Nitric oxide, NOC-12, and S-nitrosoglutathione modulate the skeletal muscle calcium release channel/ryanodine receptor by different mechanisms: An allosteric function for O2 in S-nitrosylation of the channel

Abstract

The skeletal muscle Ca2+ release channel/ryanodine receptor (RyR1) contains ∼50 thiols per subunit. These thiols have been grouped according to their reactivity/ responsiveness toward NO, O2, and glutathione, but the molecular mechanism enabling redox active molecules to modulate channel activity is poorly understood. In the case of NO, very low concentrations (submicromolar) activate RyR1 by S-nitrosylation of a single cysteine residue (Cys-3635), which resides within a calmodulin binding domain. S-Nitrosylation of Cys-3635 only takes place at physiological tissue O2 tension (PO2; i.e. ∼10 mm Hg) but not at pO2 ∼150 mm Hg. Two explanations have been offered for the loss of RyR1 responsiveness to NO at ambient pO2, i.e. Cys-3635 is oxidized by O2 versus O2 subserves an allosteric function (Eu, J. P., Sun, J. H., Xu, L., Stamler, J. S., and Meissner, G. (2000) Cell 102, 499-509). Here we report that the NO donors NOC-12 and S-nitrosoglutathione both activate RyR1 by release of NO but do so independently of pO2. Moreover, NOC-12 activates the channel by S-nitrosylation of Cys-3635 and thereby reverses channel inhibition by calmodulin. In contrast, S-nitrosoglutathione activates RyR1 by oxidation and S-nitrosylation of thiols other than Cys-3635 (and calmodulin is not involved). Our results suggest that the effect of pO2 on RyR1 S-nitrosylation is exerted through an allosteric mechanism.