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

Abstract

1The cardiac voltage-gated sodium channel Nav1.5 conducts the rapid inward sodium current crucial for 2 cardiomyocyte excitability. Loss-of-function mutations in its gene SCN5A are linked to cardiac arrhythmias 3 such as Brugada Syndrome (BrS). Several BrS-associated mutations in the Nav1.5 N-terminal domain exert 4 a dominant-negative effect (DNE) on wild-type channel function, for which mechanisms remain poorly 5 understood. We aim to contribute to the understanding of BrS pathophysiology by characterizing three 6 mutations in the Nav1.5 N-terminal domain (NTD): Y87C-here newly identified-, R104W and R121W. In 7 addition, we hypothesize that the calcium sensor protein calmodulin is a new NTD binding partner. 8Recordings of whole-cell sodium currents in TsA-201 cells expressing WT and variant Nav1.5 showed that 9 Y87C and R104W but not R121W exert a DNE on WT channels. Biotinylation assays revealed reduction 10 in fully glycosylated Nav1.5 at the cell surface and in whole-cell lysates. Localization of Nav1.5 WT channel 11 with the ER however did not change in the presence of variants, shown by transfected and stained rat 12 neonatal cardiomyocytes. We next demonstrated that calmodulin binds Nav1.5 N-terminus using in silico 13 modeling, SPOTS, pull-down and proximity ligation assays. This binding is impaired in the R121W variant 14 and in a Nav1.5 construct missing residues 80-105, a predicted calmodulin binding site. 15In conclusion, we present the first evidence that calmodulin binds to the Nav1.5 NTD, which seems to be a 16 determinant for the DNE. 17Wang et al.3

Dominant negative effects of SCN5A missense variants

“…Previous research suggested that the residues between 493 and 517 are critical for the dimerization and coupled gating of NaV1.5 at the cell surface, and another study found an enrichment of dominant negative variants at the N-terminus of the protein (8,29). We did not observe an enrichment of dominant negative variants among these previously described residues, but rather a broader distribution of variants spanning the four transmembrane domains of the protein (Figure 6A-6C).…”

Trafficking and Gating Cooperation Between Deficient Nav1.5-mutant Channels to Rescue INa

“…Unlike potassium channel genes, which encode one fourth of tetramers constituting functional channels, Na v 1.5 channels were thought to be structured as monomers, since the gene encodes the entire 4domain channel α-subunit. It was thus unexpected to report Na v 1.5 mutants with a dominant-negative effect on wildtype (WT) channels, i.e., a decrease of I Na exceeding the 50% of current density expected in case of haploinsufficiency observed when coexpressing some mutants with WT channels in a 1:1 ratio to mimic patient heterozygosity, as we and others did [2][3][4][5][6][7][8][9]. Furthermore, we demonstrated that Na v 1.5 α-subunits could interact with each other and that a trafficking-efficient mutant channel was able to drive a trafficking-deficient one to the surface membrane [3].…”

Dominant negative effects ofSCN5Amissense variants

“…Follow up studies with extensive biochemical analyses showed that the dominant negative variant L325R acted through coupled gating at the cell surface (9). Previous research suggested that the residues between 493 and 517 are critical for the dimerization and coupled gating of NaV1.5 at the cell surface, and another study found an enrichment of dominant negative variants at the N-terminus of the protein (8,29). We did not observe an enrichment of dominant negative variants among these previously described residues, but rather a broader distribution of variants spanning the four transmembrane domains of the protein (Figure 6A-6C).…”