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

Am J Physiol Cell Physiol. 2005 Mar;288(3):C692-701. doi: 10.1152/ajpcell.00460.2004. Epub 2004 Nov 17.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Brain / metabolism
  • Cell Line
  • Down-Regulation
  • Electrophysiology
  • Endosomal Sorting Complexes Required for Transport
  • Humans
  • Ion Channel Gating
  • Mice
  • Mice, Inbred C57BL
  • Molecular Sequence Data
  • Nedd4 Ubiquitin Protein Ligases
  • Peptides / metabolism
  • Protein Binding
  • Protein Isoforms / genetics
  • Protein Isoforms / metabolism*
  • Rats
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Sequence Alignment
  • Sodium Channels / genetics
  • Sodium Channels / metabolism*
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism*


  • Endosomal Sorting Complexes Required for Transport
  • Peptides
  • Protein Isoforms
  • Recombinant Fusion Proteins
  • Sodium Channels
  • NEDD4L protein, rat
  • Nedd4 Ubiquitin Protein Ligases
  • Nedd4 protein, human
  • Nedd4 protein, rat
  • Nedd4L protein, human
  • Nedd4l protein, mouse
  • WWP2 protein, human
  • Ubiquitin-Protein Ligases