Two Nedd4-binding Motifs Underlie Modulation of Sodium Channel Na(v)1.6 by p38 MAPK

Department of Neurology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 08/2010; 285(34):26149-61. DOI: 10.1074/jbc.M109.098681
Source: PubMed

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.

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Available from: Xiaoyang Cheng, Sep 26, 2015
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    • "A recent study (Gasser et al. 2010) demonstrated that a conserved PGSP motif in the intracellular loop L1 of Na v 1.6 was phosphorylated by phospho-p38 (activated stress-induced p38 MAPK), which converted this motif into a Px(pS)P Nedd4-2 binding motif (Fig. 3). It was reported that both the LPxY motif in the C-terminus and the Px(pS)P in L1 were necessary for Nedd4-2 to downregulate Na v 1.6 (Gasser et al. 2010). Thus far, the necessity of an intact PY motif in Na v s for Nedd4-dependent regulation has been demonstrated for every isoform tested. "
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    ABSTRACT: Ion channel proteins are regulated by different types of posttranslational modifications. The focus of this review is the regulation of voltage-gated sodium channels (Navs) upon their ubiquitylation. The amiloride-sensitive epithelial sodium channel (ENaC) was the first ion channel shown to be regulated upon ubiquitylation. This modification results from the binding of ubiquitin ligase from the Nedd4 family to a protein-protein interaction domain, known as the PY motif, in the ENaC subunits. Many of the Navs have similar PY motifs, which have been demonstrated to be targets of Nedd4-dependent ubiquitylation, tagging them for internalization from the cell surface. The role of Nedd4-dependent regulation of the Nav membrane density in physiology and disease remains poorly understood. Two recent studies have provided evidence that Nedd4-2 is downregulated in dorsal root ganglion (DRG) neurons in both rat and mouse models of nerve injury-induced neuropathic pain. Using two different mouse models, one with a specific knockout of Nedd4-2 in sensory neurons and another where Nedd4-2 was overexpressed with the use of viral vectors, it was demonstrated that the neuropathy-linked neuronal hyperexcitability was the result of Nav1.7 and Nav1.8 overexpression due to Nedd4-2 downregulation. These studies provided the first in vivo evidence of the role of Nedd4-2-dependent regulation of Nav channels in a disease state. This ubiquitylation pathway may be involved in the development of symptoms and diseases linked to Nav-dependent hyperexcitability, such as pain, cardiac arrhythmias, epilepsy, migraine, and myotonias.
    Handbook of experimental pharmacology 04/2014; 221:231-250. DOI:10.1007/978-3-642-41588-3_11
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    • "The N-terminus of Nav1.6 interacts with the light chain of microtubule-associated protein Map1b (Mtap1b), and co-transfection increases current density in transfected cells via increased trafficking of Nav1.6 to the cell surface (O'Brien et al., 2012b). Phosphorylation of Nav1.6 by the stress-activated MAP kinase p38 facilitates binding of E3 ubiquitin ligases and channel degradation (Sudol and Hunter, 2000; Zarrinpar and Lim, 2000; Gasser et al., 2010). Protein kinases PKA and PKC have only a small effect on channel activity (Chen et al., 2008). "
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    ABSTRACT: The sodium channel Nav1.6, encoded by the gene SCN8A, is one of the major voltage-gated channels in human brain. The sequences of sodium channels have been highly conserved during evolution, and minor changes in biophysical properties can have a major impact in vivo. Insight into the role of Nav1.6 has come from analysis of spontaneous and induced mutations of mouse Scn8a during the past 18 years. Only within the past year has the role of SCN8A in human disease become apparent from whole exome and genome sequences of patients with sporadic disease. Unique features of Nav1.6 include its contribution to persistent current, resurgent current, repetitive neuronal firing, and subcellular localization at the axon initial segment (AIS) and nodes of Ranvier. Loss of Nav1.6 activity results in reduced neuronal excitability, while gain-of-function mutations can increase neuronal excitability. Mouse Scn8a (med) mutants exhibit movement disorders including ataxia, tremor and dystonia. Thus far, more than ten human de novo mutations have been identified in patients with two types of disorders, epileptic encephalopathy and intellectual disability. We review these human mutations as well as the unique features of Nav1.6 that contribute to its role in determining neuronal excitability in vivo. A supplemental figure illustrating the positions of amino acid residues within the four domains and 24 transmembrane segments of Nav1.6 is provided to facilitate the location of novel mutations within the channel protein.
    Frontiers in Genetics 10/2013; 4:213. DOI:10.3389/fgene.2013.00213
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