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 (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 v 1.5 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 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 did not change in the presence of variants, as shown by transfected and stained rat neonatal cardiomyocytes. We demonstrated that calmodulin binds the Na v 1.5 NTD using in silico modeling, SPOTS, pull-down, and proximity ligation assays. Calmodulin binding to the R121W variant and to a Na v 1.5 construct missing residues 80-105, a predicted calmodulin-binding site, is impaired. In conclusion, we describe the new natural BrS Na v 1.5 variant Y87C and present first evidence that calmodulin binds to the Na v 1.5 NTD, which seems to be a determinant for the DNE.

Dysfunction of the cardiac sodium channel Nav1.5 (encoded by the SCN5A gene) is associated with arrhythmias and sudden cardiac death. SCN5A mutations associated with long QT syndrome type 3 (LQT3) lead to enhanced late sodium current and consequent action potential (AP) prolongation. Internalization and degradation of Nav1.5 is regulated by ubiquitylation, a post-translational mechanism that involves binding of the ubiquitin ligase Nedd4-2 to a proline-proline-serine-tyrosine sequence of Nav1.5, designated the PY-motif. We investigated the biophysical properties of the LQT3-associated SCN5A-p.Y1977N mutation located in the Nav1.5 PY-motif, both in HEK293 cells as well as in newly generated mice harboring the mouse homolog mutation Scn5a-p.Y1981N. We found that in HEK293 cells, the SCN5A-p.Y1977N mutation abolished the interaction between Nav1.5 and Nedd4-2, suppressed PY-motif-dependent ubiquitylation of Nav1.5, and consequently abrogated Nedd4-2 induced sodium current (INa) decrease. Nevertheless, homozygous mice harboring the Scn5a-p.Y1981N mutation showed no electrophysiological alterations nor changes in AP or (late) INa properties, questioning the in vivo relevance of the PY-motif. Our findings suggest the presence of compensatory mechanisms, with additional, as yet unknown, factors likely required to reduce the “ubiquitylation reserve” of Nav1.5. Future identification of such modulatory factors may identify potential triggers for arrhythmias and sudden cardiac death in the setting of LQT3 mutations.

Sudden arrhythmic death syndrome (SADS) in young individuals is a devastating and tragic event often caused by an undiagnosed inherited cardiac disease. Although post-mortem genetic testing represents a promising tool to elucidate potential diseasecausing mechanisms in such autopsy-negative death cases, a variant interpretation is still challenging, and functional consequences of identified sequence alterations often remain unclear. Recently, we have identified a novel heterozygous missense variant (N1774H) in the Na v 1.5 channel-encoding gene SCN5A in a 19-year-old female SADS victim. The aim of this study was to perform a co-segregation analysis in family members of the index case and to evaluate the functional consequences of this SCN5A variant. Functional characterization of the SCN5A N1774H variant was performed using patch-clamp techniques in TsA-201 cell line transiently expressing either wild-type or variant Na v 1.5 channels. Electrophysiological analyses revealed that variant Na v 1.5 channels show a loss-of-function in the peak current densities, but an increased late current compared to the wild-type channels, which could lead to both, loss-and gain-of-function respectively. Furthermore, clinical assessment and genetic testing of the relatives of the index case showed that all N1774H mutation carriers have prolonged QT intervals. The identification of several genotype and phenotype positive family members and the functional implication of the SCN5A N1774H variant support the evidence of the in silico predicted pathogenicity of the here reported sequence alteration.

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