Skeletal Muscle. Ca Release Control Inside the Sarcoplasmic Reticulum.

描述（由申请人提供）：Ca2+信号是细胞反应和变化的通用语言。在骨骼肌中，其感兴趣的模式涵盖多个时间尺度：毫秒- ca2 +运动决定收缩和松弛-；几秒到几分钟——持续的活动可能会导致肌肉疲劳——几小时到几周——这种模式会导致基因表达和长期适应的变化。这项研究是关于它的细胞储存；它的数量，它的浓度，[Ca2+]SR，它在每个时间尺度上控制Ca2+信号。我们询问(1)[Ca2+]SR是否以及如何控制Ca2+从储存中释放，以及(2)calsequestrin和triadin这两种战略性SR蛋白是否以及如何促进这种控制。一项技术任务，我们称之为“目标0”，是成像和测量[Ca2+]SR。这项工作已经在当前阶段完成，并将在接下来的阶段继续进行，使用新型生物传感器（由细胞自身产生的分子）和新型混合监测器，这些监测器由高性能的小型合成传感器组成，放置在被操纵的细胞中，以制造特殊的生物锚。为了回答问题1和2，我们将在测量[Ca2+]SR时分别操纵它（目标1），并迫使细胞改变钙sequestrin和triadin的禀赋（目标2）。这些目标现在在活体动物中是可行的，这要归功于一种DNA转染方法，这种方法可以与每种蛋白质一起工作，也可以用来阻止它们的合成。我们提出[Ca2+]SR -当肌肉疲劳时衰减-由SOCE维持，SOCE是一种通用的Ca2+进入途径，对于动员控制基因表达的转录因子至关重要。利用第一阶段开发的SOCE测量方法，我们提出了目标3，以确定新发现的SOCE分子在控制[Ca2+]SR中的作用。这些分子可能是抗疲劳的堡垒，并为[Ca2+]SR的实验改变提供了强大的工具，在迭代方法中解决主要问题。Ca2+信号在疾病、疲劳和衰老中恶化。快速Ca2+信号在低pp、MH易感性、中央核心和小核心以及衰老肌肉等疾病中失效。缺乏钙sequestrin的小鼠疲劳和mh样表型中，中程信号传导受到影响。长期Ca2+信号的疾病，在肌肉和免疫系统中显示出惊人的相似性，包括SCID，一种家族性免疫缺陷，结合淋巴细胞中SOCE的丧失和肌源性肌病。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。虽然在目前的项目中只有疲劳将被具体解决，但随着我们了解是什么控制了钙的储存，又是什么控制了钙的储存，关于功能缺陷、复杂的病理生理学和治疗纠正的合理设计之间的关系的问题将得到更好的解决。

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.