Direct interaction with contactin targets voltage-gated sodium channel Na(v)1.9/NaN to the cell membrane.

The mechanisms that target various sodium channels within different regions of the neuronal membrane, which they endow with different physiological properties, are not yet understood. To examine this issue we studied the voltage-gated sodium channel Na(v)1.9/NaN, which is preferentially expressed in small sensory neurons of dorsal root ganglia and trigeminal ganglia and the nonmyelinated axons that arise from them. Our results show that the cell adhesion molecule contactin binds directly to Na(v)1.9/NaN and recruits tenascin to the protein complex in vitro. Na(v)1.9/NaN and contactin co-immunoprecipitate from dorsal root ganglia and transfected Chinese hamster ovary cell line, and co-localize in the C-type neuron soma and along nonmyelinated C-fibers and at nerve endings in the skin. Co-transfection of Chinese hamster ovary cells with Na(v)1.9/NaN and contactin enhances the surface expression of the sodium channel over that of Na(v)1.9/NaN alone. Thus contactin binds directly to Na(v)1.9/NaN and participates in the surface localization of this channel along nonmyelinated axons.

It is now well established that 10 distinct genes encode different members of the voltage-gated sodium channel family (1), all sharing a common overall motif but with different physiological properties as a result of variations in their primary amino acid sequence (2). The expression of different isotypes of sodium channels within different types of neurons, or within different parts of the membrane of single neurons, endows them with distinct electrophysiological properties (3,4). However, the mechanisms that target and/or anchor various types of channels within the membranes of neurons are not fully understood. Dorsal root ganglion (DRG) 1 neurons provide a useful model for studying sodium channel expression and targeting because they express multiple sodium channels and corresponding currents (5)(6)(7)(8)(9)(10)(11). Expression of the sodium channel Na v 1.9/NaN appears to be restricted to the soma and nonmyelinated axons of small diameter (C-type) DRG neurons (10,12). The Na v 1.9/NaN channel produces a persistent sodium current with a large overlap between activation and steadystate inactivation and a relatively hyperpolarized voltage dependence (13,14), suggesting that Na v 1.9/NaN has a depolarizing influence on the cell resting potential and enhances subthreshold electrogenesis of these sensory neurons (15).
Sodium channels interact with a number of proteins that affect the subcellular localization of the channel as well as the current amplitude and/or kinetic properties. Rat brain sodium channels are heterotrimers that consist of the pore-forming ␣-subunit (ϳ260 kDa) and auxiliary ␤ (ϳ33-36 kDa)-subunits (2). Four ␤-subunits (␤1, ␤1A, ␤2, and ␤3) have been identified (16 -19) and have been shown to interact with sodium channel ␣-subunits. The ␤1and ␤3-subunits modulate the amplitude and kinetics of sodium channels expressed in Xenopus oocytes (16,18,20) and permit interaction of the sodium channel complex with the extracellular matrix proteins tenascin-C and -R (21)(22)(23)(24) and the cell adhesion molecule neurofascin 168 (25). The interaction of ␤1 and ␤3 with neurofascin 168 occurs early in development and persists through adulthood and has been proposed to contribute to targeting and retention of sodium channels at nodes of Ranvier (25).
The extracellular domain of the ␤-subunits shares significant similarity with one of the extracellular IgG domains of the cell adhesion molecule contactin (17,26). Contactin/F3/F11 (contactin hereinafter) has been shown to play an important role in organizing the paranodal region of myelinated fibers (27), a role that is thought to involve interaction with Caspr (27)(28)(29)(30)(31) and neurofascin 155 (32). Recently, Kazarinova-Noyes et al. (33) proposed that contactin, acting via ␤1-subunits, increases the expression of the Na v 1.2 ␣-subunit, a sodium channel isotype that is present at immature nodes of Ranvier (34,35). Although contactin has been reported at nodes of central nervous system axons (28), a direct interaction of contactin with sodium channels, not involving ␤-subunits, has not been reported.
Sodium channels are clustered at high density in the axon membrane at nodes of Ranvier (36,37) but, in contrast, are homogeneously distributed at a much lower density along nonmyelinated axons (38,39). Moreover, a different complement of sodium channel isotypes are expressed in small DRG neurons with nonmyelinated fibers compared with large DRG neurons with myelinated fibers (9), with the Na v 1.9/NaN channel being selectively expressed in C-type DRG neurons and their nonmyelinated axons (10,12). The mechanisms underlying targeting and retention of sodium channels at the somata and axonal membrane of nonmyelinated fibers have not been studied and may be distinct from those that localize sodium channels at nodes of Ranvier in myelinated fibers. In the present study, we used a combination of biochemical and cell biological approaches to identify proteins that may play a role in the targeting of Na v 1.9/NaN to the cell surface of DRG neurons, and we have identified contactin as a channel partner that binds directly to the channel and appears to function in this role.

Antibodies and Cell Lines
Affinity-purified polyclonal Na v 1.9/NaN-specific antibodies against the C-terminal 18-amino acid peptide of this channel were described previously (40). Polyclonal anti-contactin antibodies were generously provided by Dr. Jim Salzer (28). Monoclonal anti-contactin antibodies generously provided by Dr. Fritz G. Rathjen (41) and from a commercial source (BD Biosciences, San Diego) were both used for immunocytochemistry and produced similar results. Anti-GFP polyclonal antibodies were purchased from CLONTECH, and anti-human tenascin monoclonal antibodies (clnT2H5) were purchased from Exalpha Biologicals, Inc. (Boston). Human embryonic kidney (HEK) 293 cells and CHO fibroblasts were used in this study.

Plasmid Constructs
The bacterial expression plasmid pGEX-NaNC carrying the insert for the C-terminal polypeptide of Na v 1.9/NaN (amino acids 1588 -1765; GenBank TM accession number AF059030) was described previously (42). Na v 1.9a/NaN cDNA was cloned into the low copy number mammalian expression plasmid pLG338/Sport (43) to produce the vector pLG338/NaN. The sequence of this insert shows the following changes compared with the published Na v 1.9 sequence: a serine to proline substitution at position 962 in the cytoplasmic loop joining domains 2 and 3 (L2); a deletion of 11 amino acids (1000 -1010) at the C-terminal end of L2; and a leucine to proline substitution at position 1282 in transmembrane segment 6 (S6) of domain 3. Transfection of HEK293 cell line with this construct does not produce a sodium current above background 2 but produces a protein of the expected size of the channel when analyzed by immunoblot assay (see under "Results"). Full-length contactin cDNA (GenBank TM accession number D38492) was amplified by reverse transcription-polymerase chain reaction from rat brain template and cloned in-frame with GFP using the KpnI/XhoI sites of pEGFO-N1 fusion vector (CLONTECH) to produce pContactin-GFP plasmid. The constructs generated were verified by sequencing and expression assay. Sequencing was carried out at the HHMI/Keck Biotechnology Resource Laboratory at Yale University. Details of the constructs are available upon request.

Expression and Purification of GST Fusion Proteins
For expressing glutathione S-transferase (GST) fusion proteins, the appropriate plasmids pGEX3X and pGEX-NaN-C were transformed into Escherichia coli DH5␣ (Life Technologies, Inc.). Fusion proteins were affinity-purified on glutathione-agarose beads as described previously (44).

Rat Brain Protein Affinity Purification
Purified GST fusion proteins were dialyzed against PBS (pH 7.1) at 4°C for 24 h. Purified proteins (3 mg) were cross-linked to 1 ml of Affi-Gel 15 (Bio-Rad) in buffer A (50 mM HEPES, pH 7.6) at 4°C overnight. GST-and GST-NaNC-Affi-Gel 15 beads were then blocked with buffer B (0.1 M ethanolamine HCl, pH 8.0) on ice for 1 h. The affinity resin was then washed sequentially with 0.5 ml each of buffer C (50 mM HEPES, pH 7.6, 125 mM NaCl, 20% glycerol) containing 1 mM DTT, buffer C containing 1 mM DTT and 2.5 M urea, buffer C containing 1 mM DTT and 4 M urea, and finally buffer D (20 mM HEPES, pH 7.6, 125 mM NaCl, 10% glycerol, 1 mM DTT, 1 mM EDTA) supplemented with protease inhibitors. Bovine serum albumin (BSA)-Affi-Gel 15 beads were generated in a similar manner and were used to pre-adsorb the brain extracts prior to their application to the GST-and GST-NaNC-Affi-Gel 15 to reduce the nonspecific binding of proteins to these matrices.
Full brains from 20 adult rats were homogenized in ice-cold buffer D, and Triton X-100 was added to 1%, and the proteins were solubilized for 1 h at 4°C. The homogenate was centrifuged at 15,000 ϫ g for 10 min at 4°C three times to clear the supernatant. Nonspecific protein binding was reduced by pre-adsorbing the supernatant with 2 ml of BSA-Affi-Gel 15 beads at 4°C for 2 h. After the BSA beads were removed, 50 and 5 ml of the pre-adsorbed extracts were incubated overnight at 4°C on a shaker with 0.5 ml of GST-NaNC-Affi-Gel 15 and 0.05 ml of GST-Affi-Gel 15 (serving as control), respectively. The extract/beads were extensively washed with buffer D containing 0.5% Nonidet P-40, 1% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride followed by buffer D containing 1% Triton X-100. Proteins were eluted sequentially with 2.5 ml each of buffer D containing 1% Triton X-100 and 2.5 M urea, buffer D containing 1% Triton X-100 and 4 M urea, and finally buffer D containing 1% SDS. Eluted proteins were precipitated with 10% trichloroacetic acid and 0.05% deoxycholic acid, and the proteins were resolved by 10% SDS-PAGE. The gels were stained with Coomassie Brilliant Blue G-Colloidal solution (Sigma) according to the vendor's recommendations.

Mass Spectrometry
The visible protein bands of interest were excised and prepared for mass spectrometric analysis. The gel pieces were washed at room temperature in the following solutions: acetonitrile 50% for 5 min; 50% CH 3 CN, 50 mM NH 4 HCO 3 for 30 min; and then in 50% CH 3 CN, 10 mM NH 4 HCO 3 for 30 min. The samples were dried in a SpeedVac Concentrator (Eppendorf, Hamburg, Germany), and a trypsin solution (0.05 g of trypsin per 7 l of 10 mM NH 4 HCO 3 ) was added and allowed to incubate at 37°C for 24 h.
The supernatants of the trypsin digestion products were collected and analyzed using mass spectrometry. The standards used for the calibration of peptide masses are bradykinin (average M ϩ H, 1061.23 Da) and ACTH Clip (average M ϩ H, 2466.70 Da). One microliter of the tryptic digest was mixed with 1.0 l of ␣-cyano-4-hydroxycinnamic acid (4.5 mg/ml in 50% CH 3 CN, 0.05% trifluoroacetic acid) matrix solution, and 1 l of calibrants (100 fmol) each. The mixture was loaded onto the sample plate and then injected into the Perspective Biosystem Voyager-DE STR instrument. The spectra of the peptides were acquired in reflector/delayed extraction mode. The identity of the peptides was determined using the ProFound-Peptide Mapping search engine (www.proteometrics.com/profound_bin/WebProFound.exe) and subsequently searched against Swiss-Prot (www.expasy.ch/) or PIR site (www.nbrf.georgetown.edu/).

In Vitro Binding Assay
To examine the binding of contactin to the C terminus of Na v 1.9/NaN in vitro, glutathione-Sepharose beads loaded with GST (0.5 g), serving as control, or GST-NaN-C (0.5 g) were incubated with extract (500 g of protein) from HEK293 cells transfected with an expression plasmid encoding either GFP alone or full-length contactin-GFP, as indicated, in 150 l of buffer AM (45) supplemented with 100 mM KCl and 0.5 mg/ml bovine serum albumin (44). The bound proteins were separated by 7.5% SDS-PAGE, and GFP fusion proteins were detected by immunoblotting with anti-GFP antibodies (CLONTECH).
To test whether contactin is required for the binding of tenascin to Na v 1.9/NaN in vitro, 1 g of human tenascin (Life Technologies, Inc.) was incubated with glutathione-Sepharose beads loaded with GST (0.5 g), or GST-NaN-C (0.5 g), or GST-NaN-C (0.5 g) which was preincubated with either GFP or contactin-GFP from HEK293 cell lysates (see above). The multiprotein components were incubated in 150 l of buffer AM supplemented with 100 mM KCl and 0.5 mg/ml bovine serum albumin as described above. The bound proteins were separated by 7.5% SDS-PAGE, and tenascin was detected by immunoblotting with anti-tenascin monoclonal antibodies (clnT2H5).

Far Western Blotting
Far Western blotting was carried out according to Lee et al. (46) with minor modification. After a 1-h incubation with anti-GFP antibodies, cell extracts prepared from HEK293 cells transfected with an expression plasmid encoding either GFP or contactin-GFP fusion protein were incubated with 50 l of protein A-agarose at 4°C overnight. After washing five times, the purified GFP (control) and contactin-GFP were subjected to 12% SDS-PAGE. The proteins were electrotransferred to nitrocellulose at 85 mA for 2 h. The proteins on the blot were denatured and renatured by sequential washings in 0.1 M CZ solution (20 mM HEPES, pH 7.9, 20% glycerol, 0.1 M KCl, 5 mM MgCl 2 , 0.1 mM ZnCl 2 , 0.1 mM EDTA, 2 mM dithiothreitol) plus 0.02% polyvinylpyrrolidone and 6 M guanidine HCl for 30 min one time and then 3 times in 0.1 M CZ solution plus 0.02% polyvinylpyrrolidone for 2 h. After blocking with 5% BSA, the membranes were incubated with 50 g/ml purified GST-NaN-C followed by anti-Na v 1.9/NaN (0.2 g/ml) antibodies.

Co-immunoprecipitation of Na v 1.9/NaN and Contactin
HEK293 cells were grown in 100-mm dishes in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. After reaching 50% confluence, the cultures were co-transfected with the mammalian expression plasmids pContactin-GFP and pLG328-NaN. The cultures were harvested 48 h later, and cell extracts were prepared according to Liu et al. (47). After 1 h of incubation with Na v 1.9/NaN-specific antisera (20 g/ml), aliquots (200 l) from the cell extracts were incubated with 30 l of protein A-agarose (Life Technologies, Inc.) at 4°C overnight. After washing five times with IP buffer (48), the bound proteins were released by boiling in 20 l of 2ϫ SDS sample buffer for 3 min. The released proteins were examined by Western blotting with anti-contactin monoclonal antibodies (from Dr. Fritz G. Rathjen (41)).
For immunoprecipitation of Na v 1.9/NaN and contactin from native tissue, DRGs were homogenized in ice-cold buffer D containing 1% Triton X-100 and protease inhibitors, and the proteins were extracted for 1 h at 4°C. Protein extracts (500 g) were incubated with 5 l of control IgG (20 g/ml), or anti-Na v 1.9/NaN (20 g/ml), or anti-contactin (1:40) (from Dr Jim Salzer (28)) antibodies at 4°C for 1 h. The mixture was then incubated with 50 l of protein A-agarose at 4°C overnight. Detection of the immunoreactive proteins was carried out as described above using anti-Nav1.9/NaN antibody (0.2 g/ml).

Cell Surface Biotinylation
Dishes of transfected CHO cells were washed three times with icecold PBS, pH 8.0, and the cells were collected by centrifugation at 500 ϫ g for 5 min, and surface proteins were biotinylated with 0.5 mg/ml sulfo-NHS-LC-biotin (Pierce) in PBS, pH 8.0, at room temperature for 30 min according to the manufacturer's recommendation. These cultures were washed 3 times in PBS, and the cells were lysed in PBS, pH 8.0, containing 1% Triton X-100, 5 mM EDTA, and protease inhibitors at 4°C for 1 h. Cell extracts (500 g of proteins) were immunoprecipitated with either 5 l of anti-Na v 1.9/NaN antibodies (20 g/ml) for 1 h at 4°C followed by protein A-agarose or 100 l of streptavidin-agarose (Pierce) at 4°C overnight. The immunoprecipitated proteins were analyzed by immunoblotting with anti-Na v 1.9/NaN antibodies or peroxidase-conjugated monoclonal anti-biotin antibodies (BN-34, Sigma).

DRG Cell Culture
Cultures of DRG neurons were established as described previously (49). Briefly, lumbar ganglia (L4 and L5) from adult male Sprague-Dawley rats were excised, freed from their connective tissue sheaths, and incubated sequentially in enzyme solutions containing collagenase and then papain. The tissue was triturated in culture medium containing 1:1 Dulbecco's modified Eagle's medium and Hanks' F-12 medium and 10% fetal calf serum, 1.5 mg/ml trypsin inhibitor, 1.5 mg/ml bovine serum albumin, 100 units/ml penicillin, and 0.1 mg/ml streptomycin and plated on polyornithine/laminin-coated coverslips. The cells were maintained at 37°C in a humidified 95% air, 5% CO 2 incubator overnight or for 7 days. For cultures maintained for 7 days, glial-derived neurotrophic factor (50 ng/ml; human recombinant, PeproTech, Rocky Hill, NJ) was added to the culture medium, which was changed every other day (12).
Thin cryosections (10 m) of DRG from perfused rats were mounted on Fischer SuperFrost Plus glass slides. For teased nerve analysis, sciatic nerves from perfused rats were excised, incubated in a solution containing 0.5 mg/ml each of collagenase A and collagenase D (Roche Molecular Biochemicals) for 30 min at 37°C, desheathed, placed on Fisher SuperFrost Plus slides, and fibers gently teased apart. The sections or teased nerves were processed for immunocytochemistry as described above with the exception of the fixation incubation. Following the immunocytochemical procedures, the coverslips or slides were mounted with Aqua-polymount (Polysciences, Warrington, PA).
Control experiments included incubation without primary antibody and pre-adsorption of the antibody with 100 -500 molar excess of immunizing peptide. Only background levels of fluorescence were detected in the control experiments (data not shown).
Imaging-Cells and sections were examined with a confocal laser scanning microscope (Nikon PCM 2000) using 20ϫ NA 0.75 or 60ϫ NA 1.4 objectives. Cells and tissue were optically sectioned in the xy plane with a minimum slice thickness of 0.5 m with multiple scan averaging. Confocal images were processed with Adobe Photoshop.

Contactin and Tenascin-R Bind to the C-terminal
Polypeptide of Na v 1.9/NaN-Sodium channel Na v 1.9/NaN is preferentially expressed in small peripheral sensory neurons and their axons but is also present at low levels in the central nervous system (10). We recently demonstrated that the C-terminal polypeptide of Na v 1.9/NaN binds to FHF1B, a member of the FGF family of growth factors, using rat brain yeast two-hybrid cDNA library (42). This finding validates the use of brain homogenates in a biochemical approach to identify binding partners of Na v 1.9/NaN. The C-terminal polypeptide of Na v 1.9/NaN (amino acids 1588 -1765) was used as bait to trap adult rat brain proteins that interact with this channel. We purified a GST-NaNC fusion protein in which the C-terminal polypeptide of Na v 1.9/ NaN was fused to glutathione S-transferase, and we generated a GST-NaNC affinity chromatography column. A detergentsolubilized rat brain extract was applied to the column and in parallel to a control GST column. Both columns were washed extensively, and the bound proteins were eluted with buffer D (see "Materials and Methods") supplemented with 2.5 M urea, 4 M urea, and 1% SDS and were analyzed by SDS-PAGE. Most of the bound proteins were eluted by the 2.5 M urea wash, whereas the 1% SDS wash did not release any additional proteins (data not shown). Samples of the 2.5 and 4 M urea eluates were pooled, and the proteins were precipitated and loaded onto 7.5% SDS-polyacrylamide gel. Staining of the gel with Coomassie Brilliant Blue G-Colloidal reagent showed the presence of multiple proteins. The major signals belonged to two proteins of ϳ150 and ϳ130 kDa, respectively, that were specifically retained by the GST-NaNC column but not by the control GST column (Fig. 1, arrows). Tubulin, the most abundant protein in the brain extract, binds to both columns (Fig. 1) and represents a nonspecific interaction.
The pooled 2.5 and 4 M urea fractions were separated on a preparative SDS-PAGE, and the 150-and 130-kDa bands were excised and analyzed by mass spectrometry. Trypsin digestion products of these two proteins gave rise to reliable peptide signals (Fig. 1, B and C). Searches using ProFound software revealed that the tryptic peptide products of the 150-and 130-kDa proteins matched those of tenascin-R (Z score, 2.32) and contactin (Z score, 1.85), respectively. Tenascin-R is an extracellular matrix glycoprotein that is synthesized by glial cells especially during the period of active myelination and remyelination (for reviews see Refs. [51][52][53]. Contactin is a glycosylphosphatidylinositol-anchored neuronal adhesion molecule of the Ig superfamily (54 -56) and is a natural receptor for tenascin (57)(58)(59).
Contactin Binds Directly to the C-terminal Polypeptide of Na v 1.9/NaN in Vitro-Because sodium channel ␤-subunits share significant sequence and structural homology with one of the Ig domains of contactin (17,26), and ␤-subunits interact directly with sodium channels (60), we reasoned that contactin may bind directly to Na v 1.9/NaN. An in vitro interaction between contactin and the C-terminal polypeptide of Na v 1.9/NaN was verified using the pull-down assay. We expressed GFP alone and contactin-GFP fusion protein in HEK293 cells ( Fig.  2A, compare lane 4 and lane 1). Affinity-purified GST and GST-NaN-C immobilized to the glutathione-Sepharose beads were incubated with cell lysates containing GFP or contactin-GFP. Fig. 2A shows that GST-NaNC trapped the contactin-GFP fusion protein (lane 6). This interaction is due to the association between contactin and the C-terminal polypeptide of Na v 1.9/NaN because neither GST nor GST-NaNC binds to GFP alone (lane 2 and 3) and GST does not bind to the contactin-GFP fusion protein (lane 5).
The direct interaction of contactin with the C-terminal  6) immobilized to glutathione-Sepharose beads was incubated with extracts prepared from HEK293 cells transfected with an expression plasmid encoding either GFP alone (lanes 1-3) or contactin-GFP fusion protein (lanes 4 -6). The bound proteins and cell extracts (lanes 1 and 4) were examined by immunoblotting with anti-GFP antibodies. GFP, contactin-GFP, as well as an unknown protein X are indicated. B, direct interaction of contactin with the C terminus of Nav1.9/NaN, assayed by far Western blotting. Cell lysates prepared from untransfected (lane 1) or HEK293 cells transfected by an expression plasmid expressing either GFP (lane 2) or contactin-GFP (lane 3), respectively, were incubated with anti-GFP antibodies, followed by protein A-agarose. Purified proteins were subjected to 7.5% SDS-PAGE, and the resulting membranes were washed in a series of buffers to allow the renaturation of the proteins on the membrane and incubated with 50 g of purified GST-NaN-C/ml, followed by anti-Na v 1.9/NaN antiserum. polypeptide of Na v 1.9/NaN was tested using the far Western blotting assay (Fig. 2B). HEK293 cells were transfected with expression plasmids encoding GFP or contactin-GFP fusion protein. Anti-GFP antibodies were used to immunoprecipitate GFP and contactin-GFP from the corresponding cell lysates and from untransfected HEK293 cells that served as a negative control. The immunoprecipitated protein(s) were separated by SDS-PAGE, electrotransferred to nitrocellulose membrane, and allowed to re-nature by a series of buffer washes. The immobilized proteins were then incubated with affinity-purified GST or GST-NaNC and probed with anti-Na v 1.9/NaN antibody. As shown in Fig. 2B, the anti-Na v 1.9/NaN antibody reacted with a protein of the predicted size of contactin-GFP (lane 3) but failed to react with proteins from the untransfected HEK293 negative control (lane 1) or with GFP (lane 2). GST does not bind to GFP alone (not shown) or to FHF1B-GFP, which also binds to GST-NaNC (42). This result shows that GST-NaNC binding to the purified contactin-GFP is direct and does not require the presence of other proteins that may be present in the cell lysate.
The Interaction of Contactin with Na v 1.9/NaN C Terminus Recruits Tenascin-C to the Protein Complex-To examine the hypothesis that the interaction between contactin and Na v 1.9/ NaN recruits tenascin to the protein complex, tenascin-C, which is similar to tenascin-R and is the form that is expressed in peripheral nervous tissue (for review see Ref. 52), was tested in an in vitro multiple-protein interaction assay (Fig. 3). Purified tenascin C was incubated with immobilized GST, GST-NaNC, or GST-NaNC preincubated with lysates of cells expressing either GFP or contactin-GFP. The protein complex was subjected to SDS-PAGE, electrotransferred to nitrocellulose membrane, and probed by anti-tenascin antibody. The immunoblot assay shows that GST (lane 2), GST-NaNC (lane 3), or GST-NaNC preincubated with GFP alone (lane 4) could not capture tenascin-C, indicating a lack of direct interaction between tenascin-C and the C-terminal polypeptide of Na v 1.9/ NaN. Preincubating the C terminus of Na v 1.9/NaN with contactin-GFP, however, recruited tenascin-C to the protein complex (lane 5). These data show that contactin is required for the binding of tenascin to the sodium channel and that HEK293 cell lysates do not contain an endogenous protein, for example contactin or a sodium channel ␤-subunit, that promotes the binding of tenascin to the complex.
Contactin Co-localizes with and Binds to Na v 1.9/NaN in Vivo-The in vivo interaction of contactin and Na v 1.9/NaN was tested by a co-immunoprecipitation assay from HEK293 cells co-transfected with plasmids encoding the full-length channel and contactin-GFP (Fig. 4A). Immunoblot assays using a contactin-specific polyclonal antiserum detects contactin-GFP fusion protein in HEK293 cell lysate transfected with a plasmid encoding contactin-GFP alone (lane 2) or co-transfected with pLG338/NaN encoding the full-length Na v 1.9/NaN (lane 3). This antibody did not interact with an endogenous HEK293 protein (lane 1). The Na v 1.9/NaN-specific antibody was used to immunoprecipitate the channel complex. The precipitated channel complex was probed with the contactin-specific antibody on a Western blot (lanes 4 -6). Contactin was detected only in the samples from HEK293 cells that were co-transfected by the plasmids encoding contactin-GFP and Na v 1.9/ NaN proteins (lane 6). Contactin was specifically pulled down by the anti-Na v 1.9/NaN antibody demonstrating that it binds to this channel in vivo.
The interaction between contactin and Na v 1.9NaN was further confirmed by co-immunoprecipitation of this complex from DRG tissue. Detergent-solubilized proteins were extracted from DRGs and incubated with either anti-Na v 1.9/NaN antibodies (positive control), control IgG (negative control), or anticontactin antibodies, and the immunoprecipitated complexes were probed with anti-Na v 1.9/NaN antibodies (Fig. 4B). An immunoreactive protein with the molecular weight of Na v 1.9/ NaN was detected in the sample obtained by using anti-Na v 1.9/ NaN (lane 1) or anti-contactin (lane 3) antibodies but not from control IgG (lane 2). This assay demonstrates that contactin binds to the Na v 1.9/NaN in native tissue under physiological conditions.
Contactin Enhances Surface Expression of Na v 1.9/NaN in CHO Cells-Contactin has been shown recently to target Caspr (contactin-associated protein) to the cell surface in heterologous expression system (31) and in vivo (27). We tested the hypothesis that the interaction between contactin and Na v 1.9/ NaN enhances the surface expression of Na v 1.9/NaN. CHO

FIG. 4. Interaction between contactin and Na v 1.9/NaN in vivo.
A, contactin co-immunoprecipitates with Na v 1.9/NaN in transfected HEK293 cells. Extracts prepared from HEK293 cells transfected with pCMV-Na v 1.9/NaN plasmid (lanes 1 and 4), contactin-GFP expression plasmid (lanes 2 and 5), or with both (lanes 3 and 6) were incubated with antisera to Na v 1.9/NaN (lanes 4 -6). The immunoprecipitated (IP) proteins and cell extracts (lanes 1-3 serve as positive control) were examined by immunoblotting with an anti-contactin antibody. Contactin-GFP is indicated. B, contactin interacts with Na v 1.9/NaN in DRG neurons. Extracts (500 g) prepared from DRG were incubated with either anti-Na v 1.9/NaN, control IgG, or anti-contactin antibodies followed by protein A-agarose. The immunoprecipitated proteins were examined by immunoblotting with an anti-Nav1.9/NaN antibody. Na v 1.9/NaN is indicated. WB, Western blot. cells were co-transfected with plasmids encoding Na v 1.9/NaN and GFP or contactin-GFP. Surface proteins were labeled with biotin, and the biotinylated Na v 1.9/NaN fraction was determined on immunoblot assays (Fig. 5A). Cells that had been double transfected with either Na v 1.9/NaN and GFP (lane 1) or Na v 1.9/NaN and contactin-GFP (lane 2) were extracted with Triton X-100, and the Na v 1.9/NaN total protein was immunoprecipitated with anti-Na v 1.9/NaN antibodies (Fig. 5A). Na v 1.9/NaN expression was comparable under both transfection conditions as indicated by the comparable signal in the immunoblot assay (Fig. 5A, upper panel). The biotinylated Na v 1.9/NaN fraction was detected using peroxidase-conjugated anti-biotin antibodies (Fig. 5A, lower panel). Fig. 5A shows that only trace amounts of Na v 1.9/NaN were biotinylated when GFP was co-expressed with the channel (lane 1). By contrast, an increased pool of the channel was labeled when contactin was co-expressed with channel in the CHO cell line (lane 2). The increased surface expression of Na v 1.9/NaN was also demonstrated by an alternative approach; biotinylated surface proteins of transfected CHO cells were purified on a streptavidinagarose matrix, and the presence of Na v 1.9/NaN was determined by an immunoblot assay (Fig. 5B). Na v 1.9/NaN was easily detected in the biotinylated fraction from cells co-expressing contactin but was barely detectable in cells lacking this protein (compare lanes 1 and 2).
In parallel with the biochemical experiments, double transfected CHO cells were analyzed by immunocytochemistry to determine the effect of contactin on the surface expression of Na v 1.9/NaN (Fig. 5, C-F). CHO cells that were transfected with Na v 1.9/NaN and GFP alone and probed with anti-Na v 1.9/NaN antibodies exhibited diffuse channel immunoreactivity throughout the cytoplasm (Fig. 5C). By contrast, co-expression of Na v 1.9/NaN and contactin-GFP resulted in the enhancement of the surface expression of the channel in these cells (Fig. 5,  D-F). These data clearly show that the level of surface expression of Na v 1.9/NaN increases significantly when contactin was co-expressed with this channel in CHO cells.
Na v 1.9/NaN and Contactin Co-localize in the Soma and Along Nonmyelinated C-fibers in the Sciatic Nerve and at Nerve Endings in the Skin-To confirm a role of contactin in the FIG. 5. Contactin targets Na v 1.9/ NaN to the cell surface in transfected CHO cells. A, cells transfected with pCMV-Nav1.9/NaN and an expression plasmid encoding either GFP (lane 1) or contactin-GFP (lane 2) were grown to a confluent layer, and the cells were collected and biotinylated. After cell lysis, complexes containing Na v 1.9/NaN were immunoprecipitated (IP) and analyzed by immunoblotting for Na v 1.9/NaN (upper panel) or incubated with peroxidase-conjugated anti-biotin antibodies (lower panel). B, cells processed as in A were incubated with streptavidin-agarose, and the precipitate was examined by probing with anti-Na v 1.9/NaN antibodies. C-F, analysis of CHO cells transfected with Na v 1.9/ NaN alone or Na v 1.9/NaN and contactin by confocal microscopy. C, cytoplasmic Na v 1.9/NaN immunofluorescence is prominent within CHO cells transfected with Na v 1.9/NaN alone. In contrast, CHO cells co-transfected with Na v 1.9/NaN and contactin exhibit surface expression of Na v 1.9/NaN (D) and contactin (E) with little cytoplasmic immunofluorescence. A-C, Na v 1.9/NaN (A) and contactin (B) immunoreactivity are present in neurites extending from cell body of DRG neurons in culture for 7 days. Several 0.5-m thick optical sections are merged to ensure that the entire extent of the neurite is imaged. C, merged image of A and B demonstrates co-localization of Na v 1.9/NaN and contactin (yellow) throughout the neurite, including its distal tip. D-F, 0.5-m optical sections are merged to illustrate co-localization of Na v 1.9/NaN (D) and contactin (E) in C-fibers of teased sciatic nerve of adult rat. Both Na v 1.9/NaN and contactin are homogeneously distributed along the fibers. F, merged image of D and E demonstrates co-localization of Na v 1.9/NaN and contactin in most fibers (yellow), although some fibers express contactin (green) without Na v 1.9/NaN. Scale bars, 25 m.
trafficking of Na v 1.9/NaN to the plasmalemma, we examined the subcellular localization of Na v 1.9/NaN and contactin in DRG neurons in tissue and in culture. Approximately 70% of small (Ͻ25 m) diameter neurons in DRG cultures maintained for 7 days in the presence of exogenous glial-derived neurotrophic factor exhibit Na v 1.9/NaN immunostaining. In these cultures, 91% (88/97) of the neurons exhibited contactin immunofluorescence, and contactin was present in all neurons expressing Na v 1.9/NaN (data not shown). Na v 1.9/NaN and contactin were not diffusely localized throughout the cytoplasm; rather the immunofluorescence signal demarcated the periphery of the neuronal somata (Fig. 6, A-C). To examine the subcellular localization of Na v 1.9/NaN and contactin within the cell bodies of DRG neurons in situ, cryosections of adult rat DRG were reacted with Na v 1.9/NaN and contactin antibodies (Fig. 6, D-F). Consistent with previous observations (10,50,61), Na v 1.9/NaN is predominantly expressed in small diameter DRG neurons. Contactin immunoreactivity was present in most DRG neurons regardless of size. Co-localization of Na v 1.9/ NaN and contactin was observed at the surface of DRG neurons (Fig. 6, D-F).
The co-expression of Na v 1.9/NaN and contactin in the neurites of cultured DRG neurons (Fig. 7, A-C) suggests that these proteins are actively targeted from the cell body to neuronal processes. To determine whether similar translocation of Na v 1.9/NaN and contactin occurs in vivo, we probed teased sciatic nerves with Na v 1.9/NaN and contactin antibodies. In teased nerve preparations, many C-type fibers exhibited Na v 1.9/NaN immunostaining, and contactin was co-localized in these fibers (Fig. 7, D-F). Na v 1.9/NaN and contactin were generally distributed continuously along the fibers and did not appear to be aggregated in discrete domains along the axons. The small diameter of these fibers (Ͻ1 m) precluded, with the methodologies utilized in the present experiments, identification of axoplasmic versus axolemmal localization of Na v 1.9/ NaN and contactin. The co-expression of Na v 1.9/NaN and contactin in C-fibers continued to their terminal fields within the skin where, distal to the division of trunks of fibers within the dermis, C-type fibers exhibit Na v 1.9/NaN and contactin immunofluorescence signals (Fig. 8, A-C). DISCUSSION We show in this study that contactin binds directly to a voltage-gated sodium channel, Na v 1.9/NaN. We also show that the surface expression of this sodium channel is enhanced significantly when it is co-expressed with contactin in the CHO cell line and that contactin and Na v 1.9/NaN co-localize at the soma of small diameter DRG neurons and along their nonmyelinated C-fibers as well as at nerve endings in the skin. These observations represent the first demonstration of a direct interaction of contactin with a voltage-gated sodium channel, Na v 1.9/NaN, and suggest that contactin enhances the expression of this channel and plays a role in the membrane organization of nonmyelinated axons, which are known to express Na v 1.9/NaN. The co-purification of contactin and tenascin-R could be explained by the direct interaction of contactin with the C-terminal polypeptide of Na v 1.9/NaN and the subsequent recruitment of tenascin-R to the protein complex. Alternatively, tenascin-R could bind to the C-terminal polypeptide of Na v 1.9/NaN via another protein such as a sodium channel ␤-subunit acting as a bridge with the subsequent recruitment of contactin to this multiprotein complex. The data presented in this study provide strong evidence that contactin binds directly, although possibly transiently, to Na v 1.9/NaN and recruits tenascin-C, which is highly similar to tenascin-R (51)(52), to the channel complex. This is analogous to the role of ␤-subunits in recruiting tenascin-R and -C to interact with Na v 1.2 sodium channel complexes (21,22,62), and is consistent with the structural and sequence similarity between the external domain of ␤2-subunit and one of the Ig domains of contactin (17,26).
Contactin is a cell adhesion molecule that is a glycosylphosphatidylinositol-anchored member of the immunoglobulin gene superfamily (54 -56). Contactin is expressed in axons and dendrites of many neuronal types as well as in oligodendrocytes (63, 64) but not Schwann cells (63). Contactin has been shown to interact in cis with surface proteins such as Caspr (30) and in trans with neurofascin 155 (32). Contactin plays a role in neurite extension (65)(66)(67), axonal guidance (68), and in the organization of paranodes in myelinated fibers (27,28). Contactin has been shown to be essential for the surface expression of Caspr in transfected CHO cell lines (31) and in vivo (68). Contactin null mice do not survive past postnatal day 18, supporting an essential role for this protein in the development of normal neural circuitry (69).
The organization of the nodal region of myelinated fibers, where sodium channels are aggregated at high density (36,37), is critical for the normal generation and propagation of the nerve impulse. Na v 1.2 and Na v 1.6 sodium channels are sequentially expressed at nodes of Ranvier during normal development of the optic nerve (34,35). Sodium channels at the nodes FIG. 8. Analysis of Na v 1.9/NaN and contactin expression in C-type fibers in the skin by confocal microscopy. Bundles of C-fibers divide as they approach the epidermis (*), and co-express Na v 1.9/NaN (A) and contactin (B). C, merged image of A and B. Scale bar, 25 m. have been shown to interact with ankyrin G via the cytoplasmic tail of the ␤-subunits, and this interaction may participate in anchoring the sodium channels at nodes of Ranvier (70). Recently, Ratcliff et al. (25) provided evidence that ␤1 and ␤3 but not ␤2 interact with neurofascin 168 and suggested that this interaction may also play an important role in targeting and retaining sodium channels at the nodes. More recently, Kazarinova-Noyes et al. (33) demonstrated that contactin, acting via ␤1-subunits, increases expression of Na v 1.2-subunits. These studies, however, do not provide information about the surface expression of sodium channels along nonmyelinated fibers, where Na v 1.9/NaN is preferentially expressed (10,40), and channels are randomly distributed at a much lower density (38,39). Because sodium channels are not apparently clustered in nonmyelinated fibers, alternative mechanisms may underlie the surface expression of channels such as Na v 1.9/NaN. Contactin binds to the extracellular matrix proteins tenascin-C and -R via its immunoglobulin domains (57)(58)(59). The homology of the extracellular Ig fold of the ␤2-subunits of sodium channels to contactin (17,26) led to the demonstration that Na v 1.2 sodium channels interact with tenascin-C and -R via the ␤-subunits of the channel (21,22), and this interaction affects the gating of Na v 1.2 sodium channels when expressed in Xenopus oocytes (22). This interaction of Na v 1.2 via ␤-subunits with tenascin may immobilize the sodium channels at the membrane surface (21). The direct interaction of contactin with Na v 1.9/NaN and the recruitment of tenascin to the protein complex suggest that tenascin may participate in the stabilization of Na v 1.9/NaN at the cell surface of nonmyelinated fibers and may also modulate the gating of this channel. Although tenascin-C is widely expressed in the nervous system (for review see Ref. 51), it remains to be seen if it is also produced by the nonmyelinating Schwann cells that are in contact with the nonmyelinated C-fibers of the sciatic nerve.
Na v 1.9/NaN is known to be expressed in the somata (50) and axons (40) of small DRG neurons. The direct interaction of contactin with this sodium channel may provide a mechanism for its targeting to or anchoring at the surface of somata and non-myelinated fibers of the small diameter DRG neurons. Two glycosylated forms of contactin have been detected in DRG neurons, and the lighter form is co-purified with Caspr, whereas the heavier form is expressed on nonmyelinated fibers (28). Based on the differential distribution of the two glycosylated isoforms of contactin among myelinated and nonmyelinated fibers (28), we predict that the heavier contactin isoform is the natural partner of Na v 1.9/NaN. The cellular compartment in which contactin first interacts with Na v 1.9/NaN is not yet known. Contactin and Caspr form a complex in an endomembranous compartment (28), which appears to facilitate the expression of Caspr at the cell surface (31,68). Similarly, contactin may bind directly, although transiently, to the C terminus of Na v 1.9/NaN in a cytosolic compartment, thus facilitating the surface expression of the channel. The stability of Na v 1.9/NaN at the cell surface may not be dependent on continuing direct binding of the channel to contactin, just as the stability of Casper at the surface of transfected CHO cells does not depend in a continuing manner on interaction with contactin (31). Alternatively, contactin may interact with other segments of Na v 1.9/NaN once at the cell surface. Further experiments are needed to test these hypotheses.
In summary, our data provide the first demonstration of a direct interaction of contactin and a voltage-gated sodium channel, Na v 1.9/NaN. Our data also suggest that contactin recruits tenascin-C to the channel complex and participates in the localization of Na v 1.9/NaN channels along nonmyelinated axons of C-fibers. The biological role of contactin in relation to Na v 1.9/NaN will be further elucidated when the surface expression and gating properties of Na v 1.9/NaN in wild type, contactin-null, and Caspr null mice are compared.