Calmodulin binds to the N-terminal domain of the cardiac sodium channel Nav1.5

Abstract

ABSTRACT The cardiac voltage-gated sodium channel Na v 1.5 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 v 1.5 N-terminal domain 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 v 1.5 N-terminal domain (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 v 1.5 showed that Y87C and R104W but not R121W exert a DNE on WT channels. Biotinylation assays revealed reduction in fully glycosylated Na v 1.5 at the cell surface and in whole-cell lysates. Localization of Na v 1.5 WT channel with the ER however did not change in the presence of variants, shown by transfected and stained rat neonatal cardiomyocytes. We next demonstrated that calmodulin binds Na v 1.5 N-terminus using in silico modeling, SPOTS, pull-down and proximity ligation assays. This binding is impaired in the R121W variant and in a Na v 1.5 construct missing residues 80-105, a predicted calmodulin binding site. In conclusion, we present the first evidence that calmodulin binds to the Na v 1.5 NTD, which seems to be a determinant for the DNE.