CaMKII-Dependent Phosphorylation of RyR2 Causes Domain Unzipping and Reduced Calmodulin Binding, But Dantrolene Reverses These Effects

Abstract

Increased diastolic SR Ca2+ (Ca) leak via cardiac ryanodine receptors (RyR2) can cause cardiac arrhythmias and dysfunction in heart failure (HF). CaMKII-dependent RyR2 phosphorylation (at S2814) is thought to be a critical promoter of leaky RyR2 in HF. A conformational change in RyR2 (“domain unzipping”) observed in HF can also increase SR Ca leak. Calmodulin (CaM) quiets RyR2 gating, and the CaM affinity for RyR2 is reduced in HF. Further, we have shown reciprocal negative coupling between CaM binding to RyR2 and accessibility of the unzipping RyR peptide DPc10 (Oda et al. 2013). That is, unzipping with DPc10 reduced CaM binding and increased Ca leak, whereas CaM quieted leak and inhibited DPc10 access. Here, we tested the hypothesis that RyR2 phosphorylation by CaMKII increases Ca leak by favoring the same unzipped-conformation that exhibits reduced CaM affinity. We either activated endogenous myocyte CaMKII or used knock-in mice expressing non-phosphorylatable RyR2-S2814A or phosphomimetic RyR2-S2814D. We used FRET to directly detect binding of fluorescently labeled DPc10, FKBP12.6, and CaM to RyR2 in permeabilized cardiomyocytes. In phosphomimetic S2814D vs. S2814A myocytes, CaM-RyR2 affinity was reduced 3-fold (Kd= 72 ± 9 nM vs. 20 ± 2 nM), but FRETmax was unaltered. Access of DPc10 was also 2-fold faster in S2814D versus S2814A myocytes. Dantrolene (1 μM), which stabilizes the zipped conformation, restored CaM-RyR2 binding affinity in S2814D, slowed DPc10 wash-in, and lowered Bmax. Forced RyR2-S2814D saturation with 500 nM CaM also slowed DPc10 wash-in and reduced Bmax. We conclude that RyR2 phosphorylation by CaMKII causes pathological conformation changes (unzipping) that reduce RyR2-CaM affinity and ability to quiet SR Ca leak. Dantrolene effectively reverses these effects.