Calcium release channel dysfunction: molecular mechanisms

钙释放通道功能障碍：分子机制

• 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
• 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

 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）：钙（Ca）通过兰尼碱受体（RyR）释放对正常心脏收缩至关重要。RyR调节的缺陷导致各种心脏疾病中Ca稳态失衡和收缩功能障碍。由于最常见的心脏病（如梗死，心力衰竭）与氧化应激有关，该提案的主要目标是定义氧化应激过程中RyR功能障碍的分子机制。RyR含有大量的半胱氨酸残基，可以耦合胞质氧化还原电位和Ca稳态。然而，功能上重要的RyR的氧化还原敏感网站尚未确定。因此，氧化应激过程中RyR功能障碍的分子机制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以改善心脏疾病期间钙稳态方面的进展。因此，更直接的工作识别RyR上功能重要的氧化还原敏感半胱氨酸对于推进该领域至关重要。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激活RyR：亚基间交联。在这个建议中，我们将测试的假设，亚基间交联是负责在氧化应激过程中钙稳态失衡的RyR的功能上重要的氧化还原修饰。这一假设将使用尖端的实验技术进行测试，如RyR诱变，单RyR通道记录，高分辨率钙成像。在目标1中，我们将确定参与交联的RyR上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。在目标2中，我们将定义由交联诱导的RyR功能障碍的分子机制。与RyR结合的钙调素（CaM）在RyR活性的负调控中起重要作用。我们的初步研究表明，交联导致钙调素从RyR解离。在这里，我们将确定是否交联半胱氨酸的突变可以正常化钙稳态氧化应激过程中，通过防止钙调素-RyR解偶联。我们还将探讨稳定CaM-RyR结合是否可以保护RyR功能免受心肌细胞中的氧化应激。通过完成这些研究，我们期望为未来的治疗确定新的靶点，这些治疗可以改善与氧化应激相关的心脏疾病期间的钙稳态。