Regulation of neuronal voltage-gated sodium channels by the ubiquitin protein ligases Nedd4 and Nedd4-2

Nedd4 and Nedd4-2 are ubiquitin-protein ligases known to regulate a number of membrane proteins including receptors and ion transporters. Regulation of the epithelial Na⁺ channel by Nedd4 and Nedd4-2 is mediated via interactions between the PY motifs of the epithelial sodium channel subunits and the Nedd4/Nedd4-2 WW domains. This example serves as a model for the regulation of other PY motif-containing ion channels by Nedd4 and Nedd4-2. We found that the carboxyl termini of the six voltage-gated Na⁺ (Na_v) channels contain typical PY motifs (PPXY), and a further Na_v contains a PY motif variant (LPXY). Not only did we demonstrate by Far-Western analysis that Nedd4 and Nedd4-2 interact with the PY motif-containing Na_v channels, but we also showed that these channels have conserved WW domain binding specificity. We further showed that the carboxyl termini fusion proteins of one central nervous system and one peripheral nervous system-derived Na ⁺ channel (Na_v1.2 and Na_v1.7, respectively) are readily ubiquitinated by Nedd4-2. In Xenopus oocytes, Nedd4-2 strongly inhibited the activities of all three Na_vs (Na_v1.2, Na_v1.7, and Na_v1.8) tested. Interestingly, Nedd4 suppressed the activity of Na_v1.2 and Na_v1.7 but was a poor inhibitor of Na_v1.8. Our results provide evidence that Nedd4 and Nedd4-2 are likely to be key regulators of specific neuronal Na_v, channels in vivo.