Alterations of Nedd4-2-binding capacity in PY-motif of NaV 1.5 channel underlie long QT syndrome and Brugada syndrome

Abstract

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 Na_V 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 Na_V 1.5, which results in decreased protein expression and current through Na_V 1.5 ubiquitination. Here, we investigate the role of E3 ubiquitin ligase Nedd4-2-mediated Na_V 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 Na_V 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 Na_V 1.5 and Nedd4-2, enhancing ubiquitination and degradation of the Na_V 1.5 channel. Disruption of the new Nedd4-2-binding site of SCN5A-p.L1239P restores Na_V 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 Na_V 1.5-Nedd4-2 interaction, preventing ubiquitination and degradation of Na_V 1.5 channels.

Conclusions: Our data suggest that the PY-motif plays an essential role in modifying the expression/function of Na_V 1.5 channels through Nedd4-2-mediated ubiquitination. Alterations of Na_V 1.5-Nedd4-2 interaction represent a novel pathological mechanism for Na_V 1.5 channel diseases caused by SCN5A variants.

Keywords: Brugada syndrome; NaV1.5 channel; Nedd4-2; long QT syndrome; ubiquitination.