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Molecular Pain

Open Access

Trafficking regulates the subcellular distribution of voltage-gated sodium channels in primary sensory neurons

Molecular Pain201511:61

https://doi.org/10.1186/s12990-015-0065-7

Received: 24 July 2015

Accepted: 23 September 2015

Published: 30 September 2015

Abstract

Voltage-gated sodium channels (Navs) comprise at least nine pore-forming α subunits. Of these, Nav1.6, Nav1.7, Nav1.8 and Nav1.9 are the most frequently studied in primary sensory neurons located in the dorsal root ganglion and are mainly localized to the cytoplasm. A large pool of intracellular Navs raises the possibility that changes in Nav trafficking could alter channel function. The molecular mediators of Nav trafficking mainly consist of signals within the Navs themselves, interacting proteins and extracellular factors. The surface expression of Navs is achieved by escape from the endoplasmic reticulum and proteasome degradation, forward trafficking and plasma membrane anchoring, and it is also regulated by channel phosphorylation and ubiquitination in primary sensory neurons. Axonal transport and localization of Navs in afferent fibers involves the motor protein KIF5B and scaffold proteins, including contactin and PDZ domain containing 2. Localization of Nav1.6 to the nodes of Ranvier in myelinated fibers of primary sensory neurons requires node formation and the submembrane cytoskeletal protein complex. These findings inform our understanding of the molecular and cellular mechanisms underlying Nav trafficking in primary sensory neurons.

Keywords

Voltage-gated sodium channelPrimary sensory neuronTrafficking regulation

Background

Voltage-gated sodium channels (Navs) comprise the rising phase of action potentials and are therefore a critical factor in neuronal excitability. Navs contain α and β subunits; however, α subunits alone execute channel functions. To date, nine isoforms of the α subunit (Nav1.1–1.9), which display various channel properties and selective tissue distribution, have been discovered. Nav1.7, Nav1.8 and Nav1.9 are peripheral Navs that are highly and selectively expressed in primary sensory neurons located in the dorsal root ganglion (DRG). Recent progresses have revealed the importance of Nvs in human pain disorders, especially Nav1.7, Nav1.8 and Nav1.9 [1, 2]. In general, the function of Navs is regulated by their expression level, channel properties and subcellular distribution. Here, we focus on the regulation of the subcellular distribution of Navs in adult primary sensory neurons by Nav trafficking.

Cellular and subcellular distribution of Navs in primary sensory neurons

The cellular distribution of Navs in primary sensory neurons is mainly detected by in situ hybridization and immunohistochemistry and, more recently, by single-cell polymerase chain reaction (PCR) and RNA sequencing. The electrophysiological detection of voltage-gated sodium currents in individual neurons also significantly helps to identify functional channel expression. In adult primary sensory neurons, three tetrodotoxin-sensitive (TTX-S; Nav1.1, Nav1.6 and Nav1.7) and two tetrodotoxin-resistant (TTX-R; Nav1.8 and Nav1.9) sodium channels have been identified. High level of TTX-S Nav1.2 and Nav1.3 is embryonically expressed in DRG neurons and dramatically decreased after post-natal, whereas Nav1.3 is re-expressed under certain pathological conditions that involve peripheral nerve injuries. DRG neurons are usually divided by size into small neuron (such as <800 µm2 in mice) and large neuron (>800 µm2) subsets that primarily consist of nociceptors and mechanoreceptors, respectively. Extensive recent studies using in situ hybridization have revealed that Nav1.1 and Nav1.6 are mainly expressed in 200 kDa neurofilament subunit (NF200)-positive neurons and that high levels of Nav1.7, Nav1.8 and Nav1.9 are found in NF200-negative neurons [3, 4]. High levels of Nav1.7 and Nav1.8 are also detected in NF200/tropomyosin receptor kinase A (TrkA)-positive neurons, and Nav1.7 is additionally expressed in half of the NF200-positive and TrkA-negative neurons [4]. Recent transcriptional profiling by single-cell PCR has confirmed that Nav1.7, Nav1.8 and Nav1.9 are enriched in both the isolectin B4 (IB4)-positive and the IB4-negative SNS-Cre/TdTomato populations, while Nav1.1 and Nav1.6 are mainly expressed in Parvalbumin-Cre/TdTomato neurons [5]. Each isoform may exhibit a different distribution pattern when detected by immunohistochemistry compared with in situ hybridization. Although antibodies are generally specific and sensitive, some antibodies only recognize specific protein structures in a subset of cells under particular conditions. In addition, regulation of protein translation may cause a divergence in protein and mRNA levels and lead to further discrepancies between the cellular distributions detected by the two methods.

Although combined methods are used to discover the cellular distribution pattern of Nav channels, subcellular localization can only be detected by immunohistochemistry. In primary sensory neurons, Nav1.6, Nav1.7, Nav1.8 and Nav1.9 are the most frequently studied and display a primarily intracellular localization in the cell body, as detected by specific antibodies [611]. Nav1.6 and Nav1.7 are also localized to the nodes of Ranvier in myelinated fibers of the sciatic nerve and distributed throughout unmyelinated fibers of the sciatic nerve and dorsal root [7, 10, 12, 13]. Nav1.7 localizes to peripheral terminals in the skin and central terminals in the dorsal horn [7]. Nav1.8 is distributed in afferent fibers of the sciatic nerve and dorsal root [8, 11] and in axons of cultured DRG neurons [14] and further revealed to be localized at lipid rafts, especially in the axons of cultured small DRG neurons and sciatic nerve [15]. Nav1.9 preferentially localizes along axons of the IB4-positive unmyelinated fibers in the sciatic nerve [16].

In general, Navs must be inserted into the plasma membrane of cell bodies and axons to function in neurons. A large pool of intracellular Navs suggests the possibility that alterations in the mode of Nav trafficking could lead to quick changes in channel and neuron function. In the physiological condition, an efficient expression of Navs on the cell surface and in the axon of DRG neurons is in favor of primary sensation. However, an excessive increase of Nav trafficking to cell surface in pathological conditions including peripheral inflammation and nerve injury induces abnormally neuronal excitability, which may reduce the response threshold of DRG neurons and involve in the development of pathological pain.

Strategy to study the mechanisms that regulate Nav trafficking

Navs, similar to other membrane proteins, are synthesized in the rough endoplasmic reticulum (ER) and transported via vesicles. Regulation of the trafficking of these proteins can provide a quick and highly efficient way for cells to respond to the extracellular environment aside from transcriptional regulation. The molecular mechanisms that regulate Nav trafficking depend primarily on three components: amino acids, motifs or sequences located within the channels that mediate the regulation, interacting proteins that respond to signals and connect with the trafficking machinery, and extracellular factors that transfer changes in the extracellular environment to neurons.

Navs consist of four domains connected by three intracellular loops; each domain is formed by six transmembrane segments. Both the N-terminus and the C-terminus of Navs are located in the cytoplasm. To identify the amino acids, motifs or sequences in Navs that mediate the trafficking regulation, model molecules such as CD4, CD8α and transferring receptor 1 (TFR1), which have distinct cell surface localization, can be adapted to detect the roles that particular regions of the Navs have in subcellular distribution (Fig. 1) [11, 17, 18]. To ascertain the effect of intracellular and transmembrane segments, the orientation of the Nav intracellular sequence and transmembrane segment within the membrane should be considered when constructing the adapted molecule. The type I membrane protein CD8α is suitable for testing three intracellular loops, the C-terminus and the transmembrane segments that pass through the membrane in the extracellular to intracellular direction, whereas the type II membrane protein TFR1 is appropriate for testing the N-terminus and the transmembrane segments that pass through the membrane in the opposite direction (Fig. 1) [18]. Additionally, the specific cells used to examine distinct subcellular structures should be considered. For example, because neurons are round and their subcellular structures are occasionally obscured, COS-7 cells, which are derived from African green monkey kidney fibroblast-like cells and are flat, are usually used to analyze the subcellular localization of proteins in organelles. Importantly, the amino acids, motifs and sequences that are thought to mediate the regulation of Nav trafficking should ultimately be tested by point mutation or sequence replacement in full-length channels to evaluate their role in subcellular localization.
Fig. 1

Model molecules to identify the signals in Navs that mediate the trafficking regulation. Navs consist of four domains (I, II, III and IV) connected by three intracellular loops (L1–L3); each domain is formed by six transmembrane segments (TM; S1–S6). Both the N-terminus (N) and the C-terminus (C) of Navs are located in the cytoplasm. CD8α and TFR1, which have distinct cell-surface localization, are adapted to detect the roles that particular regions of the Navs have in subcellular distribution. The type I membrane protein CD8α is suitable for testing three intracellular loops, the C-terminus and the transmembrane segments that pass through the membrane in the extracellular to intracellular direction (S2, S4 and S6), whereas the type II membrane protein TFR1 is appropriate for testing the N-terminus and the transmembrane segments that pass through the membrane in the opposite direction (S1, S3 and S5). A Myc tag is inserted to the N-terminus of CD8α or the C-terminus of TFR1, and non-permeabilized immunostaining is performed with Myc antibody in transfected living cells to label these proteins on the plasma membrane. A Flag tag is inserted to the N-terminus of TFR1 for the permeabilized immunostaining with Flag antibody to label the protein in whole cell. The sequence of CD8α or TFR1 is replaced with corresponding region of Nav, such as Myc-CD8α(TMIVS6), Myc-CD8α-C, Flag-TFR1(TMIVS1)-Myc and N-TFR1-Myc. This figure is adapted from Li et al. [15]

Interacting proteins interact with specific amino acids, motifs or sequences within the Navs to achieve the final trafficking of channels. Although a large-scale yeast two-hybrid screen for proteins that interact with the intracellular domain of Nav1.8 has been performed [19], more of the putative interacting proteins need to be confirmed, and proteins that interact with the extracellular and transmembrane domains of Navs have yet to be discovered. A combination of mass spectrometry and immunoprecipitation with antibodies specific for Navs may provide another approach for finding novel interacting proteins that mediate the regulation of Nav trafficking. More importantly, the protein interaction could be regulated by extracellular factors that trigger signaling pathways to alter the protein activity. Large-scale screening for extracellular factors that regulate channel trafficking is limited by lack of the method for highly sensitive and efficient detection of the subcellular localization for membrane proteins.

Surface expression of Navs in primary sensory neurons

High levels of Navs are not localized on the plasma membrane of primary sensory neuron [611, 20]. The trafficking of the channels could be impeded at various points along the secretory pathway, including in the ER, Golgi complex and vesicles (Fig. 2). Nav1.8 displays a reticulum-like distribution and colocalizes with calnexin, an ER marker, in transfected COS-7 cells [11]. Using CD8α and TFR1 as model molecules to screen potential ER-localization motifs and sequences, an RXR motif in the first intracellular loop of Nav1.8 and several transmembrane segments containing acidic amino acids were found to be responsible for its ER localization [11, 18]. The β3 subunit interacts with Nav1.8 and masks the RXR motif to promote surface expression of the channel [11]. Calnexin, an ER chaperone protein, interacts with the transmembrane segments containing the acidic amino acids and induces channel degradation through the proteasome pathway [18]. p11, annexin 2 light chain, binds to aa 74–103 in the N-terminus of Nav1.8 to promote translocation of the channel to the plasma membrane [8]. Specific knockout of p11 in nociceptive DRG neurons reduces the TTX-R sodium current density and causes a dramatic loss of membrane-associated Nav1.8 [21]. As p11 has been shown to mask an ER-localization signal in TASK-1 to promote the surface expression of that channel [22], the role of p11 in promoting Nav1.8 trafficking from the ER needs to be evaluated.
Fig. 2

Main steps that regulates the subcellular distribution of Navs in primary sensory neurons. The surface expression of Navs is achieved by escape from the endoplasmic reticulum and proteasome degradation, forward trafficking and plasma membrane anchoring in primary sensory neurons. Axonal transport and localization of Navs in afferent fibers involves motor proteins and scaffold proteins. Localization of Nav1.6 to the nodes of Ranvier in myelinated fibers of primary sensory neurons requires node formation and the submembrane cytoskeletal protein complex. The molecules listed are mostly positive regulators except NEDD4-2 that may impede forward trafficking of Nav1.7. However, the hypothesized roles of molecules with question mark during various steps of Nav trafficking in primary sensory neuron need to be proved

Anchoring Navs on the plasma membrane is another key step in the regulation of their surface expression (Fig. 2). Contactin, a cell adhesion molecule, interacts with Nav1.2 and Nav1.3 [2325] and is also expressed in DRG neurons [26]. Knockout of contactin causes a reduction in the Nav1.8 and Nav1.9 currents but not in the TTX-S currents in IB4-positive DRG neurons [26]. PDZ domain containing 2 (PDZD2), a protein containing six PDZ domains, was identified as a Nav1.8 interacting protein by a yeast two-hybrid screen [19]. A subsequent study reveals that PDZD2 interacts with the second intracellular loop of Nav1.7 and Nav1.8 and that knockdown of PDZD2 causes a dramatic decrease in the Nav1.8 current [27]. However, knockout of PDZD2 does not cause a change in pain behavior and is accompanied by an increase in p11 [27]. PDZ proteins have been reported to retain and stabilize membrane proteins on the plasma membrane [2830]. Additional research is required to elucidate the roles of both contactin and PDZD2 in regulating peripheral Navs.

Nav β subunits are cell adhesion molecules and have been reported to regulate the surface expression of Navs including Nav1.7, Nav1.8 and Nav1.9 [11, 3136]. Coexpression of β1 subunit highly increases the current amplitude of Nav1.8 but not Nav1.7 in Xenopus oocytes [31]. Deficiency of β1 subunit leads to a decrease of persistent TTX-R sodium current accompanying with a reduction of surface and intracellular Nav1.9 in mouse small DRG neurons [32]. Loss of β2 subunit results in significant decrease of TTX-S sodium current concomitant with reductions in transcript and protein level of TTX-S Navs, particularly Nav1.7 [33]. These phenomena make the notion of trafficking regulation of these Navs by β subunits unsure because the change in the total protein level of channels may cause corresponding altered surface expression of these channels. However, coexpression of β3 subunit with Nav1.8 in HEK293 cells or Xenopus oocytes induces dramatically increased peak amplitude of sodium current [34, 35], in which the trafficking regulation was supported by significantly enhanced surface expression but not total protein level of Nav1.8 in coexpressed HEK293 cells [11]. Both TTX-S and TTX-R resurgent currents in small DRG neurons are enhanced by a peptide-mimetic intracellular domain of the β4 subunit [36]. To date, limited data reveal the molecular basis of β subunits in regulating the trafficking of peripheral Navs. The C-terminus of β3 subunit was examined to mediate the increased surface expression of Nav1.8 in coexpressed HEK293 cells and the C-terminal peptide of β4 subunit applied in the patch pipette was detected to enhance resurgent currents of Nav1.8 [11, 36]. Since the role of β3 subunit in masking the ER-localization motif of Nav1.8, anchoring peripheral Navs on the plasma membrane by β subunits needs further precise experiments to provide evidences.

The effects of post-translational modifications on Navs have been studied [37, 38]. Most studies have focused on the phosphorylation of these channels. Nav1.8 is phosphorylated by both protein kinase A (PKA) and protein kinase C, but only PKA-mediated Nav1.8 phosphorylation promotes the surface expression of this channel [38, 39]. Inhibition of the PKA-mediated surface expression of Nav1.8 by brefeldin A, a drug that blocks secretion upstream of the Golgi complex, reveals increased forward trafficking of this channel [38]; however, it is not clear exactly where in the secretory pathway this regulation occurs.

Recently, ubiquitination of Nav1.7 and Nav1.8 by the E3 ubiquitin ligase NEDD4-2 has been linked to regulation of the trafficking of these channels [37]. Most Navs, including Nav1.6, Nav1.7 and Nav1.8 but not Nav1.9, contain a typical PY motif (PPXY) or variant (LPXY) that interacts with NEDD4-2 and is ubiquitinated [37, 40]. In DRGs, NEDD4-2 is diffusely distributed in small neurons [37, 41]. Overexpression of NEDD4-2 in transfected HEK293 cells dramatically reduces both the amount of Nav1.7 in the plasma membrane and the Nav1.7 current without changing the abundance or the biophysical properties of this channel, while knockout of NEDD4-2 in Nav1.8-positive DRG neurons in SNS-Cre mice causes an increase in Nav1.7 current density accompanied by a non-significant change in the abundance of the channel in DRGs [37]. These lines of evidence support a role for NEDD4-2 in the negative regulation of Nav1.7 surface expression and indicate that, while channel ubiquitination may impede forward trafficking of Nav1.7 or enhance endocytosis (Fig. 2), it does not induce channel degradation or changes in channel properties. Interestingly, knockout of NEDD4-2 causes an increase in Nav1.8 current density along with a dramatic increase in the abundance of this channel in DRGs [37], indicating that loss of ubiquitination may reduce degradation of Nav1.8. Thus, the same post-translational modification has varying effects on different Navs.

The sumoylation of peripheral Navs has yet been reported in primary sensory neurons. However, the sumoylation deficiency of the collapsin response mediator protein 2 (CRMP2) reduces the surface expression of Nav1.7 in HEK293 cells and cultured cortical neurons, and dramatically decreases the peak sodium current density in DRG neurons [42]. Since CRMP2 interacts with tubulin heterodimer and promotes microtubule assembly [43], the role of CRMP2 has been proposed to regulate Nav1.7 trafficking.

Axonal transport and localization of Navs in afferent fibers of primary sensory neurons

The distribution of Navs along axons and at nerve terminals is critical for signal transduction in neurons. Nav1.7, Nav1.8 and Nav1.9 are mainly localized in small DRG neurons, which contribute to unmyelinated C fibers and thinly myelinated Aδ fibers. Transport of Navs via vesicles to the nerve terminal along long-distance axons involves several components, including motor proteins, microtubule tracks and scaffold proteins (Fig. 2). To date, a direct link between microtubule regulation and transport of Navs has not been reported. The kinesin superfamily is composed of microtubule-dependent motors, and kinesin-1 is responsible for anterograde axonal transport. Of the three kinesin-1 isoforms, KIF5A and KIF5B are abundantly expressed in DRG neurons [14]. KIF5B interacts with Nav1.8 and Nav1.9 but not Nav1.6 and Nav1.7, while KIF5A weakly interacts with Nav1.8 [14]. Overexpression of KIF5B increases the cell-surface and axonal distribution of Nav1.8 and simultaneously enhances the Nav1.8 current in the soma and axon of cultured DRG neurons, which indicates that, similar to forward trafficking, the anterograde axonal transport of Nav1.8 occurs via a mechanism involving motor proteins [14]. Knockdown of KIF5B decreases the current density of Nav1.8 in the soma of cultured DRG neurons, indicating a physiological role for KIF5B in promoting channel trafficking [14]. Whether KIF5B promotes the forward trafficking and axonal transport of Nav1.9 in primary sensory neurons has yet to be determined.

The scaffold proteins that regulate the axonal transport and localization of Navs are composed of several molecules. Knockout of the cell adhesion protein contactin causes a reduction in the expression of Nav1.8 and Nav1.9 in unmyelinated fibers of the sciatic nerve [26]. Knockout of the E3 ubiquitin ligase NEDD4-2 dramatically increases the level of Nav1.7 in the sciatic nerve [37]. Considering the critical roles that contactin and NEDD4-2 play in the trafficking of Navs, similar mechanisms may underlie axonal transport and localization of these channels. The fact that knockout of NEDD4-2 induces an increase in Nav1.8 level in DRGs but not in sciatic nerves [37] provides additional evidence to support the supposition that the NEDD4-2-mediated ubiquitination of Nav1.8 only affects the degradation and not the localization of this channel. Recent study showing an interaction between ankyrin G with an ankyrin-binding motif of Nav1.8 and a colocalization of Nav1.8 with ankyrin G at the nerve terminal of mouse hindpaw skin implies a role of ankyrin G in the axonal localization of Nav1.8 [44]. Most importantly, although annexin 2 light chain p11 has been shown to promote Nav1.8 translocation to the plasma membrane [8, 21], axonal localization of Nav1.8 in sciatic nerve and dorsal root or in cultured DRG neurons has not yet been examined in the p11 knockout mice. p11 together with PDZD2 and β1 subunit are also proposed to act as a lipid raft-sorting factor for Nav1.8 in the axons of DRG neurons because they have been shown to be partitioned into lipid rafts [15].

The abundance of Navs, including Nav1.7, Nav1.8 and Nav1.9, is increased in sciatic nerves of animal models with peripheral nerve injury and inflammation [4549]; however, the molecular mechanisms underlying the axonal transport and localization of these channels in pathological conditions are rarely investigated. In a mouse model with spared nerve ligation, downregulation of NEDD4-2 was thought to be linked to increased Nav1.7 level in the sciatic nerve because of a similar phenotype caused by knockout of NEDD4-2 [37]. Recently a potential relationship of increased axonal Nav1.8 with KIF5B comes from the result that in peripheral inflammation induced by complete Freund’s adjuvant, increased KIF5 and Nav1.8 accumulation were observed in the sciatic nerve. However, the antibody against KIF5 (Catalog Number: MAB1614; Merck Millipore—Chemicon International) was later detected to display low affinity against KIF5B but high affinity against KIF5A and KIF5C as reported by DeBoer et al. [50]. Although KIF5B participates anterograde axonal transport of Nav1.8 in the physiological condition [14], the interpretation regarding a potential correlation of the increased axonal transport of Nav1.8 with KIF5B in the pathological condition should be revised because of our unpublished result that KIF5B was not increased in the sciatic nerve of rat with peripheral inflammation using the antibody specifically against KIF5B (provided by Drs. Gerardo Morfini and Scott T. Brady). Therefore, the molecular mechanisms underlying the axonal transport and localization of Nav1.8 and Nav1.9 in pathological conditions remain to be explored.

Localization of Nav1.6 at the nodes of Ranvier in myelinated fibers of primary sensory neurons

The composition of a myelinated fiber in the peripheral nerve system includes the node of Ranvier, paranode, juxtaparanode and internode. The node of Ranvier plays a central role in impulse propagation via salutatory conduction in myelinated fibers. Usually, the channel density at the node is much higher than that in the rest of the nerve fiber [51]. Nav1.6 in adult primary sensory neurons in particular, is highly enriched at the nodes of Ranvier [52, 53].

The mechanism of Nav localization at the nodes of Ranvier has been studied for over a decade. The formation of a node of Ranvier is necessary for Nav accumulation. Knockout of a cell adhesion molecule, the 186 kDa isoform of neurofascin (NF-186) that is recruited by Schwann cell-secreted gliomedin, leads to the disruption of nodes and the absence of Nav clusters in sciatic nerves in mice [54]. NF-186 binds the submembrane cytoskeletal protein ankyrin G. Ankyrin G interacts with βVI spectrin and provides scaffolding for the recruitment of a group of functional proteins at the nodes of Ranvier (Fig. 2). Spectrins link the protein complex containing the Nav channel to the actin-based cytoskeleton at the nodes of Ranvier [55, 56]. Recently, the conditional knockout of ankyrin G in DRG neurons or retinal ganglion cells demonstrates that ankyrin G function in Nav clustering can be compensated by ankyrin R [57]. Both ankyrin G-βVI spectrin and ankyrin R-βI spectrin are recruited from a pre-existing pool of unclustered protein complexes to the nodes of Ranvier [57].

For Nav1.6 localization in the nodes of Ranvier, an ankyrin G-binding motif (VPIALGESD; corresponding to VPIAVGESD between aa 1094–1102 in murine Nav1.6) within the second intracellular loop of rat Nav1.2 [58] is sufficient for targeting CD4 chimera proteins to the nodes of Ranvier in rat DRG neuron-Schwann cell myelinating co-cultures [17]. Mutation of the conserved glutamic acid residue at E1100 within the ankyrin G-binding motif blocks Nav1.6 targeting to the nodes of Ranvier in neurons of the somatosensory cortex in in utero brain electroporation experiments [17]. Thus, the ankyrin G-binding motif is necessary and sufficient for clustering Nav1.6 at the nodes of Ranvier in both peripheral and central nerve systems.

Although knockout of the sodium channel β1 subunit causes a defect in paranodal structure in both sciatic nerves and optic nerves, Navs are still localized to the nodes of Ranvier in sciatic nerves [59]. Nav1.6 is also found in the nodes of Ranvier in sciatic nerves following knockout of the sodium channel β2 subunit [60]. Recently, a mutant form of Nav1.6, in which casein kinase phosphorylation sites within the second intracellular loop were mutated, was found to efficiently cluster at the nodes of Ranvier, indicating that regulation of casein kinase activity is not essential for node targeting [17].

Conclusion

Navs determine neuronal excitability and play a vital role in sensory transmission. Thus, Navs, specifically Nav1.7 and Nav1.8, are key drug targets for pain treatment, with pharmacological companies expending a lot of resources to screen for selective blockers of these channels. The subcellular distribution of Navs is regulated by trafficking (Fig. 2), which sometimes offers a quicker and accurate approach for changing channel function. Understanding the molecular mechanisms that promote excessive Nav trafficking in primary sensory neurons of pathological conditions may lead to the identification of pharmacological targets for pain treatment.

Summary statement

This review highlights the molecular mechanisms involved in Nav trafficking, focusing on mechanisms that regulate surface expression, axonal distribution and localization to the nodes of Ranvier in adult primary sensory neurons. It also discusses the strategies used to study these mechanisms.

Abbreviations

Nav

voltage-gated sodium channel

DRG: 

dorsal root ganglion

PCR: 

polymerase chain reaction

TTX-S: 

tetrodotoxin-sensitive

TTX-R: 

tetrodotoxin-resistant

TrkA: 

tropomyosin receptor kinase A

NF200: 

200 kDa neurofilament subunit

IB4: 

isolectin B4

ER: 

endoplasmic reticulum

TFR1: 

transferring receptor 1

PDZD2: 

PDZ domain containing 2

PKA: 

protein kinase A

NF-186: 

186 kDa isoform of neurofascin

Declarations

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (31330046 and 31271141) and National Basic Research Program of China (2014CB942802).

Compliance with ethical guidelines

Competing interests The author declares no competing interests.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

References

  1. Waxman SG. Painful Na-channelopathies: an expanding universe. Trends Mol Med. 2013;19(7):406–9.View ArticlePubMedGoogle Scholar
  2. Dib-Hajj SD, Black JA, Waxman SG. Nav1.9: a sodium channel linked to human pain. Nat Rev Neurosci. 2015;16(9):511–9.View ArticlePubMedGoogle Scholar
  3. Fukuoka T, Kobayashi K, Yamanaka H, Obata K, Dai Y, Noguchi K. Comparative study of the distribution of the α-subunits of voltage-gated sodium channels in normal and axotomized rat dorsal root ganglion neurons. J Comp Neurol. 2008;510(2):188–206.View ArticlePubMedGoogle Scholar
  4. Fukuoka T, Noguchi K. Comparative study of voltage-gated sodium channel α-subunits in non-overlapping four neuronal populations in the rat dorsal root ganglion. Neurosci Res. 2011;70(2):164–71.View ArticlePubMedGoogle Scholar
  5. Chiu IM, Barrett LB, Williams EK, Strochlic DE, Lee S, Weyer AD, et al. Transcriptional profiling at whole population and single cell levels reveals somatosensory neuron molecular diversity. Elife. 2014;3:e04660.View ArticleGoogle Scholar
  6. Amaya F, Decosterd I, Samad TA, Plumpton C, Tate S, Mannion RJ, et al. Diversity of expression of the sensory neuron-specific TTX-resistant voltage-gated sodium ion channels SNS and SNS2. Mol Cell Neurosci. 2000;15(4):331–42.View ArticlePubMedGoogle Scholar
  7. Black JA, Frezel N, Dib-Hajj SD, Waxman SG. Expression of Nav1.7 in DRG neurons extends from peripheral terminals in the skin to central preterminal branches and terminals in the dorsal horn. Mol Pain. 2012;8:82.PubMed CentralView ArticlePubMedGoogle Scholar
  8. Okuse K, Malik-Hall M, Baker MD, Poon WY, Kong H, Chao MV, et al. Annexin II light chain regulates sensory neuron-specific sodium channel expression. Nature. 2002;417(6889):653–6.View ArticlePubMedGoogle Scholar
  9. Wittmack EK, Rush AM, Craner MJ, Goldfarb M, Waxman SG, Dib-Hajj SD. Fibroblast growth factor homologous factor 2B: association with Nav1.6 and selective colocalization at nodes of Ranvier of dorsal root axons. J Neurosci. 2004;24(30):6765–75.View ArticlePubMedGoogle Scholar
  10. Xie W, Strong JA, Ye L, Mao JX, Zhang JM. Knockdown of sodium channel Nav1.6 blocks mechanical pain and abnormal bursting activity of afferent neurons in inflamed sensory ganglia. Pain. 2013;154(8):1170–80.PubMed CentralView ArticlePubMedGoogle Scholar
  11. Zhang ZN, Li Q, Liu C, Wang HB, Wang Q, Bao L. The voltage-gated Na+ channel Nav1.8 contains an ER-retention/retrieval signal antagonized by the β3 subunit. J Cell Sci. 2008;121(Pt 19):3243–52.View ArticlePubMedGoogle Scholar
  12. Black JA, Renganathan M, Waxman SG. Sodium channel Nav1.6 is expressed along nonmyelinated axons and it contributes to conduction. Brain Res Mol Brain Res. 2002;105(1–2):19–28.View ArticlePubMedGoogle Scholar
  13. Fukuoka T, Miyoshi K, Noguchi K. De novo expression of Nav1.7 in injured putative proprioceptive afferents: multiple tetrodotoxin-sensitive sodium channels are retained in the rat dorsal root after spinal nerve ligation. Neuroscience. 2015;284:693–706.View ArticlePubMedGoogle Scholar
  14. Su YY, Ye M, Li L, Liu C, Pan J, Liu WW, et al. KIF5B promotes the forward transport and axonal function of the voltage-gated sodium channel Nav1.8. J Neurosci. 2013;33(45):17884–96.View ArticlePubMedGoogle Scholar
  15. Pristerà A, Baker MD, Okuse K. Association between tetrodotoxin resistant channels and lipid rafts regulates sensory neuron excitability. PLoS One. 2012;7(8):e40079.PubMed CentralView ArticlePubMedGoogle Scholar
  16. Fjell J, Cummins TR, Fried K, Black JA, Waxman SG. In vivo NGF deprivation reduces SNS expression and TTX-R sodium currents in IB4-negative DRG neurons. J Neurophysiol. 1999;81(2):803–10.PubMedGoogle Scholar
  17. Gasser A, Ho TS, Cheng X, Chang KJ, Waxman SG, Rasband MN, et al. An ankyrinG-binding motif is necessary and sufficient for targeting Nav1.6 sodium channels to axon initial segments and nodes of Ranvier. J Neurosci. 2012;32(21):7232–43.PubMed CentralView ArticlePubMedGoogle Scholar
  18. Li Q, Su YY, Wang H, Li L, Wang Q, Bao L. Transmembrane segments prevent surface expression of sodium channel Nav1.8 and promote calnexin-dependent channel degradation. J Biol Chem. 2010;285(43):32977–87.PubMed CentralView ArticlePubMedGoogle Scholar
  19. Malik-Hall M, Poon WY, Baker MD, Wood JN, Okuse K. Sensory neuron proteins interact with the intracellular domains of sodium channel Nav1.8. Brain Res Mol Brain Res. 2003;110(2):298–304.View ArticlePubMedGoogle Scholar
  20. Schmidt J, Rossie S, Catterall WA. A large intracellular pool of inactive Na channel α subunits in developing rat brain. Proc Natl Acad Sci USA. 1985;82(14):4847–51.PubMed CentralView ArticlePubMedGoogle Scholar
  21. Foulkes T, Nassar MA, Lane T, Matthews EA, Baker MD, Gerke V, et al. Deletion of annexin 2 light chain p11 in nociceptors causes deficits in somatosensory coding and pain behavior. J Neurosci. 2006;26(41):10499–507.View ArticlePubMedGoogle Scholar
  22. Girard C, Tinel N, Terrenoire C, Romey G, Lazdunski M, Borsotto M. p11, an annexin II subunit, an auxiliary protein associated with the background K+ channel, TASK-1. EMBO J. 2002;21(17):4439–48.PubMed CentralView ArticlePubMedGoogle Scholar
  23. Kazarinova-Noyes K, Malhotra JD, McEwen DP, Mattei LN, Berglund EO, Ranscht B, et al. Contactin associates with Na+ channels and increases their functional expression. J Neurosci. 2001;21(19):7517–25.PubMedGoogle Scholar
  24. McEwen DP, Meadows LS, Chen C, Thyagarajan V, Isom LL. Sodium channel beta1 subunit-mediated modulation of Nav1.2 currents and cell surface density is dependent on interactions with contactin and ankyrin. J Biol Chem. 2004;279(16):16044–9.View ArticlePubMedGoogle Scholar
  25. Shah BS, Rush AM, Liu S, Tyrrell L, Black JA, Dib-Hajj SD, et al. Contactin associates with sodium channel Nav1.3 in native tissues and increases channel density at the cell surface. J Neurosci. 2004;24(33):7387–99.View ArticlePubMedGoogle Scholar
  26. Rush AM, Craner MJ, Kageyama T, Dib-Hajj SD, Waxman SG, Ranscht B. Contactin regulates the current density and axonal expression of tetrodotoxin-resistant but not tetrodotoxin-sensitive sodium channels in DRG neurons. Eur J Neurosci. 2005;22(1):39–49.View ArticlePubMedGoogle Scholar
  27. Shao D, Baker MD, Abrahamsen B, Rugiero F, Malik-Hall M, Poon WY, et al. A multi PDZ-domain protein PDZD2 contributes to functional expression of sensory neuron-specific sodium channel Nav1.8. Mol Cell Neurosci. 2009;42(3):219–25.PubMed CentralView ArticlePubMedGoogle Scholar
  28. Dunn HA, Ferguson SS. PDZ protein regulation of GPCR trafficking and signaling pathways. Mol Pharmacol. 2015;88(4):624–39.View ArticlePubMedGoogle Scholar
  29. Romero G, von Zastrow M, Friedman PA. Role of PDZ proteins in regulating trafficking, signaling, and function of GPCRs: means, motif, and opportunity. Adv Pharmacol. 2011;62:279–314.View ArticlePubMedGoogle Scholar
  30. Stanika RI, Flucher BE, Obermair GJ. Regulation of postsynaptic stability by the L-type calcium channel CaV1.3 and its interaction with PDZ proteins. Curr Mol Pharmacol. 2015;8(1):95–101.View ArticleGoogle Scholar
  31. Vijayaragavan K, O’Leary ME, Chahine M. Gating properties of Nav1.7 and Nav1.8 peripheral nerve sodium channels. J Neurosci. 2001;21(20):7909–18.PubMedGoogle Scholar
  32. Lopez-Santiago LF, Brackenbury WJ, Chen C, Isom LL. Na+ channel Scn1b gene regulates dorsal root ganglion nociceptor excitability in vivo. J Biol Chem. 2011;286(26):22913–23.PubMed CentralView ArticlePubMedGoogle Scholar
  33. Lopez-Santiago LF, Pertin M, Morisod X, Chen C, Hong S, Wiley J, et al. Sodium channel β2 subunits regulate tetrodotoxin-sensitive sodium channels in small dorsal root ganglion neurons and modulate the response to pain. J Neurosci. 2006;26(30):7984–94.View ArticlePubMedGoogle Scholar
  34. Shah BS, Stevens EB, Gonzalez MI, Bramwell S, Pinnock RD, Lee K, et al. β3, a novel auxiliary subunit for the voltage-gated sodium channel, is expressed preferentially in sensory neurons and is upregulated in the chronic constriction injury model of neuropathic pain. Eur J Neurosci. 2000;12(11):3985–90.View ArticlePubMedGoogle Scholar
  35. John VH, Main MJ, Powell AJ, Gladwell ZM, Hick C, Sidhu HS, et al. Heterologous expression and functional analysis of rat Nav1.8 (SNS) voltage-gated sodium channels in the dorsal root ganglion neuroblastoma cell line ND7-23. Neuropharmacology. 2004;46(3):425–38.View ArticlePubMedGoogle Scholar
  36. Tan ZY, Piekarz AD, Priest BT, Knopp KL, Krajewski JL, McDermott JS, et al. Tetrodotoxin-resistant sodium channels in sensory neurons generate slow resurgent currents that are enhanced by inflammatory mediators. J Neurosci. 2014;34(21):7190–7.PubMed CentralView ArticlePubMedGoogle Scholar
  37. Laedermann CJ, Cachemaille M, Kirschmann G, Pertin M, Gosselin RD, Chang I, et al. Dysregulation of voltage-gated sodium channels by ubiquitin ligase NEDD4-2 in neuropathic pain. J Clin Invest. 2013;123(7):3002–13.PubMed CentralView ArticlePubMedGoogle Scholar
  38. Liu C, Li Q, Su Y, Bao L. Prostaglandin E2 promotes Nav1.8 trafficking via its intracellular RRR motif through the protein kinase A pathway. Traffic. 2010;11(3):405–17.View ArticlePubMedGoogle Scholar
  39. Vijayaragavan K, Boutjdir M, Chahine M. Modulation of Nav1.7 and Nav1.8 peripheral nerve sodium channels by protein kinase A and protein kinase C. J Neurophysiol. 2004;91(4):1556–69.View ArticlePubMedGoogle Scholar
  40. Fotia AB, Ekberg J, Adams DJ, Cook DI, Poronnik P, Kumar S. Regulation of neuronal voltage-gated sodium channels by the ubiquitin-protein ligases Nedd4 and Nedd4-2. J Biol Chem. 2004;279(28):28930–5.View ArticlePubMedGoogle Scholar
  41. Cachemaille M, Laedermann CJ, Pertin M, Abriel H, Gosselin RD, Decosterd I. Neuronal expression of the ubiquitin ligase Nedd4-2 in rat dorsal root ganglia: modulation in the spared nerve injury model of neuropathic pain. Neuroscience. 2012;227:370–80.View ArticlePubMedGoogle Scholar
  42. Dustrude ET, Wilson SM, Ju W, Xiao Y, Khanna R. CRMP2 protein SUMOylation modulates NaV1.7 channel trafficking. J Biol Chem. 2013;288(34):24316–31.PubMed CentralView ArticlePubMedGoogle Scholar
  43. Fukata Y, Itoh TJ, Kimura T, Menager C, Nishimara T, Shiromizu T, et al. CRMP2 binds to tubulin heterodimers to promote microtubule assembly. Nat Cell Biol. 2002;4(8):583–91.PubMedGoogle Scholar
  44. Montersino A, Brachet A, Ferracci G, Fache MP, d’Ortoli SA, Liu W, et al. Tetrodotoxin-resistant voltage-gated sodium channel Nav1.8 constitutively interacts with ankyrin G. J Neurochem. 2014;131(1):33–41.View ArticlePubMedGoogle Scholar
  45. Coggeshall RE, Tate S, Carlton SM. Differential expression of tetrodotoxin-resistant sodium channels Nav1.8 and Nav1.9 in normal and inflamed rats. Neurosci Lett. 2004;355(1–2):45–8.View ArticlePubMedGoogle Scholar
  46. Coward K, Plumpton C, Facer P, Birch R, Carlstedt T, Tate S, et al. Immunolocalization of SNS/PN3 and NaN/SNS2 sodium channels in human pain states. Pain. 2000;85(1–2):41–50.View ArticlePubMedGoogle Scholar
  47. Black JA, Nikolajsen L, Kroner K, Jensen TS, Waxman SG. Multiple sodium channel isoforms and mitogen-activated protein kinases are present in painful human neuromas. Ann Neurol. 2008;64(6):644–53.View ArticlePubMedGoogle Scholar
  48. Kretschmer T, Happel LT, England JD, Nguyen DH, Tiel RL, Beuerman RW, et al. Accumulation of PN1 and PN3 sodium channels in painful human neuroma-evidence from immunocytochemistry. Acta Neurochir (Wien). 2002;144(8):803–10.View ArticleGoogle Scholar
  49. Yiangou Y, Birch R, Sangameswaran L, Eglen R, Anand P. SNS/PN3 and SNS2/NaN sodium channel-like immunoreactivity in human adult and neonate injured sensory nerves. FEBS Lett. 2000;467(2–3):249–52.View ArticlePubMedGoogle Scholar
  50. DeBoer SR, You Y, Szodorai A, Kaminska A, Pigino G, Nwabuisi E, et al. Conventional kinesin holoenzymes are composed of heavy and light chain homodimers. Biochemistry. 2008;47(15):4535–43.PubMed CentralView ArticlePubMedGoogle Scholar
  51. Ritchie JM, Rogart RB. The binding of labelled saxitoxin to the sodium channels in normal and denervated mammalian muscle, and in amphibian muscle. J Physiol. 1977;269(2):341–54.PubMed CentralView ArticlePubMedGoogle Scholar
  52. Caldwell JH, Schaller KL, Lasher RS, Peles E, Levinson SR. Sodium channel Nav1.6 is localized at nodes of ranvier, dendrites, and synapses. Proc Natl Acad Sci USA. 2000;97(10):5616–20.PubMed CentralView ArticlePubMedGoogle Scholar
  53. Luo S, Jaegle M, Li R, Ehring GR, Meijer D, Levinson SR. The sodium channel isoform transition at developing nodes of Ranvier in the peripheral nervous system: dependence on a genetic program and myelination-induced cluster formation. J Comp Neurol. 2014;522(18):4057–73.View ArticlePubMedGoogle Scholar
  54. Sherman DL, Tait S, Melrose S, Johnson R, Zonta B, Court FA, et al. Neurofascins are required to establish axonal domains for saltatory conduction. Neuron. 2005;48(5):737–42.View ArticlePubMedGoogle Scholar
  55. Xu K, Zhong G, Zhuang X. Actin, spectrin, and associated proteins form a periodic cytoskeletal structure in axons. Science. 2013;339(6118):452–6.View ArticlePubMedGoogle Scholar
  56. Machnicka B, Czogalla A, Hryniewicz-Jankowska A, Boguslawska DM, Grochowalska R, Heger E, et al. Spectrins: a structural platform for stabilization and activation of membrane channels, receptors and transporters. Biochim Biophys Acta. 2014;1838(2):620–34.View ArticlePubMedGoogle Scholar
  57. Ho TS, Zollinger DR, Chang KJ, Xu M, Cooper EC, Stankewich MC, et al. A hierarchy of ankyrin-spectrin complexes clusters sodium channels at nodes of Ranvier. Nat Neurosci. 2014;17(12):1664–72.PubMed CentralView ArticlePubMedGoogle Scholar
  58. Lemaillet G, Walker B, Lambert S. Identification of a conserved ankyrin-binding motif in the family of sodium channel alpha subunits. J Biol Chem. 2003;278(30):27333–9.View ArticlePubMedGoogle Scholar
  59. Chen C, Westenbroek RE, Xu X, Edwards CA, Sorenson DR, Chen Y, et al. Mice lacking sodium channel β1 subunits display defects in neuronal excitability, sodium channel expression, and nodal architecture. J Neurosci. 2004;24(16):4030–42.View ArticlePubMedGoogle Scholar
  60. Chen C, Bharucha V, Chen Y, Westenbroek RE, Brown A, Malhotra JD, et al. Reduced sodium channel density, altered voltage dependence of inactivation, and increased susceptibility to seizures in mice lacking sodium channel β2-subunits. Proc Natl Acad Sci USA. 2002;99(26):17072–7.PubMed CentralView ArticlePubMedGoogle Scholar

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