Wang, Zizun; Vermij, Sarah H.; Sottas, Valentin; Shestak, Anna; Ross-Kaschitza, Daniela; Zaklyazminskaya, Elena V; Hudmon, Andy; Pitt, Geoffrey S; Rougier, Jean-Sébastien; Abriel, Hugues (2020). Calmodulin binds to the N-terminal domain of the cardiac sodium channel Nav1.5. Channels, 14(1), pp. 268-286. Taylor & Francis 10.1080/19336950.2020.1805999
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The cardiac voltage-gated sodium channel Nav1.5 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 Nav1.5 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 Nav1.5 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 Nav1.5 showed that Y87C and R104W but not R121W exert a DNE on WT channels. Biotinylation assays revealed reduction in fully glycosylated Nav1.5 at the cell surface and in whole-cell lysates. Localization of Nav1.5 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 Nav1.5 NTD using in silico modeling, SPOTS, pull-down, and proximity ligation assays. Calmodulin binding to the R121W variant and to a Nav1.5 construct missing residues 80-105, a predicted calmodulin-binding site, is impaired. In conclusion, we describe the new natural BrS Nav1.5 variant Y87C and present first evidence that calmodulin binds to the Nav1.5 NTD, which seems to be a determinant for the DNE.
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