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2289126 
Journal Article 
Oxygen-coupled Redox Regulation of the Skeletal Muscle Ryanodine Receptor/Ca2+ Release Channel (RyR1) SITES AND NATURE OF OXIDATIVE MODIFICATION 
Sun, QiAn; Wang, B; Miyagi, M; Hess, DT; Stamler, JS 
2013 
Yes 
Journal of Biological Chemistry
ISSN: 0021-9258
EISSN: 1083-351X 
288 
32 
22961-22971 
23798702 
WOS:000330598200007 
In mammalian skeletal muscle, Ca2+ release from the sarcoplasmic reticulum (SR) through the ryanodine receptor/Ca2+-release channel RyR1 can be enhanced by S-oxidation or S-nitrosylation of separate Cys residues, which are allosterically linked. S-Oxidation of RyR1 is coupled to muscle oxygen tension (pO(2)) through O-2-dependent production of hydrogen peroxide by SR-resident NADPH oxidase 4. In isolated SR (SR vesicles), an average of six to eight Cys thiols/RyR1 monomer are reversibly oxidized at high (21% O-2) versus low pO(2) (1% O-2), but their identity among the 100 Cys residues/RyR1 monomer is unknown. Here we use isotope-coded affinity tag labeling and mass spectrometry (yielding 93% coverage of RyR1 Cys residues) to identify 13 Cys residues subject to pO(2)-coupled S-oxidation in SR vesicles. Eight additional Cys residues are oxidized at high versus low pO(2) only when NADPH levels are supplemented to enhance NADPH oxidase 4 activity. pO(2)-sensitive Cys residues were largely non-overlapping with those identified previously as hyperreactive by administration of exogenous reagents (three of 21) or as S-nitrosylated. Cys residues subject to pO(2)-coupled oxidation are distributed widely within the cytoplasmic domain of RyR1 in multiple functional domains implicated in RyR1 activity-regulating interactions with the L-type Ca2+ channel (dihydropyridine receptor) and FK506-binding protein 12 as well as in "hot spot" regions containing sites of mutation implicated in malignant hyperthermia and central core disease. pO(2)-coupled disulfide formation was identified, whereas neither S-glutathionylated nor sulfenamide-modified Cys residues were observed. Thus, physiological redox regulation of RyR1 by endogenously generated hydrogen peroxide is exerted through dynamic disulfide formation involving multiple Cys residues.