Calmodulin Regulation of Ryanodine Receptors (RyR2) Depends on RyR2 Phosphorylation at Residues S2808 and S2814

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Introduction: Calmodulin (CaM) is a calcium-binding protein that regulates target proteins directly or via their regulatory kinases. CaM binds RyR2 and forms part of the RyR2 macromolecular complex that includes Protein Kinase A (PKA), CaM-dependent kinase II (CaMKII) and the phosphatases, PP1 and PP2A. In the complex, CaM influences SR Ca2+ release either by directly binding RyR2 (inhibition) or by phosphorylation of the RyR2 at S2814 through CaMKII (activation). CaM binding to RyR2 is critical for normal cardiac function and loss of CaM inhibiting efficacy leads to pathological activation of RyR2 that produce spontaneous Ca2+ releases from the SR. These in themselves can trigger miss-timed APs, cardiac arrhythmias and sudden death. Several recent studies have reported that CaM binding affinity to RyR2 is reduced by oxidation of both CaM and RyR2. Here we test the hypothesis that the action of CaM on the activity of RyR2 also depends on the phosphorylation state of the channel. Methods and Results: CaM effect on Ca2+ sparks (and hence RyR2 activity) was tested on permeabilized cardiomyocytes from transgenic mice where CaMKII or PKA phosphorylation sites in RyR2 were ablated (S2814A and S2808A, respectively) or where CaMKII phosphorylation site in RyR2 was fully active (S2814D). RyR2 phosphorylation was confirmed by Western Blot. CaM only inhibited Ca2+ sparks frequency in cardiomyocytes with high phosphorylation levels of RyR2 in CaMKII site (S2814D), in PKA site (S2814A+cAMP or S2808A+cAMP) or in both residues (RyR2 S2814D+cAMP). In contrast, CaM did not have an inhibitory effect in cardiomyocytes with low phosphorylation levels of RyR2 (S2814A and S2808A). Conclusion: Our results show that CaM inhibitory efficacy is dependent on RyR2 phosphorylation in CaMKII and PKA sites S2814 and S2808 in mouse ventricular myocytes.