Department of Pharmaceutical Toxicology, School of Pharmacy, China Medical University, Shenyang, China.
Department of Physiology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.
Am J Physiol Cell Physiol. 2020 May 1;318(5):C991-C1004. doi: 10.1152/ajpcell.00019.2020. Epub 2020 Mar 18.
Calmodulin (CaM) mutations are associated with congenital long QT (LQT) syndrome (LQTS), which may be related to the dysregulation of the cardiac-predominant Ca channel isoform Ca1.2. Among various mutants, CaM-E141G was identified as a critical missense variant. However, the interaction of this CaM mutant with the Ca1.2 channel has not been determined. In this study, by utilizing a semiquantitative pull-down assay, we explored the interaction of CaM-E141G with CaM-binding peptide fragments of the Ca1.2 channel. Using the patch-clamp technique, we also investigated the electrophysiological effects of the mutant on Ca1.2 channel activity. We found that the maximum binding (B) of CaM-E141G to the proximal COOH-terminal region, PreIQ-IQ, PreIQ, IQ, and NT (an NH-terminal peptide) was decreased (by 17.71-59.26%) compared with that of wild-type CaM (CaM-WT). In particular, the Ca-dependent increase in B became slower with the combination of CaM-E141G + PreIQ and IQ but faster in the case of NT. Functionally, CaM-WT and CaM-E141G at 500 nM Ca decreased Ca1.2 channel activity to 24.88% and 55.99%, respectively, compared with 100 nM Ca, showing that the inhibitory effect was attenuated in CaM-E141G. The mean open time of the Ca1.2 channel was increased, and the number of blank traces with no channel opening was significantly decreased. Overall, CaM-E141G exhibits disrupted binding with the Ca1.2 channel and induces a flickering gating mode, which may result in the dysfunction of the Ca1.2 channel and, thus, the development of LQTS. The present study is the first to investigate the detailed binding properties and single-channel gating mode induced by the interaction of CaM-E141G with the Ca1.2 channel.
钙调蛋白（CaM）突变与先天性长 QT 综合征（LQTS）有关，这可能与心脏优势型 Ca 通道同工型 Ca1.2 的调节失常有关。在各种突变体中，CaM-E141G 被确定为关键的错义变异体。然而，这种 CaM 突变体与 Ca1.2 通道的相互作用尚未确定。在这项研究中，我们利用半定量下拉测定法，探讨了 CaM-E141G 与 Ca1.2 通道的 CaM 结合肽片段的相互作用。我们还使用膜片钳技术研究了突变对 Ca1.2 通道活性的电生理影响。我们发现，与野生型 CaM（CaM-WT）相比，CaM-E141G 与近端 COOH-末端区域 PreIQ-IQ、PreIQ、IQ 和 NT（NH-末端肽）的最大结合（B）降低了（17.71%-59.26%）。特别是，CaM-E141G+PreIQ 和 IQ 的组合使 Ca 依赖性 B 的增加变得更慢，但 NT 的情况则更快。在功能上，500 nM Ca 下的 CaM-WT 和 CaM-E141G 将 Ca1.2 通道活性分别降低至 24.88%和 55.99%，与 100 nM Ca 相比，表明 CaM-E141G 的抑制作用减弱。Ca1.2 通道的平均开放时间增加，无通道开放的空白痕迹数显著减少。总体而言，CaM-E141G 表现出与 Ca1.2 通道结合的破坏，并诱导闪烁门控模式，这可能导致 Ca1.2 通道功能障碍，从而导致 LQTS 的发生。本研究首次探讨了 CaM-E141G 与 Ca1.2 通道相互作用引起的详细结合特性和单通道门控模式。
