The LQT-associated calmodulin mutant E141G induces disturbed Ca²⁺-dependent binding and a flickering gating mode of the Ca_V1.2 channel.

Calmodulin (CaM) mutations are associated with congenital long QT (LQT) syndrome (LQTS), which may be related to the dysregulation of the cardiac-predominant Ca²⁺ channel isoform Ca_V1.2. Among various mutants, CaM-E141G was identified as a critical missense variant. However, the interaction of this CaM mutant with the Ca_V1.2 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 Ca_V1.2 channel. Using the patch-clamp technique, we also investigated the electrophysiological effects of the mutant on Ca_V1.2 channel activity. We found that the maximum binding (B_max) of CaM-E141G to the proximal COOH-terminal region, PreIQ-IQ, PreIQ, IQ, and NT (an NH₂-terminal peptide) was decreased (by 17.71-59.26%) compared with that of wild-type CaM (CaM-WT). In particular, the Ca²⁺-dependent increase in B_max 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 Ca²⁺ decreased Ca_V1.2 channel activity to 24.88% and 55.99%, respectively, compared with 100 nM Ca²⁺, showing that the inhibitory effect was attenuated in CaM-E141G. The mean open time of the Ca_V1.2 channel was increased, and the number of blank traces with no channel opening was significantly decreased. Overall, CaM-E141G exhibits disrupted binding with the Ca_V1.2 channel and induces a flickering gating mode, which may result in the dysfunction of the Ca_V1.2 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 Ca_V1.2 channel.

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