The LQT-associated calmodulin mutant E141G induces disturbed Ca2+-dependent binding and a vibration-like gating mode of the CaV1.2 channel.
The LQT-associated calmodulin mutant E141G induces disturbed Ca2+-dependent binding and a vibration-like gating mode of the CaV1.2 channel.
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DOI：
10.1152/ajpcell.00019.2020
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发表时间：
2020-03
期刊：
影响因子：
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通讯作者：
Jingyang Su;Qinghua Gao;Lifeng Yu;Xuanxuan Sun;Rui Feng;Dongxue Shao;Yuan Yuan-Yuan;Zhengnan Zhu;Xuefei Sun;M. Kameyama;Liying Hao
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文献类型：
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作者：
Jingyang Su;Qinghua Gao;Lifeng Yu;Xuanxuan Sun;Rui Feng;Dongxue Shao;Yuan Yuan-Yuan;Zhengnan Zhu;Xuefei Sun;M. Kameyama;Liying Hao
钙调蛋白（CaM）突变与先天性长QT综合征（LQTS）有关，这可能与心脏主要Ca2+通道异构体CaV1.2的失调有关。在各种突变体中，CaM-E141G被鉴定为关键错义变体。然而，它与CaV1.2通道的相互作用仍有待研究。在这项研究中，我们利用半定量下拉实验，探索了CaM-E141G与CaV1.2中cam结合肽片段的相互作用。我们还利用膜片钳技术研究了其对CaV1.2通道活性的电生理影响。结果表明，与野生型CaM- wt相比，CaM- e141g与近c端区最大结合量（Bmax）、PreIQ-IQ、PreIQ、IQ和NT （n端肽）均降低（降低17.71 ~ 59.26%）。特别是，当CaM- e141g与PreIQ或NT结合时，Ca2+依赖性的增加转变为Ca2+依赖性的降低。在功能上，CaM- e141g在500 nM Ca2+下降低了Ca2+/CaM对CaV1.2 （CDI）活性的抑制作用55.6%。同时，CaV1.2门控平均开启时间延长，无通道开启的空白走线数量显著减少。这些结果支持CaM-E141G与心脏CaV1.2通道的结合受到干扰，并诱导类似振动的门控模式，这可能导致CaV1.2功能障碍，从而导致LQTS的发展。本研究首次对CaM-E141G与CaV1.2相互作用诱导的单通道门控模式和结合特性进行了详细的研究。
Calmodulin (CaM) mutations are associated with congenital long QT syndrome (LQTS), which may be related to the dysregulation of the cardiac predominant Ca2+ channel isoform CaV1.2. Among various mutants, CaM-E141G was identified as a critical missense variant. However, its interaction with the CaV1.2 channel remains to be examined. In this study, by utilizing a semiquantitative pull-down assay, we explored the interaction of CaM-E141G with CaM-binding peptide fragments of CaV1.2. We also investigated its electrophysiological effects on CaV1.2 channel activity with the patch clamp technique. We found that the maximum binding (Bmax) of CaM-E141G to the proximal C-terminal region, PreIQ-IQ, PreIQ, IQ and NT (a N-terminal peptide) were all decreased (by 17.71-59.26%), compared to that of wild type CaM (CaM-WT). In particular, the Ca2+-dependent increase in the binding affinity was changed to a Ca2+-dependent decrease in the presence of a combination of CaM-E141G and PreIQ or NT. Functionally, CaM-E141G attenuated the inhibitory effects of Ca2+/CaM on the activity of CaV1.2 (CDI) by 55.6% at 500 nM Ca2+. Meanwhile the mean open time of CaV1.2 gating was elongated and the number of blank traces with no channel opening was significantly decreased. These results support the view that CaM-E141G shows disturbed binding with the cardiac CaV1.2 channel and induces a vibration-like gating mode, which may result in the dysfunction of CaV1.2 and thereby the development of LQTS. The present study, for the first time, investigated the detailed binding properties and single channel gating mode induced by the interaction of CaM-E141G with CaV1.2.