ABSTRACT

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.

摘要

心脏电压门控钠通道 Na _v 1.5 传导对心肌细胞兴奋性至关重要的快速内向钠电流。其基因SCN5A的功能丧失突变与心律失常（如布鲁格达综合征 (BrS)）有关。_{Na v} 1.5 N 末端结构域 (NTD)中的几个 BrS 相关突变对野生型通道功能产生显性负效应 (DNE)，但其机制仍知之甚少。_{我们的目标是通过表征 Na v} 1.5 NTD中的三个突变来促进对 BrS 病理生理学的理解：Y87C（此处新鉴定）、R104W 和 R121W。此外，我们假设钙传感器蛋白钙调蛋白是一种新的 NTD 结合伴侣。_{表达 WT 和变体 Na v} 1.5的 TsA-201 细胞中全细胞钠电流的记录表明，Y87C 和 R104W 但 R121W 在 WT 通道上发挥 DNE。生物素化测定显示细胞表面和全细胞裂解物中完全糖基化的 Na _v 1.5减少。Na _v 1.5 WT 通道与 ER 的定位在存在变异的情况下没有改变，如转染和染色的大鼠新生心肌细胞所示。我们使用计算机建模、SPOTS、pull-down 和邻位连接测定证明了钙调蛋白与 Na _v 1.5 NTD结合。钙调蛋白与 R121W 变体和 Na _v 1.5 构建体的结合受损，其中缺失残基 80-105（预测的钙调蛋白结合位点）。总之，我们描述了新的天然 BrS Na _v 1.5 变体 Y87C，并提出了钙调蛋白与 Na _v 1.5 NTD 结合的第一个证据，这似乎是 DNE 的决定因素。