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5 results on '"Apothéloz, Florine"'

1. Molecular determinants of voltage-gated sodium channel regulation by the Nedd4/Nedd4-like proteins.

Author

Rougier, Jean-Sébastien, van Bemmelen, Miguel X, Bruce, M Christine, Jespersen, Thomas, Gavillet, Bruno, Apothéloz, Florine, Cordonier, Sophie, Staub, Olivier, Rotin, Daniela, Abriel, Hugues, Rougier, Jean-Sébastien, van Bemmelen, Miguel X, Bruce, M Christine, Jespersen, Thomas, Gavillet, Bruno, Apothéloz, Florine, Cordonier, Sophie, Staub, Olivier, Rotin, Daniela, and Abriel, Hugues

Abstract

Udgivelsesdato: 2005-Mar, The voltage-gated Na(+) channels (Na(v)) form a family composed of 10 genes. The COOH termini of Na(v) contain a cluster of amino acids that are nearly identical among 7 of the 10 members. This COOH-terminal sequence, PPSYDSV, is a PY motif known to bind to WW domains of E3 protein-ubiquitin ligases of the Nedd4 family. We recently reported that cardiac Na(v)1.5 is regulated by Nedd4-2. In this study, we further investigated the molecular determinants of regulation of Na(v) proteins. When expressed in HEK-293 cells and studied using whole cell voltage clamping, the neuronal Na(v)1.2 and Na(v)1.3 were also downregulated by Nedd4-2. Pull-down experiments using fusion proteins bearing the PY motif of Na(v)1.2, Na(v)1.3, and Na(v)1.5 indicated that mouse brain Nedd4-2 binds to the Na(v) PY motif. Using intrinsic tryptophan fluorescence imaging of WW domains, we found that Na(v)1.5 PY motif binds preferentially to the fourth WW domain of Nedd4-2 with a K(d) of approximately 55 muM. We tested the binding properties and the ability to ubiquitinate and downregulate Na(v)1.5 of three Nedd4-like E3s: Nedd4-1, Nedd4-2, and WWP2. Despite the fact that along with Nedd4-2, Nedd4-1 and WWP2 bind to Na(v)1.5 PY motif, only Nedd4-2 robustly ubiquitinated and downregulated Na(v)1.5. Interestingly, coexpression of WWP2 competed with the effect of Nedd4-2. Finally, using brefeldin A, we found that Nedd4-2 accelerated internalization of Na(v)1.5 stably expressed in HEK-293 cells. This study shows that Nedd4-dependent ubiquitination of Na(v) channels may represent a general mechanism regulating the excitability of neurons and myocytes via modulation of channel density at the plasma membrane.

Published
2004

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

Author

van Bemmelen MX, Rougier JS, Gavillet B, Apothéloz F, Daidié D, Tateyama M, Rivolta I, Thomas MA, Kass RS, Staub O, and Abriel H

Subjects
Amino Acid Motifs, Amino Acid Sequence, Animals, Catalysis, Cell Line metabolism, Endosomal Sorting Complexes Required for Transport, Gene Expression Regulation, Humans, Ion Channel Gating, Ion Transport, Kidney, Mice, Molecular Sequence Data, Muscle Proteins genetics, Myocytes, Cardiac metabolism, NAV1.5 Voltage-Gated Sodium Channel, Nedd4 Ubiquitin Protein Ligases, Protein Interaction Mapping, Protein Isoforms physiology, Recombinant Fusion Proteins physiology, Sodium metabolism, Sodium Channels genetics, Transfection, Two-Hybrid System Techniques, Ubiquitin-Protein Ligases genetics, Muscle Proteins biosynthesis, Myocardium metabolism, Protein Processing, Post-Translational, Sodium Channels biosynthesis, Ubiquitin metabolism, Ubiquitin-Protein Ligases physiology
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.

Published
2004
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