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.
