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

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

doi:10.1152/ajpcell.00460.2004

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

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

Molecular Cardiology and Membrane Proteins

103 Citations (Scopus)

Abstract

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.

Original language	English
Journal	American Journal of Physiology: Cell Physiology
Volume	288
Issue number	3
Pages (from-to)	C692-701
ISSN	0363-6143
DOIs	https://doi.org/10.1152/ajpcell.00460.2004
Publication status	Published - 2004

Access to Document

10.1152/ajpcell.00460.2004

Cite this

@article{1db5e700acc811ddb538000ea68e967b,

title = "Molecular determinants of voltage-gated sodium channel regulation by the Nedd4/Nedd4-like proteins.",

abstract = "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.",

author = "Jean-S{\'e}bastien Rougier and {van Bemmelen}, {Miguel X} and Bruce, {M Christine} and Thomas Jespersen and Bruno Gavillet and Florine Apoth{\'e}loz and Sophie Cordonier and Olivier Staub and Daniela Rotin and Hugues Abriel",

note = "Keywords: Amino Acid Sequence; Animals; Brain; Cell Line; Down-Regulation; Electrophysiology; Humans; Ion Channel Gating; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Peptides; Protein Binding; Protein Isoforms; Rats; Recombinant Fusion Proteins; Sequence Alignment; Sodium Channels; Ubiquitin-Protein Ligases",

year = "2004",

doi = "10.1152/ajpcell.00460.2004",

language = "English",

volume = "288",

pages = "C692--701",

journal = "American Journal of Physiology: Cell Physiology",

issn = "0363-6143",

publisher = "American Physiological Society",

number = "3",

}

TY - JOUR

T1 - Molecular determinants of voltage-gated sodium channel regulation by the Nedd4/Nedd4-like proteins.

AU - Rougier, Jean-Sébastien

AU - van Bemmelen, Miguel X

AU - Bruce, M Christine

AU - Jespersen, Thomas

AU - Gavillet, Bruno

AU - Apothéloz, Florine

AU - Cordonier, Sophie

AU - Staub, Olivier

AU - Rotin, Daniela

AU - Abriel, Hugues

N1 - Keywords: Amino Acid Sequence; Animals; Brain; Cell Line; Down-Regulation; Electrophysiology; Humans; Ion Channel Gating; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Peptides; Protein Binding; Protein Isoforms; Rats; Recombinant Fusion Proteins; Sequence Alignment; Sodium Channels; Ubiquitin-Protein Ligases

PY - 2004

Y1 - 2004

N2 - 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.

AB - 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.

U2 - 10.1152/ajpcell.00460.2004

DO - 10.1152/ajpcell.00460.2004

M3 - Journal article

C2 - 15548568

SN - 0363-6143

VL - 288

SP - C692-701

JO - American Journal of Physiology: Cell Physiology

JF - American Journal of Physiology: Cell Physiology

IS - 3

ER -

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

Abstract

Access to Document

Fingerprint

Cite this