Na(v)1.5, 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(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.

van Bemmelen, M., Rougier, J., Gavillet, B., Apothéloz, F., Daidié, D., Tateyama, M., et al. (2004). Cardiac voltage-gated sodium channel Nav1.5 is regulated by Nedd4-2 mediated ubiquitination. CIRCULATION RESEARCH, 95(3), 284-291 [10.1161/01.RES.0000136816.05109.89].

Cardiac voltage-gated sodium channel Nav1.5 is regulated by Nedd4-2 mediated ubiquitination

RIVOLTA, ILARIA;
2004-08-06

Abstract

Na(v)1.5, 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(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.
Articolo in rivista - Articolo scientifico
Scientifica
Catalysis; Transfection; Protein Processing, Post-Translational; Sodium Channels; Ubiquitin-Protein Ligases; Two-Hybrid System Techniques; Protein Interaction Mapping; Mice; Molecular Sequence Data; Amino Acid Sequence; Endosomal Sorting Complexes Required for Transport; Kidney; Cell Line; Muscle Proteins; Amino Acid Motifs; Animals; Ubiquitin; Sodium; Humans; Myocardium; Myocytes, Cardiac; Ion Channel Gating; Protein Isoforms; Ion Transport; Gene Expression Regulation; Recombinant Fusion Proteins
English
van Bemmelen, M., Rougier, J., Gavillet, B., Apothéloz, F., Daidié, D., Tateyama, M., et al. (2004). Cardiac voltage-gated sodium channel Nav1.5 is regulated by Nedd4-2 mediated ubiquitination. CIRCULATION RESEARCH, 95(3), 284-291 [10.1161/01.RES.0000136816.05109.89].
van Bemmelen, M; Rougier, J; Gavillet, B; Apothéloz, F; Daidié, D; Tateyama, M; Rivolta, I; Thomas, M; Kass, R; Staub, O; Abriel, H
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/10281/22575
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