Nitrosylation and Oxidation of RYR1 in Muscle Function

描述(申请人提供)：肌浆网的骨骼肌钙释放通道(RYR1)负责在骨骼肌兴奋-收缩偶联过程中从内库释放钙。该通道受亚硝化和氧化两种作用的调节。我们建议阐明这些特定调控的机制和生理作用。半胱氨酸3635(C3635)是RYR1上S亚硝化反应的主要靶点，也是参与氧化剂反应形成亚基间二硫键的半胱氨酸之一。我们发现亚基间二硫键的另一个半胱氨酸位于相邻亚基上的1999和2075氨基酸之间。在这一应用中，我们建议：1)确定参与RYR1对NO和氧化反应的半胱氨酸，并确定通道调节剂对这些半胱氨酸氧化还原修饰的影响。2)比较RYR1中突变的半胱氨酸3635、2021和2042对活性和对氧化剂和NO供体反应的功能影响。3)建立C3635位半胱氨酸突变小鼠模型，评价RYR1半胱氨酸3635在E-C偶联和肌肉功能中的作用。我们预测，这将改变肌浆网钙离子的泄漏，从而改变肌肉的收缩特性和纤维类型分布。 RYR1基因突变增加了钙离子通过通道的泄漏，已被证明与两种人类疾病相关，恶性高热和中枢性核心病。我们的研究旨在阐明该通道的氧化和亚硝化在调节通过该通道的钙泄漏中所起的作用。阐明这些调节改变通道活动的机制可能会为恶性高热和中枢核心疾病的干预提供新的靶点。

DESCRIPTION (provided by applicant): The skeletal muscle Ca2+ release channel (RYR1) of the sarcoplasmic reticulum is responsible for the release of Ca2+ from internal stores during skeletal muscle excitation-contraction coupling. This channel is modulated by both nitrosylation and oxidation. We propose to elucidate the mechanisms and the physiological role of these specific modulations. Cysteine 3635 (C3635) is the major target of S-nitrosylation on RYR1 and is one of the cysteines involved in the formation of an inter-subunit disulfide bond in response to oxidants. We have found that the other cysteine of the inter-subunit disulfide is located between amino acids 1999 and 2075 on an adjacent subunit. In this application we propose to: 1) Identify the cysteines involved in the response of RYR1 to NO and oxidation and determine the effect of channel modulators on the redox modification of these cysteines. 2) Compare functional effects of mutating cysteines 3635, 2021 and 2042 in RYR1 on activity and response to oxidants and NO donors. 3) Evaluate the role of cysteine 3635 of RYR1 in E-C coupling and muscle function by creating a mouse with a cysteine to alanine mutation at C3635. We predict that this will alter the leak of Ca2+ from the sarcoplasmic reticulum, thereby altering muscle contractile properties and fiber type distribution. Mutations in RYR1 that increase Ca2+ leak through the channel have been shown to correlate with two human diseases, malignant hyperthermia and central core disease. Our studies are designed to elucidate the role of oxidation and nitrosylation of this channel in regulation of Ca2+ leak through this channel. The elucidation of the mechanisms whereby these modulations alter channel activity may allow the identification of new targets for intervention in malignant hyperthermia and central core disease.