Cardiac Voltage-Gated Sodium Channel Na _v 1.5 Is Regulated by Nedd4-2 Mediated Ubiquitination

Abstract

Abstract-Na v 1.5, the cardiac isoform of the voltage-gated Na ϩ channel, is critical to heart excitability and conduction. However, the mechanisms regulating its expression at the cell membrane are poorly understood. The Na v 1.5 C-terminus contains a PY-motif (xPPxY) that is known to act as binding site for Nedd4/Nedd4-like ubiquitin-protein ligases. Because Nedd4-2 is well expressed in the heart, we investigated its role in the ubiquitination and regulation of Na v 1.5. Yeast two-hybrid and GST-pulldown experiments revealed an interaction between Na v 1.5 C-terminus and Nedd4-2, which was abrogated by mutating the essential tyrosine of the PY-motif. Ubiquitination of Na v 1.5 was detected in both transfected HEK cells and heart extracts. Furthermore, Nedd4-2-dependent ubiquitination of Na v 1.5 was observed. To test for a functional role of Nedd4-2, patch-clamp experiments were performed on HEK cells expressing wild-type and mutant forms of both Na v 1.5 and Nedd4-2. Na v 1.5 current density was decreased by 65% upon Nedd4-2 cotransfection, whereas the PY-motif mutant channels were not affected. In contrast, a catalytically inactive Nedd4-2 had no effect, indicating that ubiquitination mediates this downregulation. However, Nedd4-2 did not alter the whole-cell or the single channel biophysical properties of Na v 1.5. Consistent with the functional findings, localization at the cell periphery of Na v 1.5-YFP fusion proteins was reduced upon Nedd4-2 coexpression. The Nedd4-1 isoform did not regulate Na v 1.5, suggesting that Nedd4-2 is a specific regulator of Na v 1.5. These results demonstrate that Na v 1.5 can be ubiquitinated in heart tissues and that the ubiquitin-protein ligase Nedd4-2 acts on Na v 1.5 by decreasing the channel density at the cell surface.

Regulation of the Human Ether-a-go-go-related Gene (hERG) Channel by Rab4 Protein through Neural Precursor Cell-expressed Developmentally Down-regulated Protein 4-2 (Nedd4-2)

“…Because both epithelial sodium channel and cystic fibrosis transmembrane conductance regulator are Nedd4-2 substrates (30,47), it is likely that Rab4 also regulates these two channels via Nedd4-2. In addition, cardiac and neuronal Na ϩ channels, cardiac K ϩ channel KCNQ1, and neuronal K ϩ channels KCNQ2/3/5 are among the substrates of Nedd4-2 (32)(33)(34)48). Besides the direct effects on these channels, Rab4 is expected to regulate these channels via altered Nedd4-2 expression levels.…”

Dysregulation of voltage-gated sodium channels by ubiquitin ligase NEDD4-2 in neuropathic pain

“…The external and internal solutions used were as previously described (ref. 23 and see Supplemental Methods). Data were recorded using a VE-2 amplifier (Alembic Instruments) or an Axon 700A amplifier and analyzed using pClamp software, version 8 (Molecular Devices), KaleidaGraph, version 4.03 (Synergy Software), and MATLAB (The MathWorks).…”

“…All Na v isoforms, except Na v 1.4 and Na v 1.9, possess a PY motif and are potential targets of NEDD4-2. In vitro experiments have indicated that NEDD4-2 can negatively regulate the epithelial sodium channel ENaC (22) and Na v s (23,24). The functional relevance of NEDD4-2 in sensory neurons, as well as its possible involvement in pain sensitivity, have yet to be investigated.…”

αB-Crystallin Interacts with Nav1.5 and Regulates Ubiquitination and Internalization of Cell Surface Nav1.5

“…MOG1, 14-3-3 ␤ and ankyrin-G regulate Na v 1.5 localization to the cell surface in cardiomyocytes (4,7). Nedd4-2 interacts with Na v 1.5 and regulates Na v 1.5 degradation (8). However, the Na v 1.5 protein complex is highly sophisticated.…”