Calmodulin modulates the termination threshold for cardiac ryanodine receptor-mediated Ca²⁺ release

RyR2 (cardiac ryanodine receptor)-mediated Ca²⁺ release in cardiomyocytes terminates when the sarcoplasmic reticulum Ca²⁺ content depletes to a threshold level, known as the termination threshold. Despite its importance, little is known about the mechanism that regulates the termination threshold. CaM (calmodulin), by inhibiting RyR2, has been implicated in Ca²⁺-release termination, but whether CaM modulates the termination threshold is unknown. To this end, we monitored the endoplasmic reticulum Ca²⁺ dynamics in RyR2-expressing HEK (human embryonic kidney)-293 cells transfected with WT (wild-type) CaM or mutants. We found that WT CaM or CaM mutations which abolish Ca²⁺ binding to the N-lobe (N-terminal lobe) of CaM increased the termination threshold (i.e. facilitated termination), but had no effect on the activation threshold at which spontaneous Ca²⁺ release occurs. On the other hand, CaM mutations that diminish Ca²⁺ binding to both the N-lobe and C-lobe (C-terminal lobe), or the C-lobe only, decreased the termination threshold (i.e. delayed termination) with a similar activation threshold. Furthermore, deletion of residues 3583–3603 or point mutations (W3587A/L3591D/F3603A, W3587A, or L3591D) in the CaM-binding domain of RyR2 that are known to abolish or retain CaM binding all reduced the termination threshold without having a significant impact on the activation threshold. Interestingly, the RyR2-F3603A mutation affected both the activation and termination threshold. Collectively, these data indicate that CaM facilitates the termination of Ca²⁺ release by increasing the termination threshold, and that this action of CaM depends on Ca²⁺ binding to the C-lobe, but not to the N-lobe, of CaM. The results of the present study also suggest that the CaM-binding domain of RyR2 is an important determinant of Ca²⁺-release termination and activation.