Molecular determinants of voltage-gated sodium channel regulation by the Nedd4/Nedd4-like proteins.
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.
Rougier
Jean-Sébastien
JS
Department of Pharmacology and Toxicology, Service of Cardiology, University of Lausanne, Rue du Bugnon 27, CH-1005 Lausanne, Switzerland.
van Bemmelen
Miguel X
MX
Bruce
M Christine
MC
Jespersen
Thomas
T
Gavillet
Bruno
B
Apothéloz
Florine
F
Cordonier
Sophie
S
Staub
Olivier
O
Rotin
Daniela
D
Abriel
Hugues
H
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D015536:Down-Regulation; D004594:Electrophysiology; D056827:Endosomal Sorting Complexes Required for Transport; D006801:Humans; D015640:Ion Channel Gating; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D000075702:Nedd4 Ubiquitin Protein Ligases; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D015222:Sodium Channels / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*
D056827:Endosomal Sorting Complexes Required for Transport; D010455:Peptides; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D015222:Sodium Channels; C000618479:NEDD4L protein, rat; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618484:Nedd4 protein, rat; C000618485:Nedd4L protein, human; C000618461:Nedd4l protein, mouse; C465283:WWP2 protein, human; D044767:Ubiquitin-Protein Ligases