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Two Nedd4-binding motifs underlie modulation of sodium channel Nav1.6 by p38 MAPK.

ABSTRACT: Sodium channel Na(v)1.6 is essential for neuronal excitability in central and peripheral nervous systems. Loss-of-function mutations in Na(v)1.6 underlie motor disorders, with homozygous-null mutations causing juvenile lethality. Phosphorylation of Na(v)1.6 by the stress-induced p38 MAPK at a Pro-Gly-Ser(553)-Pro motif in its intracellular loop L1 reduces Na(v)1.6 current density in a dorsal root ganglion-derived cell line, without changing its gating properties. Phosphorylated Pro-Gly-Ser(553)-Pro motif is a putative binding site to Nedd4 ubiquitin ligases, and we hypothesized that Nedd4-like ubiquitin ligases may contribute to channel ubiquitination and internalization. We report here that p38 activation in hippocampal neurons from wild-type mice, but not from Scn8a(medtg) mice that lack Na(v)1.6, reduces tetrodotoxin-S sodium currents, suggesting isoform-specific modulation of Na(v)1.6 by p38 in these neurons. Pharmacological block of endocytosis completely abolishes p38-mediated Na(v)1.6 current reduction, supporting our hypothesis that channel internalization underlies current reduction. We also report that the ubiquitin ligase Nedd4-2 interacts with Na(v)1.6 via a Pro-Ser-Tyr(1945) motif in the C terminus of the channel and reduces Na(v)1.6 current density, and we show that this regulation requires both the Pro-Gly-Ser-Pro motif in L1 and the Pro-Ser-Tyr motif in the C terminus. Similarly, both motifs are necessary for p38-mediated reduction of Na(v)1.6 current, whereas abrogating binding of the ubiquitin ligase Nedd4-2 to the Pro-Ser-Tyr motif results in stress-mediated increase in Na(v)1.6 current density. Thus, phosphorylation of the Pro-Gly-Ser-Pro motif within L1 of Na(v)1.6 is necessary for stress-induced current modulation, with positive or negative regulation depending upon the availability of the C-terminal Pro-Ser-Tyr motif to bind Nedd4-2.

SUBMITTER: Gasser A

PROVIDER: S-EPMC2924020 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Two Nedd4-binding motifs underlie modulation of sodium channel Nav1.6 by p38 MAPK.

Gasser Andreas A Cheng Xiaoyang X Gilmore Elaine S ES Tyrrell Lynda L Waxman Stephen G SG Dib-Hajj Sulayman D SD

The Journal of biological chemistry 20100608 34

Sodium channel Na(v)1.6 is essential for neuronal excitability in central and peripheral nervous systems. Loss-of-function mutations in Na(v)1.6 underlie motor disorders, with homozygous-null mutations causing juvenile lethality. Phosphorylation of Na(v)1.6 by the stress-induced p38 MAPK at a Pro-Gly-Ser(553)-Pro motif in its intracellular loop L1 reduces Na(v)1.6 current density in a dorsal root ganglion-derived cell line, without changing its gating properties. Phosphorylated Pro-Gly-Ser(553)- ...[more]

PMID: 20530479

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Project description:Mutations in brain isoforms of voltage-gated sodium channels have been identified in patients with distinct epileptic phenotypes. Clinically, these patients often do not respond well to classic anti-epileptics and many remain refractory to treatment. Exogenous as well as endogenous cannabinoids have been shown to target voltage-gated sodium channels and cannabidiol has recently received attention for its potential efficacy in the treatment of childhood epilepsies. In this study, we further investigated the ability of cannabinoids to modulate sodium currents from wild-type and epilepsy-associated mutant voltage-gated sodium channels. We first determined the biophysical consequences of epilepsy-associated missense mutations in both Nav1.1 (arginine 1648 to histidine and asparagine 1788 to lysine) and Nav1.6 (asparagine 1768 to aspartic acid and leucine 1331 to valine) by obtaining whole-cell patch clamp recordings in human embryonic kidney 293T cells with 200 μM Navβ4 peptide in the pipette solution to induce resurgent sodium currents. Resurgent sodium current is an atypical near threshold current predicted to increase neuronal excitability and has been implicated in multiple disorders of excitability. We found that both mutations in Nav1.6 dramatically increased resurgent currents while mutations in Nav1.1 did not. We then examined the effects of anandamide and cannabidiol on peak transient and resurgent currents from wild-type and mutant channels. Interestingly, we found that cannabidiol can preferentially target resurgent sodium currents over peak transient currents generated by wild-type Nav1.6 as well as the aberrant resurgent and persistent current generated by Nav1.6 mutant channels. To further validate our findings, we examined the effects of cannabidiol on endogenous sodium currents from striatal neurons, and similarly we found an inhibition of resurgent and persistent current by cannabidiol. Moreover, current clamp recordings show that cannabidiol reduces overall action potential firing of striatal neurons. These findings suggest that cannabidiol could be exerting its anticonvulsant effects, at least in part, through its actions on voltage-gated sodium channels, and resurgent current may be a promising therapeutic target for the treatment of epilepsy syndromes.

Dataset Information

Two Nedd4-binding motifs underlie modulation of sodium channel Nav1.6 by p38 MAPK.

Publications

Two Nedd4-binding motifs underlie modulation of sodium channel Nav1.6 by p38 MAPK.

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