Cardiac Voltage-Gated Sodium Channel Nav1.5 Is Regulated by Nedd4-2 Mediated Ubiquitination

• Published in: Circulation research Vol. 95; no. 3; pp. 284 - 291
• Main Authors: van Bemmelen, Miguel X, Rougier, Jean-Sébastien, Gavillet, Bruno, Apothéloz, Florine, Daidié, Dorothée, Tateyama, Michihiro, Rivolta, Ilaria, Thomas, Marc A, Kass, Robert S, Staub, Olivier, Abriel, Hugues
• Format: Journal Article
• Language: English
• Published: Hagerstown, MD American Heart Association, Inc 06.08.2004; Lippincott
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Animals; Biological and medical sciences; Catalysis; Cell Line - metabolism; Endosomal Sorting Complexes Required for Transport; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation; Humans; Ion Channel Gating; Ion Transport; Kidney; Mice; Molecular Sequence Data; Muscle Proteins - biosynthesis; Muscle Proteins - genetics; Myocardium - metabolism; Myocytes, Cardiac - metabolism; NAV1.5 Voltage-Gated Sodium Channel; Nedd4 Ubiquitin Protein Ligases; Protein Interaction Mapping; Protein Isoforms - physiology; Protein Processing, Post-Translational; Recombinant Fusion Proteins - physiology; Sodium - metabolism; Sodium Channels - biosynthesis; Sodium Channels - genetics; Transfection; Two-Hybrid System Techniques; Ubiquitin - metabolism; Ubiquitin-Protein Ligases - genetics; Ubiquitin-Protein Ligases - physiology; Vertebrates: cardiovascular system; Ubiquitin; Vertebrata; Mammalia; Sodium; sodium channels; Voltage; Nedd4; Electrophysiology; Ionic channel; Circulatory system
• ISSN: 0009-7330; 1524-4571
• DOI: 10.1161/01.RES.0000136816.05109.89

Nav1.5, the cardiac isoform of the voltage-gated Na channel, is critical to heart excitability and conduction. However, the mechanisms regulating its expression at the cell membrane are poorly understood. The Nav1.5 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 Nav1.5. Yeast two-hybrid and GST-pulldown experiments revealed an interaction between Nav1.5 C-terminus and Nedd4-2, which was abrogated by mutating the essential tyrosine of the PY-motif. Ubiquitination of Nav1.5 was detected in both transfected HEK cells and heart extracts. Furthermore, Nedd4-2–dependent ubiquitination of Nav1.5 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 Nav1.5 and Nedd4-2. Nav1.5 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 Nav1.5. Consistent with the functional findings, localization at the cell periphery of Nav1.5-YFP fusion proteins was reduced upon Nedd4-2 coexpression. The Nedd4-1 isoform did not regulate Nav1.5, suggesting that Nedd4-2 is a specific regulator of Nav1.5. These results demonstrate that Nav1.5 can be ubiquitinated in heart tissues and that the ubiquitin-protein ligase Nedd4-2 acts on Nav1.5 by decreasing the channel density at the cell surface.