌中Orai 1相互作用蛋白（“Orai 1相互作用组”）的变化。 促进CEU形成和SOCE活性的条件（急性运动，VWR和Casq 1缺乏）。 我们将利用这些发现，研究工具和尖端方法来确定分子 协调Orai 1依赖的CEU形成，稳定和拆卸的机制。我们假设 1）急性运动驱动Orai 1蛋白相互作用，触发大分子马达驱动动力学 和可逆的CEU形成和2）SR Ca 2+储存内容的挑战（延长的VWR，Casq 1缺乏） 促进Orai 1蛋白相互作用，稳定CEU。目标1将询问分子机制， 协调动态，运动依赖性CEU的形成，通过表征肌肉中的Orai 1相互作用组， 休息和剧烈运动后。目的2将描述肌肉中Orai 1相互作用组在有利于 CEU的稳定形成（长期VWR和Casq 1缺乏）。这些研究将描绘出 以及协调CEU形成/稳定和骨骼肌中SOCE功能的细胞机制。

Store-operated Ca2+ entry (SOCE) in skeletal muscle is mediated by coupling between Stim1 Ca2+ sensors in the sarcoplasmic reticulum (SR) and Ca2+-permeable Orai1 channels in the transverse tubule (TT). SOCE activity is also modulated by calsequestrin-1 (Casq1), the primary SR Ca2+ buffer in muscle. SOCE enhances muscle development, limits fatigue, and promotes fatigue-resistant type I fiber specification. On the other hand, SOCE dysfunction contributes to muscle weakness in aging, exacerbates muscular dystrophy, and mutations in Stim1, Orai1, and Casq1 all result in a myopathy characterized by the presence of tubular aggregates. Thus, Orai1-dependent SOCE activity plays a critical role in both normal muscle function and disease. We recently found that acute exercise drives the formation of new SR-TT junctions (Ca2+ entry units, CEUs). CEUs promote Orai1-dependent constitutive and store-operated Ca2+ entry that enhances SR store refilling, Ca2+ release, and force production during repetitive stimulation. Interestingly, CEUs are also constitutively present in sedentary mice that lack Casq1 and wild type mice after 1 month of voluntary wheel running (VWR). For this project, we developed knock-in mice with a V5-3xHA epitope tag on the extreme Orai1 C-terminus (Orai1V5HA/+ mice). Using these mice, we identified two distinct Orai1 isoforms (short and long) in skeletal muscle (both being heavily glycosylated), but only one isoform in spleen. Our preliminary data demonstrate the feasibility of using Orai1V5HA/+ mice to identify the Orai1 interacting proteins in muscle at rest and after acute exercise. Parallel studies will be conducted using N-terminal and C-terminal Orai1-TurboBioID tandem constructs following introduction in muscle of inducible, muscle-specific Orai1 knockout mice. The proposed “high risk/high reward” studies will employ cutting-edge, non-biased proteomic approaches (HA-IP, proximity biotinylation) to identify changes in Orai1 interacting proteins (“Orai1 interactome”) in skeletal muscle under conditions that promote CEU formation and SOCE activity (acute exercise, VWR, and Casq1 deficiency). We will use these discoveries, research tools, and cutting-edge approaches to determine the molecular mechanisms that coordinate Orai1-dependent CEU formation, stabilization and disassembly. We hypothesize that: 1) acute exercise drives Orai1 protein interactions that trigger a macromolecular motor to drive dynamic and reversible CEU formation and 2) challenges to SR Ca2+ store content (prolonged VWR, Casq1 deficiency) promote Orai1 protein interactions that stabilize CEUs. Aim 1 will interrogate the molecular mechanism that coordinates dynamic, exercise-dependent CEU formation by characterizing the Orai1 interactome in muscle at rest and after acute exercise. Aim 2 will characterize the Orai1 interactome in muscle under conditions that favor the stable formation of CEUs (long-term VWR and Casq1 deficiency). These studies will delineate the molecular and cellular mechanisms that coordinate CEU formation/stabilization and SOCE function in skeletal muscle.