Regulation of Neuronal Voltage-gated Sodium Channels by the Ubiquitin-Protein Ligases Nedd4 and Nedd4-2

Abstract

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 v 1.2 and Na v 1.7, respectively) are readily ubiquitinated by Nedd4-2. In Xenopus oocytes, Nedd4-2 strongly inhibited the activities of all three Na v s (Na v 1.2, Na v 1.7, and Na v 1.8) tested. Interestingly, Nedd4 suppressed the activity of Na v 1.2 and Na v 1.7 but was a poor inhibitor of Na v 1.8. Our results provide evidence that Nedd4 and Nedd4-2 are likely to be key regulators of specific neuronal Na v channels in vivo.

A rescuable folding defective Nav1.1 (SCN1A) sodium channel mutant causes GEFS+: Common mechanism in Nav1.1 related epilepsies?

“…In fact, Nedd-4 and FGF14-1A decreased the current density of Na v 1.1-R1916G but had no effect on the wild type, consistently with published data (Fotia et al, 2004;Lou et al, 2005;Rougier et al, 2005). Thus, the effects of R1916G depend on the contrasting action of rescuing and downregulating interactions.…”

“…The interaction with voltage gated Na + channel α subunits in general increases the internalization of the channel and possibly its degradation, but Nedd4-1 can interact with some Na + channel α subunits inducing just a moderate downregulation, or no downregulation at all (Fotia et al, 2004;Rougier et al, 2005). Na v 1.1 contains a conserved PY motif in the C-terminal cytoplasmic domain, downstream of R1916G (Fig.…”

“…Na v 1.1 contains a conserved PY motif in the C-terminal cytoplasmic domain, downstream of R1916G (Fig. 1C), and interacts with Nedd proteins (Fotia et al, 2004). Therefore, we tested Nedd4-1 because it should not induce a significant downregulation and it may be effective in rescuing Na v 1.1-R1916G.…”

Dysregulation of voltage-gated sodium channels by ubiquitin ligase NEDD4-2 in neuropathic pain

“…Since Na v 1.7 is essential for pain sensation, NEDD4-2 downregulation of Na v 1.7 in mammalian cells was investigated, as previously reported in Xenopus oocytes (24). Whole-cell Na + currents (I Na ) were recorded in HEK293 cells cotransfected with Na v 1.7 and NEDD4-2.…”

“…All Na v isoforms, except Na v 1.4 and Na v 1.9, possess a PY motif and are potential targets of NEDD4-2. In vitro experiments have indicated that NEDD4-2 can negatively regulate the epithelial sodium channel ENaC (22) and Na v s (23,24). The functional relevance of NEDD4-2 in sensory neurons, as well as its possible involvement in pain sensitivity, have yet to be investigated.…”