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）对自由基直接诱导的氧化应激和细胞氧化还原电位的间接增加都很敏感。心脏缺血和再灌注引起氧化应激，骨骼肌引起肌肉疲劳。SR控制和响应其氧化还原环境变化的分子机制，以及这如何影响Ca2+稳态是本研究计划的主要目标。SR氧化损伤的主要部位似乎是高活性的巯基，在轻度氧化条件下，它们被氧化成二硫化物。这导致Ca2+释放通道从SR打开和细胞内Ca2+浓度增加。我们的目标是测量这些硫醇在心脏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ryanodine受体的氧化还原电位。使用单通道测量，我们还将确定Ca2+释放通道如何响应SR膜细胞质和管腔两侧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氧化还原环境变化的反应速度。这项研究不仅关注了ryanodine受体对氧化还原环境的反应，而且首次从SR中鉴定了内源性NAD (P) H依赖的氧化酶，该氧化酶产生超氧化物，并刺激ryanodine受体。我们的目标是表征这种氧化酶，了解其活性如何受到肌肉活动增加时发生的变化的影响，并确定其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.