Department of Physiology and Pharmacology, Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, Alberta, Canada T2N 4N1.
Biochem J. 2013 Aug 15;454(1):123-31. doi: 10.1042/BJ20130594.
CPVT (catecholaminergic polymorphic ventricular tachycardia) is an inherited life-threatening arrhythmogenic disorder. CPVT is caused by DADs (delayed after-depolarizations) that are induced by spontaneous Ca2+ release during SR (sarcoplasmic reticulum) Ca2+ overload, a process also known as SOICR (store-overload-induced Ca2+ release). A number of mutations in the cardiac ryanodine receptor RyR2 are linked to CPVT. Many of these CPVT-associated RyR2 mutations enhance the propensity for SOICR and DADs by sensitizing RyR2 to luminal or luminal/cytosolic Ca2+ activation. Recently, a novel CPVT RyR2 mutation, G230C, was found to increase the cytosolic, but not the luminal, Ca2+ sensitivity of single RyR2 channels in lipid bilayers. This observation led to the suggestion of a SOICR-independent disease mechanism for the G230C mutation. However, the cellular impact of this mutation on SOICR is yet to be determined. To this end, we generated stable inducible HEK (human embryonic kidney)-293 cell lines expressing the RyR2 WT (wild-type) and the G230C mutant. Using single-cell Ca2+ imaging, we found that the G230C mutation markedly enhanced the propensity for SOICR and reduced the SOICR threshold. Furthermore, the G230C mutation increased the sensitivity of single RyR2 channels to both luminal and cytosolic Ca2+ activation and the Ca2+-dependent activation of [3H]ryanodine binding. In addition, the G230C mutation decreased the thermal stability of the N-terminal region (amino acids 1-547) of RyR2. These data suggest that the G230C mutation enhances the propensity for SOICR by sensitizing the channel to luminal and cytosolic Ca2+ activation, and that G230C has an intrinsic structural impact on the N-terminal domains of RyR2.
儿茶酚胺敏感性多形性室性心动过速（CPVT）是一种遗传性致命性心律失常疾病。CPVT 是由 DAD（延迟后除极）引起的，这些 DAD 是由 SR（肌浆网）Ca2+超载时自发的 Ca2+释放引起的，这个过程也称为 SOICR（储存超载诱导的 Ca2+释放）。心脏兰尼碱受体 RyR2 的许多突变与 CPVT 有关。这些 CPVT 相关的 RyR2 突变通过使 RyR2 对腔或腔/细胞质 Ca2+激活敏感，增加 SOICR 和 DAD 的倾向。最近，发现一种新的 CPVT RyR2 突变 G230C 增加了单个 RyR2 通道在脂质双层中的细胞质而非腔 Ca2+敏感性。这一观察结果导致了对 G230C 突变的 SOICR 不依赖疾病机制的建议。然而，这种突变对 SOICR 的细胞影响尚未确定。为此，我们生成了稳定诱导的表达 RyR2 WT（野生型）和 G230C 突变体的 HEK（人胚肾）-293 细胞系。使用单细胞 Ca2+成像，我们发现 G230C 突变显著增加了 SOICR 的倾向，并降低了 SOICR 的阈值。此外，G230C 突变增加了单个 RyR2 通道对腔和细胞质 Ca2+激活以及[3H]兰尼碱结合的 Ca2+依赖性激活的敏感性。此外，G230C 突变降低了 RyR2 的 N 端区域（氨基酸 1-547）的热稳定性。这些数据表明，G230C 突变通过使通道对腔和细胞质 Ca2+激活敏感，增加了 SOICR 的倾向，并且 G230C 对 RyR2 的 N 端结构域具有内在的结构影响。
