Calcium release channel dysfunction: molecular mechanisms

钙释放通道功能障碍：分子机制

基本信息

批准号：
9204856
负责人：
Aleksey V Zima
金额：
$ 37.75万
依托单位：
LOYOLA UNIVERSITY CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-01-15 至 2019-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9204856
关键词：
Affect Arrhythmia Binding Sites Blinking Calcium Calmodulin Cardiac Cardiac Myocytes Clinical Complex Confocal Microscopy Cysteine Defect Dissociation Ensure Fatal Outcome Functional disorder Future Generations Goals Heart Diseases Heart failure Homeostasis Image In Situ In Vitro Infarction Life Lipid Bilayers Measurement Measures Mediating Modification Molecular Molecular Target Muscle Cells Mutagenesis Mutation Myocardial Contraction Myocardial Infarction Optics Outcome Study Oxidation-Reduction Oxidative Stress Pathology Pilot Projects Play Post-Translational Protein Processing Process Public Health Regulation Reporting Research Proposals Resistance Resolution Role Ryanodine Receptor Calcium Release Channel Sarcoplasmic Reticulum Site Site-Directed Mutagenesis System Techniques Testing Therapeutic Therapeutic Intervention Time Treatment Efficacy UV induced Ventricular Work crosslink design disulfide bond free radical oxygen heart function improved mutant novel oxidation prevent public health relevance receptor binding receptor function receptor structure function response targeted treatment

项目摘要

DESCRIPTION (provided by applicant): Calcium (Ca) release through the ryanodine receptor (RyR) is essential for regular heart contraction. Defects in RyR regulation cause imbalance in Ca homeostasis and contractile dysfunction in a variety of cardiac diseases. Since the most common cardiac pathologies (e.g. infarction, heart failure) are associated with oxidative stress, the main goal of this proposal is to define the molecular mechanisms of RyR dysfunction during oxidative stress. The RyR contains a large number of cysteine residues that can couple the cytosolic redox potential and Ca homeostasis. However, the functionally important redox-sensing sites on the RyR have not yet been identified. As a result, the molecular mechanisms of RyR dysfunction during oxidative stress remain largely unknown. This delays our progress in designing effective therapeutic interventions that can improve Ca homeostasis during cardiac diseases. Thus, more direct work identifying functionally important redox-sensing cysteines on RyR is essential to advance the field. We have recently discovered that oxidative stress activates the RyR by forming disulfide bonds between two neighboring subunits: intersubunit crosslinking. In this proposal we will test the hypothesis that intersubunit crosslinking is the mot functionally important redox modification of RyR responsible for the imbalance in Ca homeostasis during oxidative stress. This hypothesis will be tested using cutting-edge experimental techniques, such as RyR mutagenesis, single RyR channel recordings, and high resolution Ca imaging. In aim 1 we will identify specific cysteine residues on RyR that are involved in the crosslinking. Then, we will determine if mutation of these cysteines can maintain normal RyR function and Ca homeostasis during oxidative stress. In aim 2 we will define the molecular mechanisms of RyR dysfunction induced by the crosslinking. Calmodulin (CaM) bound to the RyR plays an important role in negative control of RyR activity. Our pilot studies suggest that the crosslinking causes dissociation of CaM from the RyR. Here, we will define if mutation of crosslinking cysteines can normalize Ca homeostasis during oxidative stress by preventing the CaM-RyR uncoupling. We will also explore whether stabilizing the CaM-RyR binding can protect the RyR function against oxidative stress in cardiomyocytes. By accomplishing these studies, we expect to define novel targets for future therapies that can improve Ca homeostasis during cardiac diseases associated with oxidative stress.

描述（由申请人提供）：通过兰尼碱受体（RyR）释放钙（Ca）对于正常的心脏收缩至关重要。自最常见的心脏病以来，RyR 调节的缺陷会导致钙稳态失衡和收缩功能障碍。病理（例如梗塞、心力衰竭）与氧化应激相关，该提案的主要目标是确定氧化应激期间 RyR 功能障碍的分子机制。 RyR 含有大量的半胱氨酸残基，可以耦合细胞质氧化还原电位和 Ca 稳态，但 RyR 上功能重要的氧化还原感应位点尚未确定，因此 RyR 功能障碍的分子机制尚未确定。氧化应激在很大程度上仍然未知，这延迟了我们在设计有效的治疗干预措施方面的进展，这些干预措施可以改善心脏病期间的 Ca 稳态，因此，更直接的工作确定 RyR 上具有重要功能的氧化还原感应半胱氨酸至关重要。我们最近发现，氧化应激通过在两个相邻亚基之间形成二硫键来激活 RyR：亚基间交联。在本提案中，我们将检验亚基间交联是导致不平衡的 RyR 功能上最重要的氧化还原修饰的假设。该假设将使用尖端实验技术进行测试，例如 RyR 诱变、单 RyR 通道记录和高分辨率 Ca 成像。目标 1 我们将识别 RyR 上参与交联的特定半胱氨酸残基然后，我们将确定这些半胱氨酸的突变是否可以在氧化应激期间维持正常的 RyR 功能和 Ca 稳态。与 RyR 结合的钙调蛋白 (CaM) 引起的 RyR 功能障碍在 RyR 活性的负控制中发挥着重要作用。在这里，我们将定义交联半胱氨酸的突变是否可以通过阻止 CaM-RyR 解偶联来使氧化应激期间的 Ca 稳态正常化。我们还将探讨稳定 CaM-RyR 结合是否可以保护 RyR 功能免受心肌细胞的氧化应激。通过完成这些研究，我们期望为未来的治疗确定新的靶点，以改善与氧化应激相关的心脏病期间的钙稳态。