Redox Control of Sarcoplasmic Reticulum Calcium Release

描述(由申请人提供)：骨骼肌和心肌肌浆网(SR)都对由自由基直接诱导的氧化应激和通过细胞氧化还原电位增加间接诱导的氧化应激敏感。氧化应激由心肌缺血和再灌流引起，骨骼肌则导致肌肉疲劳。SR控制和响应氧化还原环境变化的分子机制，以及这如何影响Ca~(2+)动态平衡是本研究的主要目标。SR氧化损伤的主要部位似乎是高活性的巯基，在温和的氧化条件下，它被氧化成二硫化物。这导致SR的钙释放通道开放，细胞内钙离子浓度增加。我们的目标是测量这些硫醇在心肌和骨骼肌中的氧化还原电位，并确定细胞环境中的生理相关成分如何控制兰尼定受体的氧化还原电位。使用单通道测量，我们还将确定钙释放通道如何响应其细胞质和管腔侧SR膜上氧化还原电位的变化。此外，局部氧化还原电位的快速变化将使我们能够确定受体对其氧化还原环境变化的反应速度。这一提议不仅着眼于ryanodine受体如何响应其氧化还原环境，而且还首次从SR中发现了一种内源性NAD(P)H依赖的氧化酶，它产生超氧化物，刺激ryanodine受体。我们的目标是描述这种氧化酶的特性，了解它的活性如何受到肌肉在活动增加时发生的变化的影响，并确定它在骨骼肌疲劳中的作用。

DESCRIPTION (provided by applicant): Both skeletal and cardiac muscle sarcoplasmic reticulum (SR) are sensitive to oxidative stress induced directly by free radicals, and indirectly by an increase in the cellular redox potential. Oxidative stress results from cardiac ischemia and reperfusion, and in skeletal muscle results in muscle fatigue. The molecular mechanism by which the SR controls and responds to changes in its redox environment, and how this influences Ca2+ homeostasis is the main goal of this research proposal.The major site of oxidative damage to SR appears to be highly reactive sulfhydryl groups, which under mildly oxidative conditions, oxidize to disulfides. This causes the Ca2+ release channel from SR to open and the intracellular Ca2+ concentration to increase. It is our goal to measure the redox potential of these thiols in both cardiac and skeletal muscle, and to determine how physiologically relevant components in the cellular environment control the redox potential of the ryanodine receptor. Using single channel measurements, we will also determine how the Ca2+ release channel responds to alterations in the redox potential on both its cytoplasmic and lumenal sides of the SR membrane. Moreover, rapid changes in the local redox potential will enable us to determine how quickly the receptor responds to changes in its redox environment.Not only does this proposal focus on how the ryanodine receptor responds to its redox environment, but it also identifies for the first time an endogenous NAD (P) H dependent oxidase from SR, which produces superoxide, and which stimulates the ryanodine receptor. It is our goal to characterize this oxidase, to understand how its activity is influenced by changes that occur to the muscle during increased activity, and to determine its role in skeletal muscle fatigue.