Identification of cysteines involved in S-nitrosylation, S-glutathionylation, and oxidation to disulfides in ryanodine receptor type 1.
The skeletal muscle Ca(2+)-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, H(2)O(2) treatment of C3635A-RyR1 channels or wild-type RyR1, following their expression in human embryonic kidney cells, enhances [(3)H]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).
Aracena-Parks
Paula
P
Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030, USA.
Goonasekera
Sanjeewa A
SA
Gilman
Charles P
CP
Dirksen
Robert T
RT
Hidalgo
Cecilia
C
Hamilton
Susan L
SL
D000818:Animals; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008297:Male; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000378:metabolism; D011817:Rabbits; D026361:Reactive Nitrogen Species / Q000737:chemistry / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D004220:Disulfides; D010446:Peptide Fragments; D026361:Reactive Nitrogen Species; D019837:Ryanodine Receptor Calcium Release Channel; D014357:Trypsin; D005978:Glutathione; D003545:Cysteine