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

Abstract

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 CaV1.2. Among various mutants, CaM-E141G was identified as a critical missense variant. However, the interaction of this CaM mutant with the CaV1.2 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 CaV1.2 channel. Using the patch-clamp technique, we also investigated the electrophysiological effects of the mutant on CaV1.2 channel activity. We found that the maximum binding (Bmax) 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 Bmax 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 CaV1.2 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 CaV1.2 channel was increased, and the number of blank traces with no channel opening was significantly decreased. Overall, CaM-E141G exhibits disrupted binding with the CaV1.2 channel and induces a flickering gating mode, which may result in the dysfunction of the CaV1.2 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 CaV1.2 channel.

Calmodulin variant E140G associated with long QT syndrome impairs CaMKIIδ autophosphorylation and L-type calcium channel inactivation

“…We demonstrated a significant reduction in the CDI of Ca v 1.2 when CaM-E140G was present, which could be the result of a combination of defective Ca 2+ sensing and altered interaction with the channel binding domains. This observation is in line with previously published data on LQTS-associated CaM mutants, implying a potential common mechanism of disease involving disrupted Ca v 1.2 inactivation ( 11 , 14 , 18 , 82 , 83 ). The defect in Ca v 1.2 inactivation will elongate the ventricular action potential (AP), which would result in an increase of the QT interval, characteristic of LQTS.…”

“…These data suggest that the E140G mutation, while located in the C-lobe, induces global structural rearrangements which reduces CaM binding to NSCaTE and therefore could contribute to defects in Ca v 1.2 CDI. We measured a 2-fold increase in affinity for Ca 2+ /CaM-E140G–Ca v 1.2-IQ 1665-1685 when compared to WT, which differs from the ∼3-fold reduction in affinity previously observed by GST-pull down assay ( 82 ). However, using HEK293 cells and a FRET biosensor, Limpitikul et al.…”

Precision medicine for long QT syndrome: patient-specific iPSCs take the lead

“…Most mutations are located in calmodulin's C-terminal Ca2 + -binding domains (EF-hands III and IV), particularly in the residues directly involved in Ca2 + binding. A plethora of evidence indicate that LQTS relevant calmodulin mutations attenuate Ca2 + binding affinity and result in a defective Ca2 + -dependent inactivation of Cav1.2, causing an increased and uncontrolled Ca2 + influx (Refs 88, 89). The mutation locations in CALM1-3 reveals special topological domains that are susceptible to genetic variation and imply the significance of Ca2 + -binding affinity for proper function of calmodulin.…”

Regulation of Cardiac Cav1.2 Channels by Calmodulin

“…Mutations of CaM have been also reported to affect CDI (reviewed by Crotti et al [ 131 ]). Among them, mutations of C-lobe are most frequently found, e.g., E141G [ 132 ].…”