The central domain of cardiac ryanodine receptor governs channel activation, regulation, and stability
The central domain of cardiac ryanodine receptor governs channel activation, regulation, and stability
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DOI：
10.1074/jbc.ra120.013512
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发表时间：
2020-11-13
影响因子：
4.8
通讯作者：
Chen, S. R. Wayne
中科院分区：
文献类型：
--
作者：
Guo, Wenting;Sun, Bo;Chen, S. R. Wayne
结构分析确定了兰尼碱受体（RyR）的中央结构域作为转换器，将细胞质平台中的构象变化转换为RyR门。中央结构域也是一个调控中心，包括Ca 2 +-，ATP-和咖啡因结合位点。然而，RyR的激活和调节的中心域的作用还没有被定义。在这里，我们突变的5个残基，形成Ca 2+激活位点和10个残基带负电荷或含氧的侧链附近的Ca 2+激活位点。我们还在RyR 2的中心结构域中产生了8个疾病相关突变。我们确定了这些突变对Ca 2+，ATP和咖啡因激活和Mg 2+抑制RyR 2的影响。Ca 2+激活位点的突变显著降低了RyR 2对Ca 2+和咖啡因激活的敏感性。出乎意料的是，Ca 2+激活位点突变E3848 A显著增强了RyR 2的Ca 2+非依赖性基础活性，表明E3848 A也可能影响RyR 2闭合状态的稳定性。Ca 2+激活位点的突变也消除了ATP/咖啡因对Ca 2+非依赖性基础活性的影响，表明Ca 2+激活位点也是ATP/咖啡因作用的关键决定因素。突变残基带负电荷或含氧的侧链附近的Ca 2+激活位点显着改变Ca 2+和咖啡因的激活和降低Mg 2+的抑制。此外，中央区域内的疾病相关RyR 2突变显著增强了Ca 2+和咖啡因的激活，降低了Mg 2+的抑制。我们的数据表明，中央域在通道激活，通道调节和封闭状态稳定性中起着重要作用。
Structural analyses identified the central domain of ryanodine receptor (RyR) as a transducer converting conformational changes in the cytoplasmic platform to the RyR gate. The central domain is also a regulatory hub encompassing the Ca2+-, ATP-, and caffeine-binding sites. However, the role of the central domain in RyR activation and regulation has yet to be defined. Here, we mutated five residues that form the Ca2+ activation site and 10 residues with negatively charged or oxygen-containing side chains near the Ca2+ activation site. We also generated eight disease-associated mutations within the central domain of RyR2. We determined the effect of these mutations on Ca2+, ATP, and caffeine activation and Mg2+ inhibition of RyR2. Mutating the Ca2+ activation site markedly reduced the sensitivity of RyR2 to Ca2+ and caffeine activation. Unexpectedly, Ca2+ activation site mutation E3848A substantially enhanced the Ca2+-independent basal activity of RyR2, suggesting that E3848A may also affect the stability of the closed state of RyR2. Mutations in the Ca2+ activation site also abolished the effect of ATP/caffeine on the Ca2+-independent basal activity, suggesting that the Ca2+ activation site is also a critical determinant of ATP/caffeine action. Mutating residues with negatively charged or oxygen-containing side chains near the Ca2+ activation site significantly altered Ca2+ and caffeine activation and reduced Mg2+ inhibition. Furthermore, disease-associated RyR2 mutations within the central domain significantly enhanced Ca2+ and caffeine activation and reduced Mg2+ inhibition. Our data demonstrate that the central domain plays an important role in channel activation, channel regulation, and closed state stability.