*Libin Cardiovascular Institute of Alberta, Department of Physiology & Pharmacology, University of Calgary, Calgary, Alberta, Canada, T2N 4N1.
Biochem J. 2013 Nov 1;455(3):367-75. doi: 10.1042/BJ20130805.
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.
肌质网 Ca2+ 含量耗竭至一个阈值水平时，RyR2（心脏兰尼碱受体）介导的 Ca2+ 释放终止，该阈值被称为终止阈值。尽管其重要性不言而喻，但调控终止阈值的机制却鲜为人知。CaM（钙调蛋白）通过抑制 RyR2 参与 Ca2+ 释放终止，但 CaM 是否调节终止阈值尚不清楚。为此，我们在转染 WT（野生型）CaM 或突变体的表达 RyR2 的 HEK（人胚肾）-293 细胞中监测内质网 Ca2+ 动力学。我们发现，WT CaM 或丧失与 CaM 的 N 结构域（N 端结构域）结合 Ca2+ 的 CaM 突变增加了终止阈值（即促进终止），但对自发 Ca2+ 释放发生的激活阈值没有影响。另一方面，降低与 N 结构域和 C 结构域（C 端结构域）或仅 C 结构域结合 Ca2+ 的 CaM 突变降低了终止阈值（即延迟终止），但激活阈值相似。此外，已知废除或保留 CaM 结合的 RyR2 的 CaM 结合域中的残基 3583-3603 或点突变（W3587A/L3591D/F3603A、W3587A 或 L3591D）均降低了终止阈值，而对激活阈值没有显著影响。有趣的是，RyR2-F3603A 突变影响激活和终止阈值。总的来说，这些数据表明 CaM 通过增加终止阈值促进 Ca2+ 释放的终止，而 CaM 的这种作用依赖于 Ca2+ 与 CaM 的 C 结构域的结合，但不依赖于 N 结构域。本研究的结果还表明，RyR2 的 CaM 结合域是 Ca2+ 释放终止和激活的重要决定因素。
