AIMS Pathogenic variants of the SCN5A gene can cause Brugada syndrome (BrS) and long QT syndrome (LQTS), which predispose individuals to potentially fatal ventricular arrhythmias and sudden cardiac death. SCN5A encodes the NaV 1.5 protein, the pore forming α-subunit of the voltage-dependent cardiac Na+ channel. Using a WW domain, the E3 ubiquitin ligase Nedd4-2 binds to the PY-motif ([L/P]PxY) within the C-terminus of NaV 1.5, which results in decreased protein expression and current through NaV 1.5 ubiquitination. Here, we investigate the role of E3 ubiquitin ligase Nedd4-2-mediated NaV 1.5 degradation in the pathological mechanisms of the BrS-associated variant SCN5A-p.L1239P and LQTS-associated variant SCN5A-p.Y1977N. METHODS AND RESULTS Using a combination of molecular biology, biochemical and electrophysiological approaches, we examined the expression, function and Nedd4-2 interactions of SCN5A-p.L1239P and SCN5A-p.Y1977N. SCN5A-p.L1239P is characterized as a loss-of-function, whereas SCN5A-p.Y1977N is a gain-of-function variant of the NaV 1.5 channel. Sequence alignment shows that BrS-associated SCN5A-p.L1239P has a new Nedd4-2-binding site (from LLxY to LPxY). This new Nedd4-2-binding site increases the interaction between NaV 1.5 and Nedd4-2, enhancing ubiquitination and degradation of the NaV 1.5 channel. Disruption of the new Nedd4-2-binding site of SCN5A-p.L1239P restores NaV 1.5 expression and function. However, the LQTS-associated SCN5A-p.Y1977N disrupts the usual Nedd4-2-binding site (from PPxY to PPxN). This decreases NaV 1.5-Nedd4-2 interaction, preventing ubiquitination and degradation of NaV 1.5 channels. CONCLUSIONS Our data suggest that the PY-motif plays an essential role in modifying the expression/function of NaV 1.5 channels through Nedd4-2-mediated ubiquitination. Alterations of NaV 1.5-Nedd4-2 interaction represent a novel pathological mechanism for NaV 1.5 channel diseases caused by SCN5A variants.

中文翻译：

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NaV 1.5通道的PY基序中Nedd4-2-结合能力的改变是长QT综合征和Brugada综合征的基础。

AIMS SCN5A基因的致病性变异可能导致Brugada综合征（BrS）和长QT综合征（LQTS），这使个体容易患上致命的室性心律失常和猝死。SCN5A编码NaV 1.5蛋白，该孔形成电压依赖性心脏Na +通道的α亚基。使用WW域，E3泛素连接酶Nedd4-2在NaV 1.5的C末端与PY-基序（[L / P] PxY）结合，从而导致蛋白质表达降低和通过NaV 1.5泛素化的电流。在这里，我们调查E3泛素连接酶Nedd4-2介导的NaV 1.5降解在BrS相关变体SCN5A-p.L1239P和LQTS相关变体SCN5A-p.Y1977N的病理机制中的作用。方法与结果结合了分子生物学，生化和电生理方法，我们检查了SCN5A-p.L1239P和SCN5A-p.Y1977N的表达，功能和Nedd4-2相互作用。SCN5A-p.L1239P被描述为功能丧失，而SCN5A-p.Y1977N是NaV 1.5通道的功能获得型。序列比对显示与BrS相关的SCN5A-p.L1239P具有新的Nedd4-2-结合位点（从LLxY到LPxY）。这个新的Nedd4-2-结合位点增加了NaV 1.5和Nedd4-2之间的相互作用，增强了NaV 1.5通道的泛素化和降解。SCN5A-p.L1239P的新Nedd4-2-结合位点的破坏恢复了NaV 1.5的表达和功能。但是，与LQTS相关的SCN5A-p.Y1977N破坏了通常的Nedd4-2-结合位点（从PPxY到PPxN）。这减少了NaV 1.5-Nedd4-2的相互作用，防止了NaV 1.5通道的泛素化和降解。结论我们的数据表明，PY-基序在通过Nedd4-2介导的泛素化修饰NaV 1.5通道的表达/功能中起着至关重要的作用。NaV 1.5-Nedd4-2相互作用的变化代表由SCN5A变体引起的NaV 1.5通道疾病的新型病理机制。