Calcium release channel dysfunction: molecular mechanisms

 描述(由申请人提供)：通过兰尼定受体(RyR)释放的钙(Ca)对正常的心脏收缩是必不可少的。RyR调节缺陷导致多种心脏疾病的钙稳态失衡和收缩功能障碍。由于最常见的心脏病变(如梗塞、心力衰竭)与氧化应激有关，本研究的主要目的是确定氧化应激过程中RyR功能障碍的分子机制。RyR含有大量的半胱氨酸残基，可以偶联胞质氧化还原电位和钙稳态。然而，RyR上功能上重要的氧化还原感测位点尚未确定。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不清楚。这推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钙稳态。因此，更直接地鉴定RyR上重要的氧化还原敏感半胱氨酸对于推动这一领域的发展是至关重要的。我们最近发现，氧化应激通过在相邻的两个亚基之间形成二硫键来激活RyR：亚基间交联。在这项建议中，我们将检验这一假设，即亚基间交联是RyR功能上最重要的氧化还原修饰，导致氧化应激过程中钙稳态的失衡。这一假设将使用尖端实验技术进行验证，例如RyR突变、单RyR通道记录和高分辨率钙成像。在目标1中，我们将确定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过程中维持正常的RyR功能和钙稳态。在目标2中，我们将确定由交联剂引起的RyR功能障碍的分子机制。与RyR结合的钙调蛋白(CaM)在RyR活性的负性调控中起着重要作用。我们的初步研究表明，交联会导致CaM从RyR解离。在这里，我们将定义是否通过防止CaM-RyR解偶联，交联半胱氨酸的突变可以在氧化应激过程中使钙稳态正常化。我们还将探讨稳定CaM-RyR结合是否可以保护RyR功能免受心肌细胞氧化应激的影响。通过完成这些研究，我们希望为未来的治疗定义新的靶点，以改善与氧化应激相关的心脏病期间的钙稳态。

 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.