The Role of Cysteine 3602 in RyR2 Regulation by Calmodulin and Oxidative Stress

Abstract

In the skeletal ryanodine receptor (RyR1), cysteine 3635 (C3635) is considered to be an important residue responsible for the redox regulation of the channel. It has been shown that oxidation of C3635 causes intersubunit cross-linking and calmodulin (CaM) dissociation from RyR1. These structural modifications can lead to channel activation. While the role of RyR1 C3635 has been extensively studied, the functional significance of the corresponding cysteine 3602 (C3602) in the cardiac RyR (RyR2) remains unclear. To characterize the role of C3602 in RyR2 response to oxidative stress, we co-expressed either wild type (WT) GFP-RyR2 or the single mutant C3602A GFP-RyR2, together with the ER-targeted Ca sensor R-CEPIA1er and SERCA2a, in HEK293 cells. Cells transfected with either WT- or C3602A-RyR2 produced Ca waves that were sensitive to oxidative stress induced by diamide. Diamide equally increased the endoplasmic reticulum (ER) Ca leak in the cells expressing WT- and the C3602A-RyR2 mutant. Both WT-RyR2 and the mutant RyR2 expressed in HEK293 cells demonstrated the same level of disulfide cross-linking upon diamide treatment. Moreover, the nitric oxide donor NOC-12 had similar effect on ER Ca leak via WT and the mutant RyR2. Given the localization of C3602 within the CaM binding site in RyR2, we assessed the effect of C3602A mutation on the degree of CaM dissociation during oxidative stress. The C3602A mutation did not significantly affect CaM binding to RyR2 during oxidative stress, and the degree of this binding was comparable to the one observed in WT RyR2. Taken together, these results suggest that C3602 does not play a key role in RyR2 response to oxidative stress. Despite ∼70% homology between RyR1 and RyR2, these channels appear to exhibit important structural and functional differences, particularly with respect to redox regulation.