PKA (protein kinase A)–mediated phosphorylation of cardiac RyR2 (ryanodine receptor 2) has been extensively studied for decades, but the physiological significance of PKA phosphorylation of RyR2 remains poorly understood. Recent determination of high-resolution 3-dimensional structure of RyR2 in complex with CaM (calmodulin) reveals that the major PKA phosphorylation site in RyR2, serine-2030 (S2030), is located within a structural pathway of CaM-dependent inactivation of RyR2. This novel structural insight points to a possible role of PKA phosphorylation of RyR2 in CaM-dependent inactivation of RyR2, which underlies the termination of Ca2+ release and induction of cardiac Ca2+ alternans. To determine the role of the PKA phosphorylation site RyR2-S2030 in Ca2+ release termination in vitro and cardiac Ca2+ alternans in intact hearts.

We performed single-cell endoplasmic reticulum Ca2+ imaging to assess the impact of S2030 mutations on Ca2+ release termination in HEK293 cells. We found that mutations, S2030D, S2030G, S2030L, S2030V, and S2030W reduced the endoplasmic reticulum luminal Ca2+ level at which Ca2+ release terminates (the termination threshold), whereas S2030P and S2030R increased the termination threshold. S2030A and S2030T had no significant impact on release termination. Furthermore, CaM–wild-type increased, whereas Ca2+ binding deficient CaM mutant (CaM-M [a loss-of-function CaM mutation with all 4 EF-hand motifs mutated]), PKA, and Ca2+/CaMKII (CaM-dependent protein kinase II) reduced the termination threshold. The S2030L mutation abolished the actions of CaM–wild-type, CaM-M, and PKA, but not CaMKII, in Ca2+ release termination. To determine the role of the PKA site RyR2-S2030 in a physiological setting, we generated a novel mouse model harboring the S2030L mutation. Using confocal Ca2+ imaging, we found that isoproterenol and CaM-M suppressed pacing-induced Ca2+ alternans and accelerated Ca2+ transient recovery in intact working hearts, whereas CaM–wild-type exerted an opposite effect. The impact of isoproterenol was partially and fully reversed by the PKA inhibitor H89 and the CaMKII inhibitor KN93 individually and together, respectively. S2030L abolished the impact of CaM–wild-type, CaM-M, and H89-sensitive component, but not the KN93-sensitive component, of isoproterenol.

These data demonstrate, for the first time, that the PKA phosphorylation site RyR-S2030 is an important determinant of PKA-regulated, CaM-dependent Ca2+ release termination, and Ca2+ alternans.