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• W2016322057 abstract "Sodium channel Nav1.6 is essential for neuronal excitability in central and peripheral nervous systems. Loss-of-function mutations in Nav1.6 underlie motor disorders, with homozygous-null mutations causing juvenile lethality. Phosphorylation of Nav1.6 by the stress-induced p38 MAPK at a Pro-Gly-Ser553-Pro motif in its intracellular loop L1 reduces Nav1.6 current density in a dorsal root ganglion-derived cell line, without changing its gating properties. Phosphorylated Pro-Gly-Ser553-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 Scn8amedtg mice that lack Nav1.6, reduces tetrodotoxin-S sodium currents, suggesting isoform-specific modulation of Nav1.6 by p38 in these neurons. Pharmacological block of endocytosis completely abolishes p38-mediated Nav1.6 current reduction, supporting our hypothesis that channel internalization underlies current reduction. We also report that the ubiquitin ligase Nedd4-2 interacts with Nav1.6 via a Pro-Ser-Tyr1945 motif in the C terminus of the channel and reduces Nav1.6 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 Nav1.6 current, whereas abrogating binding of the ubiquitin ligase Nedd4-2 to the Pro-Ser-Tyr motif results in stress-mediated increase in Nav1.6 current density. Thus, phosphorylation of the Pro-Gly-Ser-Pro motif within L1 of Nav1.6 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. Sodium channel Nav1.6 is essential for neuronal excitability in central and peripheral nervous systems. Loss-of-function mutations in Nav1.6 underlie motor disorders, with homozygous-null mutations causing juvenile lethality. Phosphorylation of Nav1.6 by the stress-induced p38 MAPK at a Pro-Gly-Ser553-Pro motif in its intracellular loop L1 reduces Nav1.6 current density in a dorsal root ganglion-derived cell line, without changing its gating properties. Phosphorylated Pro-Gly-Ser553-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 Scn8amedtg mice that lack Nav1.6, reduces tetrodotoxin-S sodium currents, suggesting isoform-specific modulation of Nav1.6 by p38 in these neurons. Pharmacological block of endocytosis completely abolishes p38-mediated Nav1.6 current reduction, supporting our hypothesis that channel internalization underlies current reduction. We also report that the ubiquitin ligase Nedd4-2 interacts with Nav1.6 via a Pro-Ser-Tyr1945 motif in the C terminus of the channel and reduces Nav1.6 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 Nav1.6 current, whereas abrogating binding of the ubiquitin ligase Nedd4-2 to the Pro-Ser-Tyr motif results in stress-mediated increase in Nav1.6 current density. Thus, phosphorylation of the Pro-Gly-Ser-Pro motif within L1 of Nav1.6 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. IntroductionThe voltage-gated sodium channel (VGSC) 3The abbreviations used are: VGSCvoltage-gated sodium channelAISaxon initial segmentDRGdorsal root gangliaMAPKmitogen-activated protein kinaseNav1.6/L1intracellular loop 1 of Nav1.6Nav1.6/Cintracellular C-terminal domain of Nav1.6TTXtetrodotoxinBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolWTwild typePBSphosphate-buffered salineHRPhorseradish peroxidaseGFPgreen fluorescent proteinMOPS4-morpholinepropanesulfonic acidpFpicofaradHAhemagglutinin. Nav1.6 is abundant at axon initial segments (AIS), at mature nodes of Ranvier in myelinated fibers, and along nonmyelinated axons within the central and peripheral nervous system (1Caldwell J.H. Schaller K.L. Lasher R.S. Peles E. Levinson S.R. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 5616-5620Crossref PubMed Scopus (530) Google Scholar, 2Boiko T. Rasband M.N. Levinson S.R. Caldwell J.H. Mandel G. Trimmer J.S. Matthews G. Neuron. 2001; 30: 91-104Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 3Boiko T. Van Wart A. Caldwell J.H. Levinson S.R. Trimmer J.S. Matthews G. J. Neurosci. 2003; 23: 2306-2313Crossref PubMed Google Scholar, 4Black J.A. Renganathan M. Waxman S.G. Mol. Brain Res. 2002; 105: 19-28Crossref PubMed Scopus (116) Google Scholar). Loss-of-function mutations of Nav1.6 underlie motor disorders, and the null phenotype is lethal in mice (5Meisler M.H. Kearney J. Escayg A. MacDonald B.T. Sprunger L.K. Neuroscientist. 2001; 7: 136-145Crossref PubMed Scopus (52) Google Scholar, 6Meisler M.H. Plummer N.W. Burgess D.L. Buchner D.A. Sprunger L.K. Genetica. 2004; 122: 37-45Crossref PubMed Scopus (49) Google Scholar). Nav1.6 has been linked to axonal degeneration in multiple sclerosis (7Craner M.J. Newcombe J. Black J.A. Hartle C. Cuzner M.L. Waxman S.G. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 8168-8173Crossref PubMed Scopus (381) Google Scholar, 8Craner M.J. Hains B.C. Lo A.C. Black J.A. Waxman S.G. Brain. 2004; 127: 294-303Crossref PubMed Scopus (205) Google Scholar) and to neuronal death following traumatic brain injury (9Wolf J.A. Stys P.K. Lusardi T. Meaney D. Smith D.H. J. Neurosci. 2001; 21: 1923-1930Crossref PubMed Google Scholar). Furthermore, Nav1.6 regulates motility and phagocytosis in activated microglia and macrophages (10Black J.A. Liu S. Waxman S.G. Glia. 2008; 57: 1072-1081Crossref Scopus (104) Google Scholar, 11Carrithers M.D. Chatterjee G. Carrithers L.M. Offoha R. Iheagwara U. Rahner C. Graham M. Waxman S.G. J. Biol. Chem. 2009; 284: 8114-8126Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 12Craner M.J. Damarjian T.G. Liu S. Hains B.C. Lo A.C. Black J.A. Newcombe J. Cuzner M.L. Waxman S.G. Glia. 2005; 49: 220-229Crossref PubMed Scopus (213) Google Scholar). Thus, modulation of Nav1.6 may impact diverse functions of neurons and glial cells.Recently, we have shown that activation of the stress-induced p38 MAPK (activated p38, pp38, phosphorylated at Thr180/Tyr182) reduces Nav1.6 peak currents in a dorsal root ganglion-derived cell line (13Wittmack E.K. Rush A.M. Hudmon A. Waxman S.G. Dib-Hajj S.D. J. Neurosci. 2005; 25: 6621-6630Crossref PubMed Scopus (100) Google Scholar). Phosphorylation of Nav1.6 at a single serine residue (Ser553) within the sequence motif Pro-Gly-Ser553-Pro in loop 1 (Nav1.6/L1) by pp38 is necessary for current reduction (13Wittmack E.K. Rush A.M. Hudmon A. Waxman S.G. Dib-Hajj S.D. J. Neurosci. 2005; 25: 6621-6630Crossref PubMed Scopus (100) Google Scholar). This suggests that neuronal stress responses may involve phosphorylation of Nav1.6 and reduction of its current, resulting in attenuated neuronal excitability. However, the mechanism of pp38-mediated Nav1.6 current reduction is not yet known.Ubiquitin ligases, for example Nedd4-like proteins, can induce internalization of ion channels (14Abriel H. Staub O. Physiology. 2005; 20: 398-407Crossref PubMed Scopus (86) Google Scholar). Nedd4 family members carry different types and numbers of WW domains (amino acid segments delineated by two tryptophan residues), which determine their target specificity (15Ingham R.J. Gish G. Pawson T. Oncogene. 2004; 23: 1972-1984Crossref PubMed Scopus (391) Google Scholar). Type I WW domains bind to PXY motifs, whereas type IV WW domains interact with phosphorylated PX(pS/T)P motifs (16Sudol M. Hunter T. Cell. 2000; 103: 1001-1004Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). Furthermore, Nedd4 family members have been shown to bind to VGSCs via PXY motifs in the C termini of the channels (17Fotia A.B. Ekberg J. Adams D.J. Cook D.I. Poronnik P. Kumar S. J. Biol. Chem. 2004; 279: 28930-28935Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 18Rougier J.S. van Bemmelen M.X. Bruce M.C. Jespersen T. Gavillet B. Apothéloz F. Cordonier S. Staub O. Rotin D. Abriel H. Am. J. Physiol. Cell Physiol. 2005; 288: C692-C701Crossref PubMed Scopus (109) Google Scholar), thereby reducing peak currents in different heterologous expression systems (17Fotia A.B. Ekberg J. Adams D.J. Cook D.I. Poronnik P. Kumar S. J. Biol. Chem. 2004; 279: 28930-28935Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 18Rougier J.S. van Bemmelen M.X. Bruce M.C. Jespersen T. Gavillet B. Apothéloz F. Cordonier S. Staub O. Rotin D. Abriel H. Am. J. Physiol. Cell Physiol. 2005; 288: C692-C701Crossref PubMed Scopus (109) Google Scholar, 19van Bemmelen M.X. Rougier J.S. Gavillet B. Apothéloz F. Daidié D. Tateyama M. Rivolta I. Thomas M.A. Kass R.S. Staub O. Abriel H. Circ. Res. 2004; 95: 284-291Crossref PubMed Scopus (173) Google Scholar, 20Abriel H. Kamynina E. Horisberger J.D. Staub O. FEBS Lett. 2000; 466: 377-380Crossref PubMed Scopus (105) Google Scholar). The ubiquitin ligase Nedd4-2 contains both type I and type IV WW domains (15Ingham R.J. Gish G. Pawson T. Oncogene. 2004; 23: 1972-1984Crossref PubMed Scopus (391) Google Scholar) and interacts with the Pro-Ser-Tyr1945 motif in the C terminus of Nav1.6 (Nav1.6/C) (17Fotia A.B. Ekberg J. Adams D.J. Cook D.I. Poronnik P. Kumar S. J. Biol. Chem. 2004; 279: 28930-28935Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Notably, phosphorylation of Ser553 by pp38 could convert the Pro-Gly-Ser-Pro motif in Nav1.6/L1 into a type IV WW-binding site. Thus, we hypothesized that the WW type IV-binding motif in Nav1.6/L1, alone or together with the WW type I-binding motif in Nav1.6/C, might recruit Nedd4-2 to the channel, thereby inducing internalization and reduction of Nav1.6 current.Here, we show that stress-induced activation of p38 specifically reduces endogenous Nav1.6 current in hippocampal neurons, an effect that is abolished by a pharmacological block of endocytosis. We also show that both the Pro-Gly-Ser553-Pro motif in Nav1.6/L1 and the Pro-Ser-Tyr1945 motif in Nav1.6/C are necessary to induce internalization of a reporter protein. We also demonstrate that co-expression of Nedd4-2 and Nav1.6 reduces sodium currents, which is dependent upon both binding motifs being functional. Surprisingly, unlike its effect on wild-type channels, p38 activation increases the peak current of the Nav1.6Y1945A mutant channel. Taken together, these data suggest that a cooperative interaction of the two Nedd4-binding motifs in the L1 and C terminus is necessary for pp38-mediated regulation of Nav1.6 currents.DISCUSSIONWe have previously shown that activated p38 phosphorylates Nav1.6 exclusively at Ser553 within the Pro-Gly-Ser-Pro motif in Nav1.6/L1 (13Wittmack E.K. Rush A.M. Hudmon A. Waxman S.G. Dib-Hajj S.D. J. Neurosci. 2005; 25: 6621-6630Crossref PubMed Scopus (100) Google Scholar), which is conserved in L1 of all vertebrate orthologues identified to date (Fig. 2). Although the L1 region of rat Nav1.8 contains two similar motifs (PRSP and PQSP), but at different positions within this loop, all other VGSC family members lack PXSP motifs in their L1 sequences (see supplemental Fig. 1). In agreement with our results, the lack of PXSP motifs in other TTX-S channels expressed in the central nervous system suggests that stress-induced pp38 modulation of hippocampal neuronal sodium current is unique to Nav1.6 channels. Phosphorylation of Ser553 by pp38 reduces Nav1.6 currents in the DRG-derived cell line ND7/23 (13Wittmack E.K. Rush A.M. Hudmon A. Waxman S.G. Dib-Hajj S.D. J. Neurosci. 2005; 25: 6621-6630Crossref PubMed Scopus (100) Google Scholar), suggesting an adaptive mechanism that might limit sodium influx after injury (44Dargent B. Arsac C. Tricaud N. Couraud F. Neuroscience. 1996; 73: 209-216Crossref PubMed Scopus (31) Google Scholar, 45Paillart C. Boudier J.L. Boudier J.A. Rochat H. Couraud F. Dargent B. J. Cell Biol. 1996; 134: 499-509Crossref PubMed Scopus (25) Google Scholar). Here, we provide evidence that in hippocampal neurons (i) p38 and Nav1.6 are co-expressed (Fig. 1), (ii) p38 activation reduces native Nav1.6 currents without affecting other endogenous TTX-S sodium currents (Fig. 3), and (iii) pharmacological block of endocytosis abrogates p38-mediated current reduction (Fig. 4). Taken together, these data are consistent with a model of Nav1.6 internalization induced by p38-mediated phosphorylation of Ser553 within L1 of the channel.Although chimeric reporter proteins containing only Nav1.6/L1 or Nav1.6/C are not internalized upon p38 activation, the fusion of L1 and C terminus in one polypeptide restores, at least partially, the pp38-mediated internalization of the reporter protein from the plasma membrane (Fig. 5D). Furthermore, Nedd4-2 mediated reduction of Nav1.6 currents (Fig. 6F) depends upon both the Pro-Ser-Tyr motif in Nav1.6/C (Fig. 6F) and the Pro-Gly-Ser-Pro motif in Nav1.6/L1 (Fig. 7B). Mutating the Pro-Ser-Tyr motif in Nav1.6/C, which abolishes binding of Nedd4-2 to Nav1.6/C (Fig. 6A), surprisingly, reverses the pp38 effect on Nav1.6 by increasing the current density (Fig. 8A). Similar to the pp38-mediated current reduction of Nav1.6-WT, this effect depends upon phosphorylation of Ser553 in Nav1.6/L1 (Fig. 8B). Taken together, these data show that regulation of Nav1.6 by pp38 is a complex process that requires phosphorylation of Ser553 in Nav1.6/L1 but also depends on the binding of Nav1.6/C to Nedd4 ubiquitin ligases, and perhaps other proteins that remain to be identified.Nav1.6 is the predominant VGSC at AIS and nodes of Ranvier in myelinated neurons and is also present in unmyelinated parallel fibers (1Caldwell J.H. Schaller K.L. Lasher R.S. Peles E. Levinson S.R. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 5616-5620Crossref PubMed Scopus (530) Google Scholar, 2Boiko T. Rasband M.N. Levinson S.R. Caldwell J.H. Mandel G. Trimmer J.S. Matthews G. Neuron. 2001; 30: 91-104Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 3Boiko T. Van Wart A. Caldwell J.H. Levinson S.R. Trimmer J.S. Matthews G. J. Neurosci. 2003; 23: 2306-2313Crossref PubMed Google Scholar, 4Black J.A. Renganathan M. Waxman S.G. Mol. Brain Res. 2002; 105: 19-28Crossref PubMed Scopus (116) Google Scholar). Thus, acute regulation of Nav1.6 currents might modulate nerve impulse transmission and neuronal excitability and may allow neurons to adapt rapidly to stressful conditions. The presence of multiple VGSCs within the central nervous system neurons, which share similar biophysical and pharmacological properties, makes the analysis of Nav1.6 regulation challenging. To determine the contribution of effects on Nav1.6 to the neuronal stress response, we compared the impact of p38 activation on peak currents of endogenous TTX-S channels in hippocampal neurons from WT and Scn8amedtg mice, which lack Nav1.6 (6Meisler M.H. Plummer N.W. Burgess D.L. Buchner D.A. Sprunger L.K. Genetica. 2004; 122: 37-45Crossref PubMed Scopus (49) Google Scholar). We show here that p38 activation reduces endogenous TTX-S peak currents in hippocampal neurons from WT mice but not from Scn8amedtg mice, suggesting that other endogenous sodium channels present in these neurons are not regulated by pp38. Because activation of p38 does not alter biophysical properties of Nav1.6 currents in hippocampal neurons and in ND7/23 cells, we suggest that peak currents may be reduced by channel internalization. This hypothesis is supported by our finding that pp38-mediated current reduction in hippocampal neurons is abolished by the dynamin inhibitor Dynasore, which has been previously shown to block endocytosis in different cell types, including neurons (29Kirchhausen T. Macia E. Pelish H.E. Methods Enzymol. 2008; 438: 77-93Crossref PubMed Scopus (300) Google Scholar, 30Macia E. Ehrlich M. Massol R. Boucrot E. Brunner C. Kirchhausen T. Dev. Cell. 2006; 10: 839-850Abstract Full Text Full Text PDF PubMed Scopus (1472) Google Scholar).Sodium channel density at the plasma membrane is known to be regulated by cytosolic binding partners, which link channels to various internalization pathways. For example, SCLT1 (sodium channel-CLaThrin-linker 1; previously known as CAP1A), an isoform-specific adaptor protein, binds to both clathrin and Nav1.8 and facilitates internalization of the channel (46Liu C. Cummins T.R. Tyrrell L. Black J.A. Waxman S.G. Dib-Hajj S.D. Mol. Cell. Neurosci. 2005; 28: 636-649Crossref PubMed Scopus (33) Google Scholar). In contrast, Nedd4 family ubiquitin ligases are ubiquitously expressed and can induce internalization of several sodium channels, including ENaC (47Staub O. Dho S. Henry P. Correa J. Ishikawa T. McGlade J. Rotin D. EMBO J. 1996; 15: 2371-2380Crossref PubMed Scopus (735) Google Scholar, 48Staub O. Gautschi I. Ishikawa T. Breitschopf K. Ciechanover A. Schild L. Rotin D. EMBO J. 1997; 16: 6325-6336Crossref PubMed Scopus (597) Google Scholar) and some VGSCs (17Fotia A.B. Ekberg J. Adams D.J. Cook D.I. Poronnik P. Kumar S. J. Biol. Chem. 2004; 279: 28930-28935Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 18Rougier J.S. van Bemmelen M.X. Bruce M.C. Jespersen T. Gavillet B. Apothéloz F. Cordonier S. Staub O. Rotin D. Abriel H. Am. J. Physiol. Cell Physiol. 2005; 288: C692-C701Crossref PubMed Scopus (109) Google Scholar, 19van Bemmelen M.X. Rougier J.S. Gavillet B. Apothéloz F. Daidié D. Tateyama M. Rivolta I. Thomas M.A. Kass R.S. Staub O. Abriel H. Circ. Res. 2004; 95: 284-291Crossref PubMed Scopus (173) Google Scholar, 20Abriel H. Kamynina E. Horisberger J.D. Staub O. FEBS Lett. 2000; 466: 377-380Crossref PubMed Scopus (105) Google Scholar). Previously, Nedd4 and Nedd4-2 were shown to bind to the Nav1.6/C region, but the functional impact of this interaction, as well as possible interactions with other intracellular regions of the channel, were not reported (17Fotia A.B. Ekberg J. Adams D.J. Cook D.I. Poronnik P. Kumar S. J. Biol. Chem. 2004; 279: 28930-28935Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Here, we present data that clearly demonstrate a functional effect of the interaction of Nedd4-2 and full-length Nav1.6, resulting in a reduction of peak sodium current.Ubiquitin ligases induce internalization of target proteins with the outcome dependent on whether mono- or poly-ubiquitination takes place (reviewed in Ref. 14Abriel H. Staub O. Physiology. 2005; 20: 398-407Crossref PubMed Scopus (86) Google Scholar). Nedd4 family ubiquitin ligases (31Chen C. Matesic L.E. Cancer Metastasis Rev. 2007; 26: 587-604Crossref PubMed Scopus (170) Google Scholar) contain different numbers of type I WW domains (16Sudol M. Hunter T. Cell. 2000; 103: 1001-1004Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar) that bind to PXY sequence motifs, which are present in the C termini of all VGSC family members except Nav1.4 and Nav1.9 channels (see supplemental Fig. 2). Here, we confirm that the C terminus of Nav1.6 binds to Nedd4-2 (Fig. 6A), and we demonstrate that co-expression of Nav1.6 with Nedd4-2 results in a robust reduction of Nav1.6 peak currents (Figs. 6D and 7B). This functional effect depends on the Pro-Ser-Tyr motif in Nav1.6/C (Fig. 6D), similar to Nav1.5 (19van Bemmelen M.X. Rougier J.S. Gavillet B. Apothéloz F. Daidié D. Tateyama M. Rivolta I. Thomas M.A. Kass R.S. Staub O. Abriel H. Circ. Res. 2004; 95: 284-291Crossref PubMed Scopus (173) Google Scholar, 20Abriel H. Kamynina E. Horisberger J.D. Staub O. FEBS Lett. 2000; 466: 377-380Crossref PubMed Scopus (105) Google Scholar) and Nav1.8 (17Fotia A.B. Ekberg J. Adams D.J. Cook D.I. Poronnik P. Kumar S. J. Biol. Chem. 2004; 279: 28930-28935Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Interestingly, we found that Nedd4-2-mediated current reduction of Nav1.6 also depends on a functional Pro-Gly-Ser-Pro motif in Nav1.6/L1, because regulation by Nedd4-2 is abolished in the mutant channel Nav1.6S553A. Although we did not activate p38 by anisomycin treatment in these experiments, these data suggest that phosphorylation of Ser553 in WT Nav1.6 takes place, which may be due to basal pp38 activity or activities of other MAPK that are able to phosphorylate SP motifs (e.g. ERK1/2 or JNK). Consistent with this hypothesis is our finding that purified recombinant Nav1.6/L1B produced in bacteria, which is not expected to be phosphorylated at Ser553, does not bind to Nedd4-2 (Fig. 6A), either because of too low affinity or because of phosphorylation-dependent folding of L1 that becomes permissive to Nedd4 binding. Unmasking of weak interactions by overexpression of Nedd4-2 in transfected cells or the marked strengthening of PGpSP affinity to Nedd4 may explain our data.Previously, we hypothesized (13Wittmack E.K. Rush A.M. Hudmon A. Waxman S.G. Dib-Hajj S.D. J. Neurosci. 2005; 25: 6621-6630Crossref PubMed Scopus (100) Google Scholar) that proteins containing type IV WW domains bind to the Pro-Gly-Ser-Pro motif in Nav1.6/L1 after phosphorylation of Ser553 by pp38. Thus, Nedd4-2 might be recruited to the channel after stress-induced p38 activation, causing ubiquitination and internalization of Nav1.6. However, a CD4-L1-EGFP reporter protein carrying the Pro-Gly-Ser-Pro motif was not internalized after p38 activation (Fig. 5), which indicates that Ser553 within the Pro-Gly-Ser-Pro motif is necessary but not sufficient to transduce the effects of pp38. Similarly, p38 activation had no effects on a reporter protein containing only the Nav1.6/C region. In contrast, a reporter protein carrying both Nav1.6/L1 and Nav1.6/C showed a 15% reduction in cell surface localization upon p38 activation (Fig. 5). This suggests that the interaction between the phosphorylated Nav1.6/L1 region and Nedd4 family proteins alone may not be robust enough to facilitate ubiquitination and subsequent internalization of the reporter protein. The additional Pro-Ser-Tyr motif of Nav1.6/C in the CD4-L1-C-EGFP protein could allow a co-operative binding of Nedd4 proteins to the phosphorylated Pro-Gly-Ser-Pro motif in L1, thus inducing ubiquitination and internalization of the protein. However, the reduction of Nav1.6 peak currents in ND7/23 cells upon p38 activation, which was in the range of 40–50% (Fig. 8) (13Wittmack E.K. Rush A.M. Hudmon A. Waxman S.G. Dib-Hajj S.D. J. Neurosci. 2005; 25: 6621-6630Crossref PubMed Scopus (100) Google Scholar), was markedly higher than the 15% reduction of surface expression of the reporter protein. A possible explanation for this disparity could be that additional target sites for ubiquitination may be located outside of the Nav1.6/L1 and Nav1.6/C regions of the channel, resulting in an impaired ubiquitination of the chimeric protein compared with the full-length channel. Consistent with this hypothesis, ENaC sodium channel subunits, which are regulated by Nedd4 family proteins upon binding to C-terminal PXY motifs, are ubiquitinated at sites within their N termini (48Staub O. Gautschi I. Ishikawa T. Breitschopf K. Ciechanover A. Schild L. Rotin D. EMBO J. 1997; 16: 6325-6336Crossref PubMed Scopus (597) Google Scholar). Alternatively, the folded structure of the Nav1.6 fragments in the CD4-L1-C-EGFP reporter protein may not be as well suited for interaction with Nedd4 proteins as a result of their topology within the full-length channel protein.Our data show that both the Pro-Ser-Tyr motif in Nav1.6/C and the Pro-Gly-Ser-Pro motif in Nav1.6/L1 play important roles in the regulation of this channel. In agreement with published data on VGSCs Nav1.5 (19van Bemmelen M.X. Rougier J.S. Gavillet B. Apothéloz F. Daidié D. Tateyama M. Rivolta I. Thomas M.A. Kass R.S. Staub O. Abriel H. Circ. Res. 2004; 95: 284-291Crossref PubMed Scopus (173) Google Scholar, 20Abriel H. Kamynina E. Horisberger J.D. Staub O. FEBS Lett. 2000; 466: 377-380Crossref PubMed Scopus (105) Google Scholar) and Nav1.8 (17Fotia A.B. Ekberg J. Adams D.J. Cook D.I. Poronnik P. Kumar S. J. Biol. Chem. 2004; 279: 28930-28935Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), the mutant channel Nav1.6Y1945A is no longer regulated by Nedd4-2 (Fig. 6B). Surprisingly, activation of p38 increased the peak current of Nav1.6Y1945A (Fig. 8A), in contrast to the pp38-induced reduction of Nav1.6-WT peak current. Although the mechanistic details are not well understood, the increase in the peak current of Nav1.6Y1945A by pp38 is reminiscent of the pp38-induced increase in Nav1.8 current density in DRG neurons (43Hudmon A. Choi J.S. Tyrrell L. Black J.A. Rush A.M. Waxman S.G. Dib-Hajj S.D. J. Neurosci. 2008; 28: 3190-3201Crossref PubMed Scopus (145) Google Scholar). However, the increase of the peak current of Nav1.6Y1945A by pp38 was abolished by the substitution S553A in the Pro-Gly-Ser-Pro motif in Nav1.6/L1 (Fig. 8B). This indicates that both possible outcomes of the dynamic regulation of Nav1.6 by pp38, a decrease in current density of Nav1.6-WT and an increase in current density of Nav1.6Y1945A, depends upon phosphorylation of Ser553 within Nav1.6/L1.Taken together, the data presented here reveal a complex regulation of Nav1.6 by pp38. Stress-activated p38 induces phosphorylation of Nav1.6, which causes a reduction in Nav1.6 peak currents in native neurons and neuronal cell lines (this study and Ref. 13Wittmack E.K. Rush A.M. Hudmon A. Waxman S.G. Dib-Hajj S.D. J. Neurosci. 2005; 25: 6621-6630Crossref PubMed Scopus (100) Google Scholar). Block of endocytosis abolishes this current reduction, suggesting that phosphorylation of Nav1.6 reduces the number of available channels by internalization. Thus neurons may modulate their excitability as an adaptive response toward cellular stress. However, our data suggest a dual function of the Pro-Gly-Ser-Pro motif within Nav1.6/L1, which can act as a switch that transduces pp38 effects in a positive or negative direction, depending on the ability of Nedd4-2 to bind the Pro-Ser-Tyr motif in the C terminus of the channel. IntroductionThe voltage-gated sodium channel (VGSC) 3The abbreviations used are: VGSCvoltage-gated sodium channelAISaxon initial segmentDRGdorsal root gangliaMAPKmitogen-activated protein kinaseNav1.6/L1intracellular loop 1 of Nav1.6Nav1.6/Cintracellular C-terminal domain of Nav1.6TTXtetrodotoxinBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolWTwild typePBSphosphate-buffered salineHRPhorseradish peroxidaseGFPgreen fluorescent proteinMOPS4-morpholinepropanesulfonic acidpFpicofaradHAhemagglutinin. Nav1.6 is abundant at axon initial segments (AIS), at mature nodes of Ranvier in myelinated fibers, and along nonmyelinated axons within the central and peripheral nervous system (1Caldwell J.H. Schaller K.L. Lasher R.S. Peles E. Levinson S.R. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 5616-5620Crossref PubMed Scopus (530) Google Scholar, 2Boiko T. Rasband M.N. Levinson S.R. Caldwell J.H. Mandel G. Trimmer J.S. Matthews G. Neuron. 2001; 30: 91-104Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 3Boiko T. Van Wart A. Caldwell J.H. Levinson S.R. Trimmer J.S. Matthews G. J. Neurosci. 2003; 23: 2306-2313Crossref PubMed Google Scholar, 4Black J.A. Renganathan M. Waxman S.G. Mol. Brain Res. 2002; 105: 19-28Crossref PubMed Scopus (116) Google Scholar). Loss-of-function mutations of Nav1.6 underlie motor disorders, and the null phenotype is lethal in mice (5Meisler M.H. Kearney J. Escayg A. MacDonald B.T. Sprunger L.K. Neuroscientist. 2001; 7: 136-145Crossref PubMed Scopus (52) Google Scholar, 6Meisler M.H. Plummer N.W. Burgess D.L. Buchner D.A. Sprunger L.K. Genetica. 2004; 122: 37-45Crossref PubMed Scopus (49) Google Scholar). Nav1.6 has been linked to axonal degeneration in multiple sclerosis (7Craner M.J. Newcombe J. Black J.A. Hartle C. Cuzner M.L. Waxman S.G. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 8168-8173Crossref PubMed Scopus (381) Google Scholar, 8Craner M.J. Hains B.C. Lo A.C. Black J.A. Waxman S.G. Brain. 2004; 127: 294-303Crossref PubMed Scopus (205) Google Scholar) and to neuronal death following traumatic brain injury (9Wolf J.A. Stys P.K. Lusardi T. Meaney D. Smith D.H. J. Neurosci. 2001; 21: 1923-1930Crossref PubMed Google Scholar). Furthermore, Nav1.6 regulates motility and phagocytosis in activated microglia and macrophages (10Black J.A. Liu S. Waxman S.G. Glia. 2008; 57: 1072-1081Crossref Scopus (104) Google Scholar, 11Carrithers M.D. Chatterjee G. Carrithers L.M. Offoha R. Iheagwara U. Rahner C. Graham M. Waxman S.G. J. Biol. Chem. 2009; 284: 8114-8126Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 12Craner M.J. Damarjian T.G. Liu S. Hains B.C. Lo A.C. Black J.A. Newcombe J. Cuzner M.L. Waxman S.G. Glia. 2005; 49: 220-229Crossref PubMed Scopus (213) Google Scholar). Thus, modulation of Nav1.6 may impact diverse functions of neurons and glial cells.Recently, we have shown that activation of the stress-induced p38 MAPK (activated p38, pp38, phosphorylated at Thr180/Tyr182) reduces Nav1.6 peak currents in a dorsal root ganglion-derived cell line (13Wittmack E.K. Rush A.M. Hudmon A. Waxman S.G. Dib-Hajj S.D. J. Neurosci. 2005; 25: 6621-6630Crossref PubMed Scopus (100) Google Scholar). Phosphorylation of Nav1.6 at a single serine residue (Ser553) within the sequence motif Pro-Gly-Ser553-Pro in loop 1 (Nav1.6/L1) by pp38 is necessary for current reduction (13Wittmack E.K. Rush A.M. Hudmon A. Waxman S.G. Dib-Hajj S.D. J. Neurosci. 2005; 25: 6621-6630Crossref PubMed Scopus (100) Google Scholar). This suggests that neuronal stress responses may involve phosphorylation of Nav1.6 and reduction of its current, resulting in attenuated neuronal excitability. However, the mechanism of pp38-mediated Nav1.6 current reduction is not yet known.Ubiquitin ligases, for example Nedd4-like proteins, can induce internalization of ion channels (14Abriel H. Staub O. Physiology. 2005; 20: 398-407Crossref PubMed Scopus (86) Google Scholar). Nedd4 family members carry different types and numbers of WW domains (amino acid segments delineated by two tryptophan residues), which determine their target specificity (15Ingham R.J. Gish G. Pawson T. Oncogene. 2004; 23: 1972-1984Crossref PubMed Scopus (391) Google Scholar). Type I WW domains bind to PXY motifs, whereas type IV WW domains interact with phosphorylated PX(pS/T)P motifs (16Sudol M. Hunter T. Cell. 2000; 103: 1001-1004Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). Furthermore, Nedd4 family members have been shown to bind to VGSCs via PXY motifs in the C termini of the channels (17Fotia A.B. Ekberg J. Adams D.J. Cook D.I. Poronnik P. Kumar S. J. Biol. Chem. 2004; 279: 28930-28935Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 18Rougier J.S. van Bemmelen M.X. Bruce M.C. Jespersen T. Gavillet B. Apothéloz F. Cordonier S. Staub O. Rotin D. Abriel H. Am. J. Physiol. Cell Physiol. 2005; 288: C692-C701Crossref PubMed Scopus (109) Google Scholar), thereby reducing peak currents in different heterologous expression systems (17Fotia A.B. Ekberg J. Adams D.J. Cook D.I. Poronnik P. Kumar S. J. Biol. Chem. 2004; 279: 28930-28935Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 18Rougier J.S. van Bemmelen M.X. Bruce M.C. Jespersen T. Gavillet B. Apothéloz F. Cordonier S. Staub O. Rotin D. Abriel H. Am. J. Physiol. Cell Physiol. 2005; 288: C692-C701Crossref PubMed Scopus (109) Google Scholar, 19van Bemmelen M.X. Rougier J.S. Gavillet B. Apothéloz F. Daidié D. Tateyama M. Rivolta I. Thomas M.A. Kass R.S. Staub O. Abriel H. Circ. Res. 2004; 95: 284-291Crossref PubMed Scopus (173) Google Scholar, 20Abriel H. Kamynina E. Horisberger J.D. Staub O. FEBS Lett. 2000; 466: 377-380Crossref PubMed Scopus (105) Google Scholar). The ubiquitin ligase Nedd4-2 contains both type I and type IV WW domains (15Ingham R.J. Gish G. Pawson T. Oncogene. 2004; 23: 1972-1984Crossref PubMed Scopus (391) Google Scholar) and interacts with the Pro-Ser-Tyr1945 motif in the C terminus of Nav1.6 (Nav1.6/C) (17Fotia A.B. Ekberg J. Adams D.J. Cook D.I. Poronnik P. Kumar S. J. Biol. Chem. 2004; 279: 28930-28935Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Notably, phosphorylation of Ser553 by pp38 could convert the Pro-Gly-Ser-Pro motif in Nav1.6/L1 into a type IV WW-binding site. Thus, we hypothesized that the WW type IV-binding motif in Nav1.6/L1, alone or together with the WW type I-binding motif in Nav1.6/C, might recruit Nedd4-2 to the channel, thereby inducing internalization and reduction of Nav1.6 current.Here, we show that stress-induced activation of p38 specifically reduces endogenous Nav1.6 current in hippocampal neurons, an effect that is abolished by a pharmacological block of endocytosis. We also show that both the Pro-Gly-Ser553-Pro motif in Nav1.6/L1 and the Pro-Ser-Tyr1945 motif in Nav1.6/C are necessary to induce internalization of a reporter protein. We also demonstrate that co-expression of Nedd4-2 and Nav1.6 reduces sodium currents, which is dependent upon both binding motifs being functional. Surprisingly, unlike its effect on wild-type channels, p38 activation increases the peak current of the Nav1.6Y1945A mutant channel. Taken together, these data suggest that a cooperative interaction of the two Nedd4-binding motifs in the L1 and C terminus is necessary for pp38-mediated regulation of Nav1.6 currents." @default.
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• W2016322057 title "Two Nedd4-binding Motifs Underlie Modulation of Sodium Channel Nav1.6 by p38 MAPK" @default.
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