Identification of Cysteines Involved in S-Nitrosylation, S-Glutathionylation, and Oxidation to Disulfides in Ryanodine Receptor Type 1

• Published in: The Journal of biological chemistry Vol. 281; no. 52; pp. 40354 - 40368
• Main Authors: Aracena-Parks, Paula, Goonasekera, Sanjeewa A., Gilman, Charles P., Dirksen, Robert T., Hidalgo, Cecilia, Hamilton, Susan L.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 29-12-2006; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Cell Line; Cysteine - chemistry; Cysteine - metabolism; Disulfides - chemistry; Disulfides - metabolism; Glutathione - chemistry; Glutathione - metabolism; Humans; Hydrolysis; Male; Oxidation-Reduction; Peptide Fragments - metabolism; Rabbits; Reactive Nitrogen Species - chemistry; Reactive Nitrogen Species - metabolism; Ryanodine Receptor Calcium Release Channel - chemistry; Ryanodine Receptor Calcium Release Channel - metabolism; Sarcoplasmic Reticulum - chemistry; Sarcoplasmic Reticulum - metabolism; Trypsin - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M600876200

The skeletal muscle Ca2+-release channel (ryanodine receptor type 1 (RyR1)) is a redox sensor, susceptible to reversible S-nitrosylation, S-glutathionylation, and disulfide oxidation. So far, Cys-3635 remains the only cysteine residue identified as functionally relevant to the redox sensing properties of the channel. We demonstrate that expression of the C3635A-RyR1 mutant in RyR1-null myotubes alters the sensitivity of the ryanodine receptor to activation by voltage, indicating that Cys-3635 is involved in voltage-gated excitation-contraction coupling. However, H2O2 treatment of C3635A-RyR1 channels or wild-type RyR1, following their expression in human embryonic kidney cells, enhances [3H]ryanodine binding to the same extent, suggesting that cysteines other than Cys-3635 are responsible for the oxidative enhancement of channel activity. Using a combination of Western blotting and sulfhydryl-directed fluorescent labeling, we found that two large regions of RyR1 (amino acids 1-2401 and 3120-4475), previously shown to be involved in disulfide bond formation, are also major sites of both S-nitrosylation and S-glutathionylation. Using selective isotopecoded affinity tag labeling of RyR1 and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy, we identified, out of the 100 cysteines in each RyR1 subunit, 9 that are endogenously modified (Cys-36, Cys-315, Cys-811, Cys-906, Cys-1591, Cys-2326, Cys-2363, Cys-3193, and Cys-3635) and another 3 residues that were only modified with exogenous redox agents (Cys-253, Cys-1040, and Cys-1303). We also identified the types of redox modification each of these cysteines can undergo. In summary, we have identified a discrete subset of cysteines that are likely to be involved in the functional response of RyR1 to different redox modifications (S-nitrosylation, S-glutathionylation, and oxidation to disulfides).