Characterization of the Exercise-induced Orai1 Proteome in Skeletal Muscle

骨骼肌钙离子通道中STIM1受体偶联介导的钙离子内流 横管内的肌浆网(SR)和钙离子通道Orai1。SOCE 钙调蛋白-1(Casq1)是肌肉中主要的肌浆网钙缓冲物质，其活性也受到调节。SOCE增强 促进肌肉发育，限制疲劳，促进抗疲劳的I型纤维规格。另一方面， SOCE功能障碍会导致衰老时的肌肉无力，加剧肌肉营养不良，并导致突变 在STIM1、Orai1和Casq1中，都会导致以管状聚集体的存在为特征的肌病。因此， 依赖于ORAI1的SOCE活性在正常肌肉功能和疾病中都起着关键作用。 我们最近发现，急性运动会导致新的SR-TT连接(钙离子进入单位，CEU)的形成。 CEU促进依赖于Orai1的构成和存储操作的钙进入，从而增强SR存储重新填充， CA2的释放，并在重复刺激过程中强制产生。有趣的是，CEU也是结构性的 存在于缺乏Casq1的久坐小鼠和自愿车轮跑(VWR)1个月后的野生型小鼠中。 在这个项目中，我们开发了在极端Orai1C末端带有V5-3xHA表位标签的敲入小鼠 (Orai1V5HA/小鼠)。利用这些小鼠，我们在骨骼中鉴定出两种不同的Orai1亚型(短和长) 肌肉(都是高度糖基化的)，但在脾中只有一种异构体。我们的初步数据表明 应用Orai1V5HA/小鼠鉴定安静和急性运动后肌肉中Orai1相互作用蛋白的可行性 锻炼身体。将使用N端和C端Orai1-TurboBioID串联进行平行研究 在可诱导的、肌肉特异性的Orai1基因敲除小鼠的肌肉中引入后的结构。建议数 “高风险/高回报”研究将采用尖端的、无偏见的蛋白质组方法(HA-IP、邻近性 生物素化)以确定骨骼肌中Orai1相互作用蛋白(“Orai1相互作用体”)的变化 促进CEU形成和SOCE活动的条件(急性运动、VWR和Casq1缺乏症)。 我们将使用这些发现、研究工具和尖端方法来确定分子 协调依赖Orai1的CEU的形成、稳定和拆解的机制。我们假设 那就是：1)急性运动驱动Orai1蛋白相互作用，从而触发大分子马达驱动动力 和可逆的CEU形成以及2)对SR钙储存内容的挑战(延长VWR，Casq1缺乏) 促进Orai1蛋白相互作用，稳定CEU。目标1将询问分子机制 通过描述肌肉中Orai1相互作用体的特征，协调动态的、运动依赖的CEU的形成 休息和剧烈运动后。目标2将描述肌肉中Orai1相互作用体的特征 CEU的稳定形成(长期VWR和Casq1缺乏)。这些研究将描绘出 以及协调骨骼肌中CEU的形成/稳定和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.