Tetrodotoxin-resistant voltage-gated sodium channel Nav1.8 constitutively interacts with ankyrin G

Authors


Abstract

The tetrodotoxin-resistant (TTX-R) voltage-gated sodium channel Nav1.8 is predominantly expressed in peripheral afferent neurons, but in case of neuronal injury an ectopic and detrimental expression of Nav1.8 occurs in neurons of the CNS. In CNS neurons, Nav1.2 and Nav1.6 channels accumulate at the axon initial segment, the site of the generation of the action potential, through a direct interaction with the scaffolding protein ankyrin G (ankG). This interaction is regulated by protein kinase CK2 phosphorylation. In this study, we quantitatively analyzed the interaction between Nav1.8 and ankG. GST pull-down assay and surface plasmon resonance technology revealed that Nav1.8 strongly and constitutively interacts with ankG, in comparison to what observed for Nav1.2. An ion channel bearing the ankyrin-binding motif of Nav1.8 displaced the endogenous Nav1 accumulation at the axon initial segment of hippocampal neurons. Finally, Nav1.8 and ankG co-localized in skin nerves fibers. Altogether, these results indicate that Nav1.8 carries all the information required for its localization at ankG micro-domains. The constitutive binding of Nav1.8 with ankG could contribute to the pathological aspects of illnesses where Nav1.8 is ectopically expressed in CNS neurons.

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The peripheral voltage-gated sodium channel Nav1.8 can be abnormally expressed in central nervous system (CNS) neurons in cases of neuronal injury. We here demonstrated that Nav1.8 binds strongly and constitutively to the scaffolding protein ankyrin G. This indicates that Nav1.8 concentrates at the ankyrin G micro-domains and could disturb the electrophysiological signature of CNS neurons where it is ectopicaly expressed.

Abbreviations used
ABD

ankyrin-binding domain

AIS

axon initial segment

ankG

ankyrin G

GST

gluthation-S-transferase

GFP

green fluorescent protein

MBD

membrane-binding domain

SPR

surface plasmon resonance

Voltage-gated sodium channels α subunits (Nav1) are present in most excitable cell membranes and play a crucial role in generating action potentials (APs). Nine paralogous Nav1 have been identified in the mammalian genome and seven of them are specifically expressed in neurons (Meisler et al. 2010). These neuronal channels share a strong sensitivity to the puffer fish toxin tetrodotoxin (TTX) except two, namely Nav1.8 and Nav1.9, which are TTX-resistant (TTX-R) (Rush et al. 2007). Nav1.8 has been initially reported to be preferentially expressed in small-diameter unmyelinated primary afferents and specialized for the detection of noxious stimuli (Abrahamsen et al. 2008). More recently, an exhaustive study revealed that Nav1.8 is expressed in almost all peripheral primary afferent neurons including neurons of dorsal root ganglia and trigeminal ganglia (TG), and along numerous A and C types of nerve fibers in the skin (Shields et al. 2012). As Nav1.8, Nav1.9 is expressed in dorsal root ganglia and TG neurons and in a subset of primary afferent sensory neurons of the myenteric and submucosal plexuses in the small intestine (Rugiero et al. 2003; Kwong et al. 2008). Interestingly, several studies have shown that the expression level and/or the distribution of Nav1.8 can be modified after neuronal injury. In particular, an ectopic expression of Nav1.8 in CNS neurons occurs in human multiple sclerosis (MS) or in mouse model of MS (Black et al. 2000). Furthermore, an ectopic expression of Nav1.8 has been observed at the nodes of Ranvier of motor axons with dysmyelinating phenotype such as Charcot–Marie-Tooth (CMT) disease (Ulzheimer et al. 2004; Devaux and Scherer 2005). The ectopic expression of Nav1.8 in non-sensory neurons induces an ectopic electrical activity implicated in the pathogenic motor symptoms found in CMT disease (Moldovan et al. 2011).

The key function of Nav1 channels expressed in CNS neurons is the generation of AP at the axon initial segment (AIS) where they are clustered (Clark et al. 2009). This accumulation results from a direct interaction with the scaffold protein ankyrin G (ankG) through an ankyrin-binding domain (ABD) found in the intracellular loop II-III of Nav1 channels (Garrido et al. 2003; Lemaillet et al. 2003; Gasser et al. 2012). Within the ABD, several studies have underlined the critical role of a conserved glutamate residue (E1111 of rat Nav1.2) in the binding of Nav1 to ankG. Importantly, this interaction is regulated by the phosphorylation of four serine residues found in close proximity of E1111 by protein kinase CK2 (CK2) (Brechet et al. 2008; Brachet et al. 2010). As shown in Fig. 1a, the sequence alignment of ABD of neuronal Nav1 is not fully conserved between TTX sensible (TTX-S) and TTX-R channels particularly with respect to serine residues. We thus analyzed the ability of ABD of TTX-R channels to bind ankG, using combination of biochemical and biophysical techniques. We observed that TTX-R channels strongly interact with ankG but surprisingly in a constitutive manner, in contrast to what observed for CNS Nav1.2. Moreover, this association is sufficient to target a chimeric protein bearing the ABD of Nav1.8 to the AIS, suggesting a possible mechanism involved in Nav1.8 aberrant segregation during its ectopic expression in CNS neurons. Finally, we also observed a co-localization of Nav1.8 and ankG in nerve fibers of mouse skin, suggesting that the interaction between these two proteins also occurs in normal tissue.

Figure 1.

Nav1.8 and Nav1.9 interact with ankyrin G. (a) Part of sequence alignment of rat neuronal TTX-S and TTX-R Nav1 channels obtained with ClustalW (2.1) algorithm. Amino-acid number is indicated for each sequence. Sequence defined by Hill et al. (2008) to be a core ankyrin-binding motif is underlined. (b) Rat brain ankyrin G (total) was solubilized (input) and incubated with purified recombinant GST proteins as indicated on the figure. Proteins retained on glutation-sepharose beads were analyzed by western blotting with an anti-ankyrin G (ankG) antibody. (c and d) 270 kDa ankG-GFP was expressed in COS-1 cells, solubilized (input) and incubated with purified recombinant GST proteins as indicated in the figure. Proteins retained on glutation-sepharose beads were analyzed by western blotting with an anti-AnkG antibody.

Materials and methods

Antibodies

Mouse monoclonal antibodies to myc (1 : 100; Roche Diagnostic, Mannheim, Germany), ankyrinG (1 : 500, N106/36 NeuroMab), Nav1.8 (1 : 200, N134/12 NeuroMab) and sodium channels (pan Nav; 1 : 100; Sigma-Aldrich, St Louis, MI, USA), rabbit polyclonal antibodies to myc (1 : 1000; Abcam, Cambridge, MA, USA), PGP9.5 (1 : 500; RB-9202; Thermo, Waltham, MA, USA), goat antibody to gluthation-S-transferase (GST) and chicken polyclonal antibodies to myc and MAP2 (1 : 250 and 1 : 10 000, respectively; Abcam) were used. Secondary goat antibodies conjugated to Alexa Fluor 488, 555, 633 (Invitrogen, Carlsbad, CA, USA) or Cy5 (Jackson Immuno-Research, West Grove, PA, USA) were used at 1 : 400–800 dilutions. For immunoblotting a donkey, secondary antibody coupled to horseradish peroxidase (1 : 1000 dilution; GE Healthcare, Piscataway, NJ, USA) directed against rabbit or mouse IgG was used.

Animals

All procedures were in agreement with the European Communities Council directive (86/609/EEC). Experiments were performed on pregnant female Wistar rats (Janvier labs, Laval, France). Animals were sacrificed by decapitation and embryo brains were used for primary neuronal cell culture. For some animals, the brain was removed and used to prepare crude rat brain membrane fraction as described below.

DNA constructs

The nucleotide sequence of rat Nav1.8 fragment coding from amino acid (aa) 969 to 1148 was obtained by PCR amplification (Expand High Fidelity Taq polymerase, Roche Molecular Biochemicals) on pCB6-CD4-Nav1.8 II–III (Fache et al. 2004) and inserted into the pCDNA3-myc-Kv2.1-Nav1.2 1080–1203 (Brechet et al. 2008) to produce the pCDNA3-myc-Kv2.1-Nav1.8 969–1148. The pGEX-Nav1.8 969–1148 was generated by subcloning from pCDNA3-myc-Kv2.1-Nav1.8 969–1148. Site-directed mutants were obtained using Quick Change XL site directed mutagenesis kit (Stratagene, La Jolla, CA, USA). The rat Nav1.9 867–1029 nucleotide sequence was chemically synthetized and inserted into pCRR2.1-TOPO (Eurofins mwg/operon, Ebersberg, Germany). This sequence was then subcloned into EcoR1/Not1 sites of the pGEX-4T2 vector to produce GST-Nav1.9 867–1029. Histidin-tagged membrane-binding domain (MBD)-ankG (amino acids 1-981) was generated by PCR from Rat 270 kDa ankyrin G-green fluorescent protein (GFP) plasmid (gift from V. Bennett, Duke University Medical Center, Durham, NC, USA) and cloned into BamH1/Not1 sites of the derived pET-28(a) vector (Novagen, Merk-Millipore, Paris, France). All constructs were verified by DNA sequencing (Beckman Coulter Genomics, Danvers, MA, USA).

Recombinant protein expression and purification

His-MBD AnkG and GST-tagged protein were expressed in Escherichia coli strain BL21 or Rosetta2 (DE3) and purified by Ni-NTA agarose, respectively (Qiagen, Valencia, CA, USA). or Glutathione-Sepharose 4B (GE Healthcare) according to the manufacturer's instructions. Protein concentrations were determined using Bradford assay. Purified histidin-tagged human 220-kDa ankyrin B and rat 190-kDa ankyrin G were provided by V. Bennett (Duke University Medical Center, Durham, NC, USA).

Surface plasmon resonance experiments

Surface plasmon resonance (SPR) experiments were performed at 25°C on a Biacore 3000 instrument with CM5 sensor chips (GE Healthcare Life Sciences) as previously described (Brechet et al. 2008). Briefly, GST or GST fusion proteins (4–8 fmol) were immobilized in flow cells, via anti-GST polyclonal goat antibody. When indicated the CK2 phosphorylation was directly performed onto chips. Purified ankG (190 kDa), ankB (220 kDa) or His-MBD ankG were injected at a flow rate of 20 μL/min for 3 min. The non-specific signal (GST) was subtracted from the total signal (GST fusion protein) and control run injections (buffer) were performed in the same conditions before ankyrin injections and subtracted for data analysis. Except for the Nav1.8 E1005A mutant, the Biacore single-cycle kinetic method was used to provide kinetic data (Palau and Di Primo 2013). The rate constants and the KD (koff/kon) were calculated in BiaeEvaluation 4.1 software (GE Healthcare) by global fitting using a single-site interaction model. For each experiment, kon was measured in the initial part of the association phase using 5 increasing analyte concentrations injected successively, and koff was determined during the period of dissociation after the last analyte injection. In the case of Nav1.8 E1005A mutant, the Biacore multi-cycle kinetic method was used. Saturation-binding curves were plotted using the equilibrium response value at the plateau level of all sensorgrams and KD was calculated as half of the maximal response.

Cell culture, transfection, and immunofluorescence

Primary hippocampal neurons were prepared as described previously (Garrido et al. 2003). Neurons were transfected after 8 days in vitro using Lipofectamine 2000 (Life Technologies, Grand Island, NY, USA). Transfected cells were processed for immunofluorescence 24 h after transfection. To detect the steady-state surface distribution of the Kv2.1-Nav1.8 chimera in transfected neurons, non-specific binding was blocked with 0.22% gelatin in 0.1 M phosphate buffer after paraformaldehyde fixation. Cells were incubated for 1 h with an anti-myc antibody. Endogenous proteins (ankG, MAP2, and Nav1) were immunodetected after a permeabilization step (0.1% of Triton X-100 and 0.22% gelatin in phosphate buffer). Corresponding secondary antibodies conjugated to Alexa Fluor or Cy5 were incubated for 1 h. Coverslips were mounted in Fluor Save reagent (EMD).

Immunohistochemistry

Glabrous skin was dissected from the hind paws of non-perfused male adult mice (3-month-old), and processed according to Zylka et al. protocol (Zylka et al. 2005). Briefly, free-floating sections of 40 μm were immuno-stained with Nav1.8, ankG and PGP9.5, counterstained for nuclei with Hoechst 33342 (1 μg/mL) and dry-mounted in ProLong® Gold (Life Technologies). Image acquisition was performed on a Zeiss LSM780 confocal microscope (Zeiss, Jena, Germany) equipped with a 63× 1.4 N.A. oil immersion objective. Three dimensional z-stacks were collected automatically as frame by frame sequential image series. Image editing was performed using ImageJ software (http://rsb.info.nih.gov/ij/) and was limited to Sigma Plus Filter, linear contrast enhancement, and gamma adjustment.

Confocal microscopy on primary hippocampal neurons and quantification

Immunofluorescence slides were imaged using a confocal microscope (TCS-SPE; LAS-AF software; Leica, Wetzlar, Germany). Confocal images were acquired with 63×/1.32 NA, or 63×/1.40 NA oil objectives (Leica). Fluorescence was collected as z stacks with sequential wavelength acquisition. Quantification was performed using ImageJ. Regions of interest corresponding to AIS were manually selected on ankG images and reported on other channels for intensity measurements. All intensities were corrected for background labeling. For illustration purposes, image editing was performed using ImageJ or Photoshop 7.0 (Adobe, Dublin, Ireland) and was limited to rolling-ball background subtraction, linear contrast enhancement, and gamma adjustment.

Pull-down assays

A crude rat brain membrane fraction was prepared as described previously (Vacher et al. 2011). COS-1 cells were transfected with ankG-270 kDa-GFP plasmid using Lipofectamine 2000 following the manufacturer's instructions (Life Technologies). Rat brain membrane proteins or transfected COS-1 cells were solubilized for 30 min under agitation at 4°C in Tris-buffered saline (Tris-HCl 50 mM pH 7.4, NaCl 150 mM), containing 1 mM EDTA, inhibitors of proteases (cOmplete EDTA-free; Roche), 1% Triton X-100, 0.1% sodium dodecyl sulfate, 0.1% sodium deoxycholate (DOC) and 0.1% bovine serum albumin. Unsoluble proteins were removed by centrifugation at 14 000 g for 15 min. Two nanomoles of GST-fusion protein were incubated with 40 μL of glutathione-Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ, USA), for 1 h at 4°C in phosphate-saline buffer. After three washes in phosphate-saline buffer, beads were incubated with gentle agitation overnight at 4° with 1 mL of solubilized rat brain proteins or COS-1 cells lysate. After extensive washes, proteins retained on the beads were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blotting.

Statistic analyses

Results are expressed as means ± SEM. Statistical analyses were performed using Prism 5 (Graphpad Software, La Jolla, CA, USA). Significance of the differences was assessed using a two-tailed t-test for two groups or one-way anova followed by Newman–Keuls post-test for more than two groups.

Results

The TTX-R Nav1 channels binds to ankG via a motif homologous to the ABD of Nav1.2

The ABD of TTX-S Nav1 directly interacts with a highly conserved domain in ankG and ankB called the MBD. Several studies have underlined the critical role of the glutamate E1111 (in rat Nav1.2) and four serine residues close to E1111 of ABD in the regulation of Nav1/ankG interaction (Brechet et al. 2008; Gasser et al. 2012). All these serine residues are highly conserved in the mammalian sodium channels types expressed in the CNS, namely Nav1.1, Nav1.2, Nav1.3 and Nav1.6, but also in Nav1.7, a sodium channel expressed in the peripheral nervous system (PNS). In contrast, only two serine residues putatively phosphorylated by CK2 are located in rat Nav1.8 motif (NetPhosK prediction: score 0.70) and no site in rat Nav1.9 (Fig. 1a). In view of these differences, we performed pull-down experiments to examine the ability of ankG extracted from rat brain, to interact with purified gluthation-S-transferase (GST) recombinant proteins bearing the ABD of either Nav1.2 (Nav1.2 ABD) or Nav1.8 (Nav1.8 ABD). The bacterially produced ABD of Nav1.8 strongly interacted with rat brain ankG when compared to the ABD of Nav1.2 (Fig. 1b). Sequence alignment indicated that the glutamate E1005 of Nav1.8 is homologous to the crucial glutamate E1111 of Nav1.2 (Brechet et al. 2008). Moreover, serine S1006 of Nav1.8 is a potential phosphorylation site for protein kinase CK2. In order to test the importance of these two residues in the association with ankG, Nav1.8E1005 and Nav1.8S1006, were converted into alanine (Nav1.8 E1005A and Nav1.8 S1006A), respectively. The replacement of E1005 by alanine abolished Nav1.8 ABD binding to rat brain ankG (Fig. 1b), validating the crucial role of this aspartic acid in the association. In contrast, the interaction was not perturbed when Nav1.8 S1006 was substituted by alanine (Fig. 1b). Several ankG isoforms, mainly of 480, 270 and 190 kDa, are expressed in CNS and 480/270 isoforms are specifically localized at the AIS (Kordeli et al. 1995). All these isoforms shares the same MBD but the major isoform visualized by western blotting in pull-down experiments had an apparent molecular weight around 190 kDa (Fig. 1b). To confirm our results for the AIS-specific ankG, 270 kDa ankG isoform tagged with GFP (270 kDa ankG-GFP) was expressed in COS-1 cells and subsequently used for in vitro binding experiments. Under these conditions, Nav1.8 ABD strongly interacted with 270 kDa ankG-GFP, unlike Nav1.2 ABD (Fig. 1c). This interaction was abolished by Nav1.8 E1005A mutation, unlike Nav1.8 S1006A mutation (Fig. 1d). Noteworthy, Nav1.9 ABD (GST-Nav1.9 867–1029) also associates with 270 kDa ankG-GFP (Fig. 1c). Altogether our data highlight the ability of the ABD of Nav1.8 and Nav1.9 to interact with ankG isoforms.

Constitutive interaction between TTX-resistant Nav1 and axonal ankyrins

In the view of the differences in the ability of Nav1.2, Nav1.8 and Nav1.9 to associate with ankG (Fig. 1), we further quantitatively analyzed the binding properties, using SPR technology. Nav1.2-ABD, Nav1.8-ABD, and Nav1.9-ABD were purified and immobilized on the sensor surface by immunoaffinity. When sequential increasing concentrations of purified ankG-190 kDa were injected over flow cells, no significant reactivity was observed for Nav1.2-ABD (Fig. 2a). In contrast, under the same experimental conditions, an increasing binding signal was observed for Nav1.8-ABD (Fig. 2a). Kinetic analysis of the interaction between ankG-190 kDa and GST-Nav1.8-ABD gave a KD of 29 ± 19 nM (Table 1). We previously demonstrated that the association of Nav1.2 with ankyrin G is regulated by protein kinase CK2 phosphorylation (Brechet et al. 2008). When CK2 phosphorylation was performed on immobilized Nav1.2-ABD before ankG-190 kDa injection, a specific and robust-binding signal was observed (KD = 1.0 ± 1.0 nM) (Fig. 2a and Table 1). This indicates that the impact of phosphorylation on Nav1.2-ABD binding to ankG-190 kDa was strong, from no significant reactivity to very high affinity. In the case of Nav1.8-ABD, CK2 phosphorylation increased 15 times the binding affinity (KD = 1.9 ± 1.8 nM) as compared to what was observed before phosphorylation (Table 1). We then compared the interaction of ankG-190 kDa with Nav1.9-ABD versus Nav1.8-ABD (Fig. 2a). Values obtained for Nav1.9 gave a KD of 160 ± 50 nM. The lower binding affinity observed with Nav1.9-ABD versus Nav1.8-ABD is mainly due to a decrease in the association rate (24-fold) (Table 1). Similar binding signals and kinetic parameters were obtained when Nav1.9-ABD was phosphorylated by CK2 (Table 1). When ankG-190 kDa was substituted by one other neuronal ankyrin, namely the ankB-220 kDa, similar binding signals and KD were obtained for the ABD tested (Fig. 2b and Table 1). To determine if the TTX-R Nav1 binds to the same domain of ankyrins than other neuronal Nav1, namely the MBD, we measured the ability of native and mutated Nav1.8-ABD to interact with MBD of ankG (MBD-ankG) by SPR technology as above. Kinetic analysis showed that the binding characteristics of Nav1.8-ABD, before and after CK2 phosphorylation, on the MBD-ankG were very similar to values found for ankG-190 kDa or ankB-220 kDa (Table 1). As expected, no significance difference in the KD values determined in the absence or in the presence of in situ CK2 phosphorylation was observed for Nav1.8 S1006A mutant (Table 1). As suggested by pull-down experiments, no or a very weak association occurred between Nav1.8 E1005A mutant and MBD-ankG (Table 1). Altogether these results showed that the TTX-R Nav1 constitutively associate with axonal ankyrins.

Table 1. Kinetic parameters of interaction between ABD of Nav1.2, Nav1.8, Nav1.9, and ankyrins
 Immobilized GST construction n CK2 k on M −1 s −1 k off s −1 K D nM
  1. Values are the mean ± SD calculated from n independent experiments using the single-cycle kinetics method except for Nav1.8 E1005A (see 'Materials and methods'). ND, No sufficient specific binding to determine kinetic parameters. NA: Not applicable.

AnkG-190 kDaNav1.2 ABD5  ND
8+18 ± 22 × 1059.3 ± 8.3 × 10−41.0 ± 1.0
Nav1.8 ABD717 ± 27 × 10418 ± 22 × 10−429 ± 19
9+12 ± 24 × 10510 ± 14 × 10−41.9 ± 1.8
Nav1.9 ABD57 ± 3 × 10310 ± 5 × 10−4160 ± 50
2+9 ± 5 × 10313 ± 4 × 10−4168 ± 83
AnkB-220 kDaNav1.2 ABD4  ND
5+30 ± 30 × 1042.8 ± 3.0 × 10−41.2 ± 0.7
Nav1.8 ABD63.0 ± 3.0 × 1043.7 ± 1.5 × 10−424 ± 13
5+87 ± 13 × 1047.3 ± 12 × 10−41.0 ± 0.7
AnkG-MBDNav1.8 ABD95.2 ± 2.0 × 10437 ± 9 × 10−480 ± 16
8+48 ± 45 × 10529 ± 3 × 10−41.2 ± 0.8
Nav1.8 S1006A64.5 ± 2.0 × 10433 ± 7 × 10−474 ± 22
8+9.8 ± 3.0 × 10424 ± 6 × 10−426 ± 1
Nav1.8 E1005A6  ND
8+NANA250 ± 150
Figure 2.

Constitutive interaction between TTX-resistant Nav1 and axonal ankyrins. Representative sensorgrams: GST tagged ankyrin-binding domain (ABD) of Nav1.8 (black curves), Nav1.2 (red curves) or Nav1.9 (blue curves) were immobilized onto the sensor-chips and subjected or not to an in situ CK2 phosphorylation (as indicated). Increasing amount of ankyrin G (ankG)-190 kDa (a) or ankB-220 kDa (b) (as indicated on the graph) was injected over immobilized constructs. R.U: resonance unit.

Chimeric protein containing the ABD of Nav1.8 is segregated at the AIS and displaces endogenous Nav1 in hippocampal neurons

In neurons, the AIS and the nodes of Ranvier (noR) are specialized subdomains where the direct interaction between ankG and Nav1 is essential in their clustering and subsequently in initiation and propagation of APs. We have already demonstrated that the replacement of the C-terminus of Myc-tagged Kv2.1 (which is critical for the somato-dendritic distribution of Kv2.1) by the ABD of Nav1.2 induced a clustering of the chimeric protein Kv2.1-Nav1.2 at the AIS (Brechet et al. 2008). To test if the in vitro interaction described above occurs in a cellular context, we constructed a chimeric ion channel in which the C-terminus of Myc-tagged Kv2.1 was replaced by the ABD sequence of Nav1.8 (Kv2.1-Nav1.8). Kv2.1-Nav1.8 was expressed in cultured hippocampal neurons and its surface distribution was analyzed. As shown in Fig. 3, Kv2.1-Nav1.8 was restricted to the AIS identified by ankG staining in the large majority of transfected cells. Thus, the ABD of Nav1.8 interacts with endogenous ankG in live neurons as Nav1.2 ABD does. Nav1.8 E1005A and Nav1.8 S1006A mutations were generated in Kv2.1-Nav1.8 chimera and the surface expression of the constructs were quantified. In accordance with SPR experiments, the suppression of E1005 reduced drastically the surface segregation of Kv2.1-Nav1.8 at the AIS, whereas the surface distribution of Kv2.1-Nav1.8 S1006A was similar to the one observed for Kv2.1-Nav1.8 (Fig. 3a and c). We next quantified endogenous Nav1 and ankG at the AIS in neurons expressing Kv2.1-Nav1.8. A 68% reduction of Nav1 staining was found in neuron expressing Kv2.1-Nav1.8 compared to non-transfected cells, whereas ankG staining remained unchanged (Fig. 3b and d). Interestingly, endogenous Nav1 clustering was also perturbed in neurons expressing Kv2.1-Nav1.8 S1006A (Fig. 3d). These results demonstrated that Kv2.1-Nav1.8 chimera acts as a dominant negative on endogenous Nav1 clustering independently of CK2 phosphorylation.

Figure 3.

Ankyrin-binding domain (ABD) of Nav1.8 induces segregation of the Kv2.1-Nav1.8 chimera at the axon initial segment (AIS) of hippocampal neurons and acts as a dominant negative on endogenous Nav1. channels. (a) Cell surface distribution of Kv2.1-Nav1.8 and E1005A mutant transfected in 8 DIV hippocampal neurons. Transfected neurons were detected with an anti-myc antibody (green) before permeabilization, the somatodendritic domain and AIS were, respectively, identified by MAP2 staining (blue) and ankyrin G (ankG) staining (red). Scale bar: 10 μm. (b) 8 DIV hippocampal neuron transfected with Kv2.1-Nav1.8 (asterisk) and immunolabeled for myc, ankG and Nav1. Scale bar: 10 μm. (c) Histograms of the cell surface distribution of Kv2.1-Nav1.8 and mutants. The percentage of myc-positive neurons was classified into three categories: segregated at the AIS (AIS); distributed at the soma and proximal dendrites with enrichment at the AIS (AIS+SD) or uniformly distributed (non-polarized). 100% represents the total population of transfected neurons. Bars indicate mean ± SEM from three to five experiments; n = 113 to 130 neurons, one-way anova posttest comparison with Kv2.1-Nav1.8 distribution, **p < 0.01 *p < 0.05. (d) Quantification of Nav1 and ankG staining intensity in untransfected cells (NT) and transfected cells with Kv2.1-Nav1.8 or S1006A mutant. Fluorescence intensities were normalized by taking as 100% the intensity measured in the NT cells. Bars indicate mean ± SEM from three experiments; n = 62 neurons. Mann–Whitney test: **p < 0.01; ***p < 0.001.

Nav1.8 and ankG are co-localized in the skin nerve fibers

The exhaustive distribution of the Nav1.8 expression in the skin have been done using Nav1.8-Cre mice crossed with tdTomato Cre-reporter mice (Shields et al. 2012). In this tissue, ankG and ankB are also specifically distributed along nervous fibers (Engelhardt et al. 2012). We performed immunohistochemical localization of ankG and Nav1.8 in mouse skin. In glabrous skin of the hind footpad, we noted abundant Nav1.8 labeling distributed along dermal-epidermal boundary (Fig. 4). A substantial part of this labeling co-localized with PDP9.5, a specific marker of nerve fibers, consistent with the idea that Nav1.8 is expressed in a large population of afferents in mouse skin. Remarkably, ankG labeling co-localized with Nav1.8 in nerve fibers of mouse skin, indicating that these two proteins could interact in normal tissues (Fig. 4).

Figure 4.

Colocalization of Nav1.8 and ankyrin G (ankG) in skin nerve fibers. The overall skin structure of two different mice was visualized by nucleus staining (Hoechst, blue) and nerve fibers were labeled by PGP9.5 immunostaining (magenta). Nav1.8 (red) and ankG (green) co-localized in nerve fibers. Scale bar: 15 μm.

Discussion

In the present study, we demonstrated that the loop II-III of TTX-R sodium channels contains an ABD capable of binding constitutively to ankG. Interestingly, the amino acid sequence of this domain is weakly conserved between TTX-R and TTX-S neuronal channels. This is also true for the core motif of this domain which is shared by the Nav1 and KCNQ2/3 potassium channels (Hill et al. 2008). Despite this absence of conservation, binding to ankG still occurs, suggesting that in addition to the critical glutamate residue (boxed in Fig. 1a), the negatively charged environment of this motif seems sufficient to maintain its functional properties. In the case of TTX-S Nav1, the presence of negative charges is increased by CK2 phosphorylation of 4 serine residues (Brechet et al. 2008), but in the case of Nav1.8 the presence of many acidic residues (12 D or E in 25 residues) is sufficient to provide a high affinity between the Nav1.8 ABD and MBD of ankyrins without phosphorylation. As a consequence, Nav1.8 is constitutively immobilized in ankG positive domains.

It is interesting to note that the respective affinity of phosphorylated Nav1.8 ABD or Nav1.2 ABD for axonal ankyrins (ankG or ankB) are similar (Table 1 and Brechet et al. 2008). This suggests that TTX-R and TTX-S sodium channels may compete for ankyrins and thus for functional targeting and anchoring. Recent studies have demonstrated that ankB-specific small sequences suppressed, by intramolecular interaction, the ability of MBD of ankB to interact in vivo with membrane partners of ankG (He et al. 2013). This could explain why the endogenous Nav1 interact specifically with ankG in neurons and why Kv2.1-Nav1.8 constructs are specifically targeted to the AIS and not in the distal part of the axon where ankB is present.

In CNS neurons, ankG, and Nav1 co-localize in AIS and noR where APs are, respectively, initiated and regenerated. In contrast, in neurons expressing Nav1.8 such as the somatosensory neurons, APs is generated in peripheral axon terminals in unknown sites. Recent studies demonstrated in the skin of various mammals that axon terminals of somatosensory neurons are enriched in ankB, but not in ankG, neither in Nav1 (Engelhardt et al. 2012). AnkG and Nav1 are instead accumulated in axonal micro-domains located proximal to the somatosensory terminals. Moreover, we showed here that Nav1.8 colocalized with ankG in nerve fibers of mouse skin. The interaction between TTX-R Nav1 and ankG we describe here, could be crucial for the formation and maintenance of Nav1-enriched axonal micro-domains of the PNS neurons. It would be very interesting to know whether these micro-domains are also enriched with other ankG partners found in the AIS and noR such as KCNQ2/3, Neurofascin 186 or ß4-spectrin (Leterrier and Dargent 2014) and whether these micro-domains are implicated in APs generation.

Several studies have observed that Nav1.8 is ectopically expressed in CNS neurons in pathologies such as CMT or MS (Black et al. 2000; Ulzheimer et al. 2004; Devaux and Scherer 2005). Moreover, an overexpression of Nav1.8 is observed in injured peripheral nerves with painful neuromas (Black et al. 2008). It has been proposed that the up-regulation of Nav1.8 expression is mediated by a nerve growth factor signaling pathway (Damarjian et al. 2004). Moreover, the particular electrophysiological properties of Nav1.8, when ectopically or over expressed, could have a critical role in pathological processes (Moldovan et al. 2011). Here, we demonstrated that Nav1.8 interacts constitutively with ankG, unlike CNS Nav1 for which the interaction is regulated by local phosphorylation. It is tempting to speculate that the ability of Nav1.8 to constitutively bind to ankG may dramatically alter the Nav1 composition at the AIS of CNS neurons, and consequently the electrophysiological signature of the targeted neurons. Altogether, these observations could explain the detrimental role of Nav1.8 in CNS neurons when this channel is ectopically expressed.

Acknowledgments and conflict of interest disclosure

We are grateful to Raymond Miquelis and Christian Leveque for their help with SPR kinetic analysis and Aziz Moqrich for his help with experiment with mouse skin. We thank Van Bennett for providing 190-kDa ankyrin G and 220-kDa ankyrin B. We thank Drs Christophe Leterrier, Marie-Jeanne Papandreou, Hélène Vacher, and Nadine Clerc for critically reading the manuscript; Armand Tasmadjian for technical help. This work was supported by the Centre National pour la Recherche Scientifique, and by Grant R06048AA from the Fondation pour la Recherche Médicale (to A.M.); and Grant R09070AA from the National Multiple Sclerosis Society.

All experiments were conducted in compliance with the ARRIVE guidelines. None of the authors has any conflicts of interest.

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