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

Abstract

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 wildtype 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). Ca 2ϩ

NADPH oxidases in lung biology and pathology: Host defense enzymes, and more

“…NOX-dependent Ca 2+ signaling may be mediated by increased voltage-dependent Ca 2+ channel opening, as has been demonstrated in angiotensin II signaling in neural cells (50), in B cell receptor-mediated signaling (51), and during NOX-mediated plant root hair outgrowth (52). In addition, NOXmediated ROS may also induce oxidative cysteine modifications within Ca 2+ channel, as was demonstrated for with respect to activation of the ryanodine receptor (53,54).…”

Intracellular Cysteine 346 Is Essentially Involved in Regulating Panx1 Channel Activity

“…Examples of ion channels, which were found to be regulated by modulation of cysteines, are the olfactory cyclic nucleotide-gated channel (39,40), the ryanodine receptor type 1 (41,42), Na ϩ channels (43), and the NMDA receptor (44,45). To uncover putative mechanisms of hemichannel regulation of Panx1, we investigated the role of single intracellular cysteine residues.…”

How widespread is stable protein S-nitrosylation as an end-effector of protein regulation?

“…Several reviews have attempted to 'pull apart' the complex oxidative regulation of RyR1. Out of 101 cysteines RyR1 contains at least 12 known to be either S-nitrosated, S-glutathiolated or involved in the formation of inter-or intramolecular disulfides [121]. Though all forms of oxidative modification were associated with channel opening, S-nitrosation is thought to enhance Ca 2+ activation, whereas S-glutathiolation decreased the inhibitory effect of Mg 2+ without altering Ca 2+ activation [116].…”