Calmodulin binds to the N-terminal domain of the cardiac sodium channel Na_v1.5

The cardiac voltage-gated sodium channel Na_v1.5 conducts the rapid inward sodium current crucial for cardiomyocyte excitability. Loss-of-function mutations in its gene SCN5A are linked to cardiac arrhythmias such as Brugada Syndrome (BrS). Several BrS-associated mutations in the Na_v1.5 N-terminal domain (NTD) exert a dominant-negative effect (DNE) on wild-type channel function, for which mechanisms remain poorly understood. We aim to contribute to the understanding of BrS pathophysiology by characterizing three mutations in the Na_v1.5 NTD: Y87C-here newly identified-, R104W, and R121W. In addition, we hypothesize that the calcium sensor protein calmodulin is a new NTD binding partner. Recordings of whole-cell sodium currents in TsA-201 cells expressing WT and variant Na_v1.5 showed that Y87C and R104W but not R121W exert a DNE on WT channels. Biotinylation assays revealed reduction in fully glycosylated Na_v1.5 at the cell surface and in whole-cell lysates. Localization of Na_v1.5 WT channel with the ER did not change in the presence of variants, as shown by transfected and stained rat neonatal cardiomyocytes. We demonstrated that calmodulin binds the Na_v1.5 NTD using in silico modeling, SPOTS, pull-down, and proximity ligation assays. Calmodulin binding to the R121W variant and to a Na_v1.5 construct missing residues 80-105, a predicted calmodulin-binding site, is impaired. In conclusion, we describe the new natural BrS Na_v1.5 variant Y87C and present first evidence that calmodulin binds to the Na_v1.5 NTD, which seems to be a determinant for the DNE.