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Molecular Mechanism and Functional Role of SOCE in Skeletal Muscle

SOCE在骨骼肌中的分子机制和功能作用

基本信息

批准号：
8477131
负责人：
Robert T Dirksen
金额：
$ 30.58万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-08-01 至 2015-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8477131
关键词：
Adult Autoimmune Diseases Biochemical Biological Biological Assay Calcium Cell membrane Cells Central Core Myopathy Chronic Clinical Coupling Dantrolene Data Deterioration Development Diagnostic Disease Dominant-Negative Mutation Electron Microscopy Embryo Exhibits Fatigue Fiber Fluorescence Fluorescence Resonance Energy Transfer Frequencies Functional disorder Genes Halothane Heating Human Image Immunologic Deficiency Syndromes In Vitro Incidence Knock-in Mouse Link Location Malignant hyperpyrexia due to anesthesia Mitochondrial Myopathies Molecular Molecular Target Morphology Mus Muscle Muscle Contraction Muscle Fibers Muscle function Mutation Myopathy Pathway interactions Patients Performance Physiological Plasma Play Predisposition Proteins Role RyR1 Ryanodine Receptor Calcium Release Channel STIM1 gene Sarcolemma Sarcoplasmic Reticulum Seminal Signal Transduction Skeletal Muscle Sudden Death T-Lymphocyte Testing Time Transgenic Mice Transgenic Organisms Triad Acrylic Resin base extracellular frontier in vivo innovation loss of function loss of function mutation mouse model muscular structure novel novel strategies protein S precursor protein function public health relevance sensor skeletal technological innovation

项目摘要

DESCRIPTION (provided by applicant): It has long been known that skeletal muscle twitch contraction persists in the absence of extracellular Ca2+. Nevertheless, recent studies, including our own, have identified a rapidly activated store-operated Ca2+ entry (SOCE) pathway in skeletal muscle used to replenish previously depleted intracellular Ca2+ stores. We provide strong preliminary data to demonstrate that STIM1 proteins function as the calcium sensor of the sarcoplasmic reticulum (SR) and Orai1 proteins as the calcium permeable sarcolemmal channel in adult skeletal muscle fibers. In addition, STIM1 and Orai1 proteins are known to be expressed at high levels in skeletal muscle and a debilitating myopathy is consistently observed in human severe combined immuno-deficiency patients possessing either loss-of-function STIM1 or Orai1 mutations. Together, these results demonstrate that SOCE likely plays a previously unappreciated role in skeletal muscle function and disease; indicating that STIM1- Orai1 coupling represents an exciting and untapped frontier in skeletal muscle Ca2+ signaling. Based on these findings, and by analogy to SOCE in non-excitable cells, we hypothesize that "STIM1 and Orai1 proteins pre-localize to the triad junction to coordinate uniquely rapid SOCE activation that limits SR Ca2+ store depletion during repetitive tetanic stimulation. Chronic SOCE contributes to muscle fiber hyperexcitability and deterioration in a mouse model of malignant hyperthermia (MH) and central core disease (CCD)." We will test the validity of this hypothesis by characterizing the: 1) molecular mechanism of rapid SOCE activation in adult skeletal muscle (Aim 1), 2) role of SOCE in maintaining SR Ca2+ content and release during repetitive high frequency tetanic stimulation (Aim 2), and 3) degree to which SOCE dysfunction contributes/modifies muscle disease. An important conceptual innovation of this project is the hypothesis that a SOCE activity limits activity-dependent skeletal muscle performance and serves as an important modifier of muscle disease. An important technological innovation is our recent development of skeletal muscle-specific dominant negative Orai1 transgenic mice and use of these mice to probe the (patho)physiological role of SOCE in skeletal muscle. This project will employ a battery of molecular/cell biological, bi-molecular fluorescence complementation, fluorescence energy resonance transfer, electrophysiological, confocal imaging, biochemical, transgenic, electron microscopy, in vitro muscle contraction, and in vivo muscle performance assays to characterize the mechanism of STIM1-Orai1 activation and the functional role of SOCE in skeletal muscle. The molecular mechanisms characterized during this venture will have implications not only for muscle function, but will also extend more broadly to disorders of Ca2+ dysregulation across a diversity of related clinical arenas including immunodeficiency, autoimmune disease, fatigue, and myopathy. Thus, discoveries resulting from this proposal will provide promise for the development of novel approaches, treatments, and diagnostics for a wide range of diseases in Ca2+ signaling.

描述（由申请人提供）：长期以来，已知骨骼肌抽搐收缩在细胞外Ca2+不存在的情况下持续存在。然而，最近的研究，包括我们自己的，已经确定了一个快速激活的商店操作的钙离子进入（SOCE）途径在骨骼肌用于补充以前耗尽的细胞内钙商店。我们提供了强有力的初步数据表明，STIM1蛋白的功能作为钙传感器的肌浆网（SR）和Orai1蛋白作为钙渗透性肌膜通道在成人骨骼肌纤维。此外，已知STIM1和Orai1蛋白在骨骼肌中以高水平表达，并且在具有功能丧失STIM1或Orai1突变的人类严重联合免疫缺陷患者中始终观察到衰弱性肌病。总之，这些结果表明，SOCE可能在骨骼肌功能和疾病中起着以前未被认识到的作用;表明STIM1-Orai1偶联代表了骨骼肌Ca2+信号传导中令人兴奋和未开发的前沿。基于这些发现，并通过类比非兴奋性细胞中的SOCE，我们假设"STIM 1和Orai 1蛋白预定位于三联体连接以协调独特的快速SOCE激活，从而限制重复强直刺激期间SR Ca2+库耗尽。慢性SOCE导致恶性高热（MH）和中央核心病（CCD）小鼠模型中的肌纤维过度兴奋和恶化。“我们将通过以下特征来测试这一假设的有效性：1）成人骨骼肌中快速SOCE激活的分子机制（目标1），2）SOCE在维持SR Ca 2+含量和重复高频强直刺激期间释放的作用（目标2），以及3）SOCE功能障碍有助于/改变肌肉疾病的程度。该项目的一个重要概念创新是假设SOCE活动限制活动依赖性骨骼肌性能，并作为肌肉疾病的重要修饰剂。一个重要的技术创新是我们最近开发的骨骼肌特异性显性负Orai1转基因小鼠和使用这些小鼠探测（病理）生理作用的骨骼肌中的SOCE。该项目将采用一系列分子/细胞生物学、双分子荧光互补、荧光能量共振转移、电生理学、共聚焦成像、生物化学、转基因、电子显微镜、体外肌肉收缩和体内肌肉性能测定来表征STIM1-Orai1激活的机制和SOCE在骨骼肌中的功能作用。在这项研究中发现的分子机制不仅对肌肉功能有影响，而且还将更广泛地扩展到各种相关临床领域的Ca2+失调，包括免疫缺陷，自身免疫性疾病，疲劳和肌病。因此，从这一建议中得到的发现将为开发新的方法，治疗和诊断各种疾病的Ca2+信号提供希望。