Skeletal Muscle. Ca Release Control Inside the Sarcoplasmic Reticulum.

描述(申请人提供)：CA2信号是细胞用来做出反应和改变的通用语言。在骨骼肌中，它的兴趣模式涵盖多个时间尺度：毫秒--决定收缩和放松的钙运动--；几秒到几分钟--持续活动可能导致肌源性疲劳--以及几小时到几周--导致基因表达和长期适应变化的模式--。这项研究是关于它的细胞内存储；它的数量和浓度，[Ca~(2+)]SR，在每个时间尺度上调节钙信号。我们询问(1)[Ca~(2+)]SR是否以及如何控制存储器中的Ca~(2+)释放，以及(2)两个位于战略位置的SR蛋白Calequestrin和Triadin是否以及如何参与这一控制。一项技术任务，我们称之为“目标0”，是成像和测量[钙]SR。这是在当前阶段完成的，并将在下一阶段继续，使用新型生物传感器-由细胞本身制造的分子-以及新的混合监测器，由放置在细胞中的高性能小型合成传感器组成，这些传感器被操纵以制造特殊的生物锚。为了回答问题1和2，我们将在测量的同时分别操作[Ca~(2+)]SR(目标1)，并迫使细胞改变其钙固缩蛋白和Triadin的天赋(目标2)。这些目标现在在活着的动物身上是可行的，这要归功于一种DNA转基因方法，这种方法可以处理每一种蛋白质，也可以用来阻止它们的合成。我们认为，当肌肉疲劳时，[Ca~(2+)]SR会衰退，这是由SOCE维持的，SOCE是一种普遍的Ca~(2+)进入途径，对动员控制基因表达的转录因子至关重要。使用第一阶段开发的SOCE方法，我们提出目标3来定义新发现的SOCE分子在控制[Ca~(2+)]SR中的作用。这些分子可能是抵抗疲劳的堡垒，并为在迭代方法中对主要问题进行[钙]SR的实验改变提供强大的工具。CA2信号在疾病、疲劳和衰老时会恶化。快速钙信号在PP过低、MH易感性、中央核心区和微核区以及肌肉老化等疾病中失灵。中期信号在疲劳和缺乏钙固醇素的MH样表型小鼠中受到影响。长期钙信号在肌肉和免疫系统中显示出惊人的相似之处的疾病包括SCID，这是一种家族性免疫缺陷，结合了淋巴细胞中SOCE的丢失和肌源性肌病。我们的工作将通过评估特定分子的作用及其相互作用来促进对这些缺陷的理解。虽然在本项目中将只具体讨论疲劳，但随着我们了解什么控制储存钙，以及储存的钙控制什么，关于功能缺陷、复杂的病理生理学和治疗矫正的合理设计之间的关系的问题将被更好地解决。

DESCRIPTION (provided by applicant): Ca2+ signaling is a universal language used by cells to react and change. In skeletal muscle its patterns of interest cover multiple time scales: milliseconds -Ca2+ movements that determine contraction and relaxation-; seconds to minutes -when sustained activity may lead to myogenic fatigue- and hours to weeks -patterns that cause changes in gene expression and long-term adaptation-. This study is about inside its cellular store; its quantity, and its concentration, [Ca2+]SR, which conditions Ca2+ signals in every time scale. We ask (1) whether and how [Ca2+]SR controls Ca2+ release from the store, and (2) whether and how calsequestrin and triadin, two strategically located SR proteins, contribute to this control. A technical task, which we call "aim 0", is to image and measure [Ca2+]SR. This was accomplished in the current period and will continue in the next, using novel biosensors -molecules made by the cells themselves- and new hybrid monitors, consisting of high performance small synthetic sensors placed into cells manipulated to make special bio-anchors. To answer questions 1 and 2, we will respectively manipulate [Ca2+]SR while we measure it (aim 1) and force cells to change their endowment of calsequestrin and triadin (aim 2). These goals are now feasible in living animals thanks to a DNA transfection method that works with every protein and can be used also to prevent their synthesis. We propose that [Ca2+]SR -which decays when muscles fatigue- is sustained by SOCE, a universal Ca2+ entry pathway, crucial for mobilizing transcription factors that control gene expression. Using SOCE measures developed in the first period, we propose as aim 3 to define the role of newly discovered molecules of SOCE in the control of [Ca2+]SR. These molecules could be bulwarks against fatigue, and provide powerful tools for experimental alterations of [Ca2+]SR in iterative approaches to the main questions. Ca2+ signals deteriorate in disease, fatigue and aging. Fast Ca2+ signals fail in diseases like hypo-PP, MH susceptibility and central core and minicore, as well as in ageing muscle. Mid-range signaling is affected in fatigue and in an MH-like phenotype of mice lacking calsequestrin. Diseases of long term Ca2+ signals, which show striking parallels in muscle and the immune system, include SCID, a familial immune defect that combines loss of SOCE in lymphocytes and a myogenic myopathy. Our work will advance understanding of these deficits by evaluating roles of specific molecules and their interactions. While only fatigue will be specifically addressed in the present project, questions on the relationships among deficits of function, the intricate pathophysiology and the rational design of therapeutic corrections will be addressed better as we understand what controls stored calcium, and what the stored calcium controls.