Skeletal Muscle. Ca2+ release control inside the SR

骨骼肌。

• 批准号: 6915217
• 负责人: Eduardo Rios
• 金额: $ 32.72万
• 依托单位: RUSH UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6915217
• 关键词: Rana; biophysics; buffers; calcium binding protein; calcium channel; calcium flux; calcium ion; confocal scanning microscopy; cytoplasm; gene expression; laboratory mouse; laboratory rat; mature animal; model design /development; molecular dynamics; muscle contraction; protein quantitation /detection; protein structure function; recombinant DNA; sarcoplasmic reticulum; striated muscles; sulfates; tissue /cell culture; transfection; voltage /patch clamp

DESCRIPTION (provided by applicant): Ca2+ signaling links plasma membrane events to metabolic change. Excitation-contraction (EC) coupling -- which in muscle transduces the action potential to increase in intracellular Ca2+ concentration, and contraction-- was the first recognized example of Ca2+ signaling. Its key molecules (voltage sensors or DHPrs and release channels or RyRs) are increasingly found in other cells and tissues. Its involvement is recognized in an increasing number of inheritable diseases. Its changes, upon prolonged exercise, crucially contribute to muscle fatigue. EC coupling has traditionally been probed by measuring Ca2+ transients, based on the consensus that control of Ca2+ release is exerted by ligands (like Ca2+, or loops in the DHPr) at the cytosolic face of the RyRs. Evidence is now increasing for crucial regulation from within the SR. This is a proposal for the systematic study of such control. Four working hypotheses will be tested, which elaborate the idea of regulation from within the SR: 1) Ca2+ release activation is largely determined by SR Ca2+ content. 2) Ca2+ release termination is largely determined by local Ca2+ depletion within the SR. 3) A novel concerted mode of channel activation is controlled by SR Ca. 4) The intra-SR Ca2+-binding protein calsequestrin has a dynamic role in regulation of Ca2+ release, which may involve interactions with other SR proteins. These hypotheses will be tested by 1) monitoring Ca2+ release on single cells, at the cell-averaged and at the local level (where it results in Ca2+ sparks), while manipulating and monitoring the Ca2+ content of the SR. 2) Introducing extrinsic Ca2+ buffers in the SR, one of which, sulfate, has already proved to have extraordinary effects. 3) Changing the concentration and functionality of calsequestrin, by its over- or under-expression in cells in culture, or by gene transfer to adult muscle. 4) To better understand molecular requirements, effects of calsequestrin will be sought on the control of single RyRs by Ca2+. 5) An existing model of Ca2+ release will help test the validity of various quantitative versions of these hypotheses. Technical innovation is substantial in aims 1 (simultaneous recording of intra-SR and cytosolic [Ca2+]), 2 (distinction of buffers and limit-buffers), 3 (transfer of calsequestrin genes to adult muscle) and 4 (combined application of calsequestrin and photoreleased Ca to channels in bilayers).

描述（由申请人提供）：Ca2+信号传导将质膜事件与代谢变化联系起来。兴奋-收缩 (EC) 耦合——在肌肉中转导动作电位以增加细胞内 Ca2+ 浓度和收缩——是 Ca2+ 信号传导的第一个公认的例子。其关键分子（电压传感器或 DHPr 和释放通道或 RyR）越来越多地出现在其他细胞和组织中。人们认识到它与越来越多的遗传性疾病有关。长时间运动后其变化对肌肉疲劳至关重要。传统上，EC 耦合是通过测量 Ca2+ 瞬变来探测的，这是基于对 Ca2+ 释放的控制是由 RyR 胞质面的配体（如 Ca2+ 或 DHPr 中的环）控制的共识。现在越来越多的证据表明 SR 内部的关键监管。这是对这种控制进行系统研究的建议。将测试四个工作假设，这些假设详细阐述了 SR 内部的调节思想： 1) Ca2+ 释放激活很大程度上取决于 SR Ca2+ 含量。 2) Ca2+ 释放终止很大程度上取决于 SR 内的局部 Ca2+ 耗尽。 3) 一种新颖的通道激活协调模式由 SR Ca 控制。 4) SR内Ca2+结合蛋白calsequestrin在调节Ca2+释放中具有动态作用，这可能涉及与其他SR蛋白的相互作用。这些假设将通过以下方式进行检验：1）在细胞平均和局部水平（导致 Ca2+ 火花）监测单个细胞上的 Ca2+ 释放，同时操纵和监测 SR 的 Ca2+ 含量。 2）在SR中引入外源性Ca2+缓冲液，其中一种硫酸盐已被证明具有非凡的效果。 3) 通过在培养细胞中过表达或表达不足，或通过基因转移至成体肌肉，改变钙结合蛋白的浓度和功能。 4) 为了更好地了解分子需求，将寻求 calsequestrin 对 Ca2+ 对单个 RyR 的控制的影响。 5) 现有的 Ca2+ 释放模型将有助于测试这些假设的各种定量版本的有效性。技术创新在目标 1（同时记录 SR 内和胞质 [Ca2+]）、2（缓冲液和限制缓冲液的区别）、3（将 calsequestrin 基因转移到成人肌肉）和 4（将 calsequestrin 和光释放 Ca 联合应用到双层通道）方面具有重大意义。