Nitrosylation of RyR2 Prevents Activation of Ca Waves Induced by Redox-Mediated Intersubunit Cross-Linking

Abstract

We have recently discovered a novel post-translational modification of type-2 ryanodine receptor (cardiac RyR2) induced by oxidative stress: intersubunit cross-linking (XL). Because RyR gating is associated with large-scale intersubunit dynamics, we hypothesized that XL is functionally the most important redox modification of RyR2. We have developed a simple cell model system to define the functional importance of RyR2 XL. We found that co-expressing recombinant human RyR2 and the SR Ca-ATPase (SERCA2a) in HEK293 cells generates Ca oscillations with similar kinetics to cardiac Ca waves. The frequency of these “cardiac-like” Ca waves was significantly increased in the presence of oxidants, and this effect was associated with RyR2 XL. By analyzing intra-luminal [Ca] dynamics, we concluded that activation of Ca waves by oxidants was due to increased RyR2-mediated Ca leak (but not Ca uptake). Pretreating HEK293 cells co-expressing RyR2/SERCA2a with nitric oxide (NO) donors (30 μM GSNO or SNAP) effectively prevented RyR2 XL and the augmentation of Ca waves induced by oxidants. NO donors by themselves did not affect Ca waves in control conditions. It has been shown in the homologous type-1 RyR (skeletal RyR1) that cysteine 3635 is involved in intersubunit XL as well as in S-nitrosylation. Accordingly, we tested whether mutation of the homologous cysteine in cardiac RyR2 (Cys-3602) abolishes XL. We found that mutation of this cysteine to alanine did not prevent intersubunit XL or the augmentation of Ca waves during oxidative stress. Thus, despite high homology between RyR1 and RyR2, it appears that these channels exhibit important structural and functional differences in redox regulation. Importantly, these results illustrate that S-nitrosylation of RyR2 can be an effective approach to prevent RyR2 XL and, thus, SR Ca mishandling during oxidative stress.