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(Circulation Research. 2004;95:284.)
© 2004 American Heart Association, Inc.

Cellular Biology

Cardiac Voltage-Gated Sodium Channel Na_v1.5 Is Regulated by Nedd4-2 Mediated Ubiquitination

Miguel X. van Bemmelen^*, Jean-Sébastien Rougier^*, Bruno Gavillet, Florine Apothéloz, Dorothée Daidié, Michihiro Tateyama, Ilaria Rivolta, Marc A. Thomas, Robert S. Kass, Olivier Staub, Hugues Abriel

From the Department of Pharmacology and Toxicology (M.X.v.B., J.-S.R., B.G., F.A., D.D., M.A.T., O.S., H.A.) and Service of Cardiology (H.A.), University of Lausanne, Switzerland; and Department of Pharmacology (M.T., I.R., R.S.K.), Columbia University, New York.

Correspondence to H. Abriel, MD, PhD, SNF-Professor, Department of Pharmacology and Toxicology, Service of Cardiology, University of Lausanne Bugnon, 27, 1005 Lausanne, Switzerland. E-mail Hugues.Abriel{at}ipharm.unil.ch

Na_v1.5, 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 Na_v1.5 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_v1.5. Yeast two-hybrid and GST-pulldown experiments revealed an interaction between Na_v1.5 C-terminus and Nedd4-2, which was abrogated by mutating the essential tyrosine of the PY-motif. Ubiquitination of Na_v1.5 was detected in both transfected HEK cells and heart extracts. Furthermore, Nedd4-2–dependent ubiquitination of Na_v1.5 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_v1.5 and Nedd4-2. Na_v1.5 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_v1.5. Consistent with the functional findings, localization at the cell periphery of Na_v1.5-YFP fusion proteins was reduced upon Nedd4-2 coexpression. The Nedd4-1 isoform did not regulate Na_v1.5, suggesting that Nedd4-2 is a specific regulator of Na_v1.5. These results demonstrate that Na_v1.5 can be ubiquitinated in heart tissues and that the ubiquitin-protein ligase Nedd4-2 acts on Na_v1.5 by decreasing the channel density at the cell surface.

Key Words: sodium channels • ubiquitin • Nedd4 • electrophysiology

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