Two Nedd4-binding motifs underlie modulation of sodium channel Nav1.6 by p38 MAPK.
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.
Department of Neurology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Andreas
A
Gasser
Xiaoyang
X
Cheng
Elaine S
ES
Gilmore
Lynda
L
Tyrrell
Stephen G
SG
Waxman
Sulayman D
SD
Dib-Hajj
D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D004594:Electrophysiology; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism*; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D062557:NAV1.6 Voltage-Gated Sodium Channel; D000075702:Nedd4 Ubiquitin Protein Ligases; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009474:Neurons / Q000502:physiology; D010766:Phosphorylation; D015222:Sodium Channels / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
D056827:Endosomal Sorting Complexes Required for Transport; D062557:NAV1.6 Voltage-Gated Sodium Channel; D009419:Nerve Tissue Proteins; C491046:Scn8a protein, mouse; D015222:Sodium Channels; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618485:Nedd4L protein, human; C000618461:Nedd4l protein, mouse; D044767:Ubiquitin-Protein Ligases; D048051:p38 Mitogen-Activated Protein Kinases