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Originally published In Press as doi:10.1074/jbc.M405990200 on October 4, 2004

J. Biol. Chem., Vol. 279, Issue 50, 52744-52752, December 10, 2004
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Heterophilic Interactions of Sodium Channel {beta}1 Subunits with Axonal and Glial Cell Adhesion Molecules*

Dyke P. McEwen{ddagger} and Lori L. Isom§

From the Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109-0632

Received for publication, May 28, 2004 , and in revised form, August 4, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Voltage-gated sodium channels localize at high density in axon initial segments and nodes of Ranvier in myelinated axons. Sodium channels consist of a pore-forming {alpha} subunit and at least one {beta} subunit. {beta}1 is a member of the immunoglobulin superfamily of cell adhesion molecules and interacts homophilically and heterophilically with contactin and Nf186. In this study, we characterized {beta}1 interactions with contactin and Nf186 in greater detail and investigated interactions of {beta}1 with NrCAM, Nf155, and sodium channel {beta}2 and {beta}3 subunits. Using Fc fusion proteins and immunocytochemical techniques, we show that {beta}1 interacts with the fibronectin-like domains of contactin. {beta}1 also interacts with NrCAM, Nf155, sodium channel {beta}2, and Nf186 but not with sodium channel {beta}3. The interaction of the extracellular domains of {beta}1 and {beta}2 requires the region 169TEEEGKTDGEGNA181 located in the intracellular domain of {beta}2. Interaction of {beta}1 with Nf186 results in increased Nav1.2 cell surface density over {alpha} alone, similar to that shown previously for contactin and {beta}2. We propose that {beta}1 is the critical communication link between sodium channels, nodal cell adhesion molecules, and ankyrinG.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The axo-glial complex in myelinated axons is composed of a nodal gap region that contains high density clusters of voltage-gated sodium channels and a juxtaparanodal region that contains voltage-gated potassium channels (1–6). The nodal gap and the juxtaparanode are separated by the paranode, a region containing septate-like junctions composed of cell adhesion molecules (CAMs)1 that act as diffusion barriers to ion channel movement (2, 6–8). This specific arrangement of voltage-gated sodium and potassium channels results in rapid and efficient saltatory conduction of action potentials (9–12).

Voltage-gated sodium channels are composed of a pore-forming {alpha} subunit, from the gene family containing Nav1.1 through Nav1.9 and at least one {beta} subunit from the gene family containing {beta}1, {beta}1A, {beta}2, {beta}3, and {beta}4 (10, 13–16). Sodium channels are clustered at high density in axon initial segments and nodes of Ranvier of myelinated axons, where they co-localize with members of the Ig superfamily of CAMs such as contactin, NrCAM, and neurofascin 186 (Nf186) (2, 17, 18).

Sodium channel {beta}1 and {beta}2 subunits are also members of the Ig superfamily of CAMs. {beta}1 and {beta}2 colocalize with sodium channel {alpha} subunits at nodes of Ranvier, and {beta}1 interacts with contactin and with Nf186 in vitro (9, 17, 19, 20). Nf186, NrCAM, {beta}1, and {beta}2 each interact in vitro with a key cytoskeletal anchoring protein, ankyrinG, that is also localized to nodes of Ranvier (21–23). {beta}1- or {beta}2-mediated homophilic cell-adhesive interactions result in ankyrin recruitment in Drosophila S2 cells, and the interaction of sodium channel {alpha} subunits with ankyrinG is greatly enhanced in the presence of {beta}1 subunits in vitro (22, 24). We propose that the sodium channel signaling complex at nodes of Ranvier includes the CAMs contactin, NrCAM, and Nf186 as well as ankyrinG and that sodium channels interact with these proteins via {beta}1 subunits.

In sciatic nerve, Nf186 and NrCAM are early markers of nodal formation that cluster at presumptive nodes prior to sodium channels and ankyrinG (2, 25). In the peripheral nervous system (PNS), Schwann cell contact at the beginning stages of myelin ensheathement is required for sodium channel clustering (26, 27). In optic nerve, developing nodes of Ranvier are defined by clustering of ankyinG prior to the arrival of CAMs and sodium channels (28, 29), and this process is independent of paranodal axoglial cell adhesion (29). In the central nervous system (CNS), conditioned medium from optic nerve glia induces clustering of sodium channels in retinal ganglion cell axons in vitro (30). In vivo, the presence of oligodendrocytes is necessary for sodium channel clustering and nodal formation in the CNS (30).

Contactin is expressed at paranodes in the PNS, where it interacts with Caspr on the axonal membrane and neurofascin 155 (Nf155) on the Schwann cell membrane to form septate-like junctions that separate the nodal gap from the juxtaparanode (31–33). In the CNS, contactin is expressed in the nodal gap, where it interacts with sodium channels (17, 32), as well as in the paranode. Contactin increases the level of sodium channel expression at the cell surface from 4- to 6-fold in vitro (17), and this interaction is dependent on presence of {beta}1 subunits (9, 17). In {beta}1 (-/-) mice, contactin and sodium channels co-localize at optic nerve nodes of Ranvier but do not associate, confirming that interaction of these two molecules is dependent on the presence of {beta}1 (9). The number of mature nodes of Ranvier is significantly reduced in {beta}1 (-/-) optic nerves, and we have proposed that the loss of sodium channel-contactin interactions at the node may be responsible for this effect (9).

The purpose of the present study was to test the hypothesis that sodium channel {beta}1 subunits participate in heterophilic cell-adhesive interactions with CAMs found in the axo-glial complex. We show that {beta}1, expressed in the absence of sodium channel {alpha} subunits, interacts with NrCAM, Nf186, contactin, and Nf155. We observed an interaction of the {beta}1 and {beta}2 subunit extracellular domains only in the presence of a region of the {beta}2 intracellular domain, suggesting that cytoskeletal interactions may affect {beta}2-mediated heterophilic cell adhesion. We did not detect {beta}1 interactions with sodium channel {beta}3 subunits in our system. {beta}1-Nf186 interactions result in increased sodium channel {alpha} subunit cell surface expression in vitro, similar to what we showed previously for contactin and {beta}2. In contrast, although {beta}1 is required for NrCAM interactions with Nav1.2, coexpression of {beta}1 and NrCAM did not increase {alpha} subunit cell surface expression. These results suggest that {beta}1 subunits interact with multiple CAMs to form the basis of the sodium channel complex at nodes of Ranvier. In addition, the present results showing {beta}1 interaction with Nf155, coupled with our previous observation that {beta}1 (-/-) mice lack septate-like junctions at the periphery of the nodal gap in a subset of CNS and PNS axons (9), support our hypothesis that {beta}1 subunits may also participate in axo-glial communication at the nodal-paranodal boundary.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies and Constructs—Rabbit polyclonal antisera to sodium channel {beta}1 and {beta}2 subunits were described previously (19, 22, 24). A polyclonal anti-{beta}3 antibody was a gift from Dr. William A. Catterall (University of Washington, Seattle, WA) and has been described previously (20). Cy3-conjugated anti-human-Fc antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Antimouse HA.11 was obtained from Covance Research Products, Inc. (Berkeley, CA). cDNA encoding Nf155 and anti-Nf155 antibodies were gifts from Dr. Peter Brophy (University of Edinburgh, Edinburgh, Scotland) (33). cDNAs encoding NrCAM and NrCAM-Fc (Nr-Fc) and anti-NrCAM antibodies were provided by Dr. Martin Grumet (The State University of New Jersey, Piscataway, NJ) and were described previously (18). The Nf186 cDNA construct was a gift from Dr. Vann Bennett (Duke University, Durham, NC) (34). Nf155-Fc and Cn-Fc cDNA constructs were provided by Dr. Elior Peles (Weizmann Institute, Rehovot, Israel) (35, 36). The Cn and CnIg-Fc constructs were gifts from Dr. Genviève Rougon (Laboratoire de Génétique et Physiologie du Développement, Marseille, France) (37). cDNAs encoding {beta}1 and {beta}2 were described previously (24). Alexa Fluor-conjugated secondary antibodies were obtained from Molecular Probes, Inc. (Eugene, OR). Anti-mouse horseradish peroxidase-conjugated secondary antibodies were obtained from Cell Signaling (Beverly, MA), and anti-rabbit horseradish peroxidase-conjugated secondary antibodies were obtained from ICN Pharmaceuticals (Aurora, OH).

Generation of {beta} Subunit Fc Fusion Proteins—{beta}1, {beta}2, or {beta}3 cDNAs (24, 38–40) encoding each extracellular domain up to but not including the transmembrane segment were amplified by polymerase chain reaction using Taq polymerase (Invitrogen) and oligonucleotide primers that contained appropriate restriction sites to facilitate directional cloning into the polylinker region of pIG1 (provided by Dr. Genviève Rougon). A splice donor site was incorporated at the 3' end of the PCR product as previously described (37, 41). Briefly, the 3' end of each {beta} subunit cDNA fragment was defined by nucleotides A and G, which represent the first half of the introduced splice donor site (AG/GT). The amplified region with the splice donor site was then fused to the genomic sequence of the Fc region (hinge, CH2, and CH3) of the human IgG1, which bears a 3' acceptor site into the pIG1 vector. The PCR fragment and the intron were sequenced and were determined to be free of mutations. Following transfection, the resulting pre-mRNAs were spliced by the cells to yield mature mRNAs that produce chimeric molecules containing the Ig-like domains of the {beta} subunits and the hinge, CH2, and CH3 regions of the human IgG1. The resulting chimeras are called {beta}1-Fc, {beta}2-Fc, and {beta}3-Fc, respectively.

Generation of {beta}2 Truncation Mutants—Truncation mutants of the {beta}2 intracellular domain were generated using polymerase chain reaction. {beta}2{Delta}181–186 lacks the terminal six amino acids in the carboxyl domain, {beta}2{Delta}174–186 lacks the terminal 13 amino acids, {beta}2{Delta}169–186 lacks the terminal 18 amino acids, and {beta}2{Delta}164–186 lacks the terminal 23 amino acids. Each truncation mutant was generated by substituting a termination signal (TAG) for Ala181, Lys174, Thr169, or Thr164, respectively. PCR products were then subcloned into the pCR2.1 cloning vector (Invitrogen), sequenced, and then subcloned into the BamHI and EcoRI sites of pcDNA3.1Zeo(-) (Invitrogen).

Binding and Immunofluorescence—Fc fusion protein binding assays were performed as previously described (35, 36). 1610 Chinese hamster lung fibroblasts (CHL) (CRL-1657; American Type Culture Collection, Manassas, VA) grown in 8-well chamber slides (BD Biosciences) were transiently transfected with 0.5 µg of a cDNA encoding a particular CAM, as indicated in the figure legends. Independent CHL cultures were transfected in parallel with 1 µg of an Fc fusion cDNA, as indicated in the figure legends. 24–36 h post-transfection, conditioned medium containing a 0.5–1 µg/ml concentration of the Fc fusion protein was incubated overnight with the now confluent 8-well chamber slides containing CHL cells transfected with the CAM of interest. The next morning, cells were washed with PBS and fixed with 4% paraformaldehyde. The cells were washed twice with PBS, followed by blocking with PBS containing 1% glycine and 10% goat serum (PBGG) for 30 min at room temperature as previously described (35). Primary antibodies were diluted in PBGG and incubated for 2 h at room temperature. Fc fusion proteins were detected using a Cy3-conjugated anti-human IgG Fc fragment-specific antibody (Jackson ImmunoResearch Laboratories). Cells were washed three times with PBS and incubated with secondary antibodies diluted in PBGG for 45 min at room temperature. Cells were washed once in PBS and mounted using Pro Long® anti-fade kit (Molecular Probes). Immunofluorescence was detected and analyzed using an Olympus BX51 inverted light microscope using a 40x UPlanApo objective lens (Olympus, Melville, NY) located in the Microscopy and Image Analysis Laboratory at the University of Michigan. Images were captured using an Olympus DP70 camera and processed using DP Controller version 1.1.1.65 [EC] and DP Manager version 1.1.1.71 [EC] software.

Stable Transfection and Characterization of Cell Lines—CHL cells stably expressing Nav1.2, Nav1.2/{beta}1, or Nav1.2/Cn/{beta}1 have been described previously (17, 42, 43). 1 µg of cDNA encoding NrCAM, Nf155, or Nf186 was transfected into Nav1.2, Nav1.2/{beta}1, and Nav1.2/Cn/{beta}1 cells, respectively, at 80% confluence using Fugene 6 according to the manufacturer's instructions (Roche Applied Science). 24 h later, the cells were replated in the presence of Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum (Invitrogen), 1% penicillin/streptomycin (Invitrogen), and 400 µg/ml Zeocin (Invitrogen). Clonal colonies were expanded, and expression was verified by Western blot using anti-HA.11 to detect Nf186 (1:1000), anti-Nf155 (1:2500), or anti-NrCAM (1:200) antibodies.

[3H]Saxitoxin (STX) Binding Analysis—Whole cell [3H]STX saturation binding analysis of each cell line was performed using a vacuum filtration method as previously described (42) at a concentration of 5 nM [3H]STX with the addition of 10 µM unlabeled tetrodotoxin (Calbiochem) to assess nonspecific binding. [3H]STX (28 Ci/mmol) was obtained from Amersham Biosciences. Binding data were normalized to protein concentrations using Advanced Protein Assay Reagent (Cytoskeleton, Inc., Denver, CO).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Production of Fc Fusion Proteins and Binding Assays—To investigate potential {beta}1-interacting CAMs, we used soluble Fc fusion proteins encoding the extracellular domains of contactin, Nf155, NrCAM, sodium channel {beta}2, or sodium channel {beta}3 (Fig. 1A) (18, 36, 37, 44) as well as full-length Nf186, full-length {beta}2, and the truncation mutant {beta}2STOP (22, 34). Immunoblots of cell supernatants from transiently transfected CHL fibroblasts indicated expression of all of the soluble Fc fusion proteins used (Fig. 1B).



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  		FIG. 1.
Characterization of Fc fusion proteins. A, schematic representation of the soluble Fc fusion proteins. The circles represent Ig loop domains, whereas the squares represent the fibronectin-like repeats. The presence of the Fc domain results in the dimerization of the soluble proteins. B, Western blot of CHL conditioned medium showing the expression of the various Fc fusion constructs in cell supernatants. Blots were probed with antibodies to the respective proteins with the following dilutions: anti-Nf155 (1:2500), anti-Cn (1:500), anti-NrCAM (1:200), {beta}1EX (1:500), {beta}2EC (1:200), and anti-{beta}3 (1:500). Molecular masses are shown in kilodaltons. C, parental CHL cells do not bind the Fc fusion proteins. Conditioned medium from CHL cells transiently transfected with the following fusion constructs was incubated with untransfected CHL cells as follows. a, pIG1; b, Cn-Fc; c, {beta}1 and pIG1; d, Nr-Fc; e, {beta}1-Fc; f, {beta}2-Fc; g, Nf155-Fc; h, {beta}3-Fc. Cells were stained with Cy3-conjugated anti-human Fc (red, to detect Fc) and 4',6-diamidino-2-phenylindole (blue, insets a, b, d, f, g, and h) or rabbit anti-{beta}1 subunit antibodies (green, inset c). A differential interference contrast image of CHL cells is shown in inset e. Scale bar, 50 µm.

 
For each binding experiment, CHL cells were transiently transfected with the indicated CAM as described in the figure legends. To investigate binding interactions, cells were incubated with conditioned medium containing a particular Fc fusion protein. 36 h post-transfection and 12 h postincubation, the cells were fixed and stained for the expressed CAM using specific antibodies and for the Fc fusion protein of interest using a Cy3-conjugated anti-human IgG antibody. None of the Fc fusion proteins showed binding to untransfected CHL cells above background (Fig. 1C). CHL cells transfected with {beta}1 only did not bind the Fc protein backbone (Fig. 1C, c). Thus, any interaction that was observed between CHL cells transfected with {beta}1 and incubated with the Fc fusion proteins was interpreted as a specific interaction between {beta}1 and the Fc fusion protein.

{beta}1 Interacts with the Fibronectin-like Domains of Contactin—We have shown previously that contactin is part of the sodium channel complex at CNS nodes of Ranvier and may be involved in nodal formation during development and remyelination (17). The association of contactin with sodium channels in vitro is dependent on the presence of {beta}1, and this interaction results in increased sodium channel {alpha} subunit expression at the cell surface (17). The interaction between {beta}1 and contactin requires the Ig loop domain but not the juxtamembrane, transmembrane, or intracellular domains of {beta}1 (24). We further investigated this interaction by examining the regions of contactin that are important for interaction with {beta}1. {beta}1 interacts with Cn-Fc, a fusion protein containing full-length contactin (Fig. 2A). These results are similar to the interaction previously described for contactin with Nf155 and confirmed in Fig. 2C (36). In contrast, a Fc fusion construct consisting only of the six Ig domains of contactin and not the fibronectin-like domains, CnIg-Fc, did not bind {beta}1 (Fig. 2B) but did interact with NrCAM as previously described (45) (Fig. 2D). These results suggest that the fibronectin-like repeats of contactin are required for its interaction with {beta}1.



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  		FIG. 2.
{beta}1 binds Cn-Fc but does not bind CnIg-Fc. Binding was performed to detect interactions between {beta}1 and either Cn-Fc or CnIg-Fc. Binding interactions between Cn-Fc and Nf155 and between CnIg-Fc and NrCAM are shown as positive controls. Staining for anti-{beta}1EX, anti-Nf155, or anti-NrCAM is in green (insets), and staining for Cy3 is in red (to detect Fc). Cells were not permeabilized, indicating cell surface expression of all proteins. A, CHL cells were transfected with {beta}1 (green, inset) and incubated with Cn-Fc (red). B, CHL cells were transfected with {beta}1 (green, inset) and incubated with CnIg-Fc (red). C, CHL cells were transfected with Nf155 (green, inset) and incubated with Cn-Fc (red). D, CHL cells were transfected with NrCAM (green, inset) and incubated with CnIg-Fc (red). The arrows indicate examples of {beta}1-expressing cells positive for Cn-Fc (A and C) or CnIg-Fc (D) binding. The arrowheads indicate examples of {beta}1-expressing cells negative for CnIg-Fc binding (B). Scale bars, 50 µm.

 
{beta}1 Associates with Nf186, Nf155, and NrCAM—We next investigated whether the neurofascin isoforms interact with {beta}1. Previous studies have shown that Nf186 is important for determining node of Ranvier location (2, 18). Ratcliffe et al. (20) showed that the extracellular domain of {beta}1 interacts with Nf186 in a heterologous system using coimmunoprecipitation techniques. As shown in Fig. 3A, {beta}1-Fc incubated with CHL cells that were transfected with full-length NF186 showed fluorescent binding, supporting the previous evidence that {beta}1 and NF186 interact in this configuration (Fig. 3A).



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  		FIG. 3.
{beta}1 binds Nf186 and Nf155. Binding was performed to detect interactions between {beta}1 and either Nf186 or Nf155. Cells were stained with anti-HA.11 to detect Nf186 (A, inset), anti-{beta}1EX (B, inset), or anti-Cn (C, inset) and Cy3 (red, to detect Fc). A, CHL cells were transfected with Nf186 (green, inset) and incubated with {beta}1-Fc (red). B, CHL cells were transfected with {beta}1 (green, inset) and incubated with Nf155-Fc (red). C, CHL cells were transfected with Cn (green, inset) and incubated with Nf155-Fc as a positive control. The arrows indicate examples of Nf186- or {beta}1-expressing cells positive for binding. Scale bars, 50 µm.

 
The 155-kDa form of neurofascin is expressed in oligodendrocytes and is important for the formation of axo-glial septate-like junctions separating nodes of Ranvier from the juxtaparanodes (6–8, 33). Axo-glial septate-like junctions are formed through interactions between contactin and Caspr on the axonal membrane (6–8, 33). Nf155 may be the glial receptor for the contactin-Caspr complex, although recent evidence has shown that Caspr may negatively regulate interactions between contactin and Nf155 (36). Our next step was to test for {beta}1 interactions with Nf155. We were interested in investigating interactions between {beta}1 and Nf155, as {beta}1 (-/-) mice show disrupted paranodal loops. As shown in Fig. 3B, the extracellular domain of {beta}1 interacts with the extracellular domain of Nf155 (Nf155-Fc), suggesting that {beta}1 and Nf155 may interact in a trans manner between adjacent cells. As a positive control, and consistent with previous data, we show that Nf155-Fc interacts with contactin (Fig. 3C) (36).

We next investigated whether the nodal protein NrCAM could interact with {beta}1. When {beta}1-transfected cells were incubated with Nr-Fc, we detected only a weak interaction between these two proteins (Fig. 4A). Using the opposite configuration, we detected a strong signal when NrCAM-transfected CHL cells were incubated with {beta}1-Fc (Fig. 4B). Our results suggest that the NrCAM intracellular domain may influence its ability to participate in extracellular cell-adhesive interactions with {beta}1.



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  		FIG. 4.
{beta}1 interacts with NrCAM. CHL cells were transfected with {beta}1 or NrCAM and incubated with either Nr-Fc or {beta}1-Fc, respectively. Cells were stained with anti-{beta}1EX (A, inset) or anti-NrCAM (B, inset) and Cy3 (red, to detect Fc). A, CHL cells transfected with {beta}1 (green, inset) and incubated with Nr-Fc (red). B, CHL cells transfected with NrCAM (green, inset) and incubated with {beta}1-Fc (red). The arrows indicate examples of NrCAM (B)-expressing cells positive for {beta}1-Fc binding. The arrowheads indicate examples of {beta}1-expressing cells negative for Nr-Fc binding (A). Scale bars, 50 µm.

 
{beta}1 Interacts with Sodium Channel {beta}2 but Not {beta}3 Subunits—{beta}1 participates in homophilic ({beta}1-{beta}1) interactions that result in ankyrin recruitment to points of cell-cell contact in Drosophila S2 cells and in mammalian fibroblasts (22, 23). However, heterophilic interactions between {beta} subunits (i.e. {beta}1-{beta}3 or {beta}1-{beta}2) were not tested in those studies. In the next series of experiments, we tested whether {beta}1 interacts heterophilically with either {beta}2 or {beta}3.

As a positive control, CHL cells transfected with full-length {beta}1 were incubated with {beta}1-Fc. We observed {beta}1-Fc binding in this assay (Fig. 5A), consistent with our previous results (22). In contrast, we could not detect an interaction between {beta}1- and {beta}3-Fc in parallel experiments (Fig. 5B), suggesting that {beta}1 and {beta}3 do not interact. To perform the reverse experiment, CHL cells were transfected with full-length {beta}3 and then incubated with {beta}1-Fc (Fig. 5C). Again, no interaction between {beta}1 and {beta}3 was observed in this configuration. Ratcliffe et al. (20) showed previously that {beta}3 interacts heterophilically with Nf186. Consequently, as a positive control, CHL cells were transfected with Nf186 and incubated with {beta}3-Fc. Under these conditions, {beta}3-Fc interacted with Nf186 (Fig. 5D). Thus, our {beta}1-Fc and {beta}3-Fc constructs were functional in terms of binding interactions, but they did not interact with each other in our system.



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  		FIG. 5.
{beta}1 interacts with itself but does not interact with {beta}3. Binding was performed to detect interactions between {beta}1 and {beta}3 subunits. Binding interactions between {beta}3 and Nf186 are shown as a positive control. A, CHL cells transfected with {beta}1 (green, inset) and incubated with {beta}1-Fc (red), indicating homophilic {beta}1-{beta}1 interactions. B, CHL cells transfected with {beta}1 (green, inset) and incubated with {beta}3-Fc (red). C, CHL cells transfected with {beta}3(green, inset) and incubated with {beta}1-Fc (red). D, CHL cells transfected with Nf186 and detected with HA.11 (green, to detect Nf186, inset) and incubated with {beta}3-Fc (red). The arrows indicate examples of {beta}1- or Nf186-expressing cells positive for {beta}1-Fc (A) or {beta}3-Fc (D) binding, respectively. The arrowheads indicate examples of {beta}3- or {beta}1-expressing cells negative for {beta}1-Fc (B) or {beta}3-Fc (C) binding, respectively. Scale bars, 50 µm.

 
When CHL cells were transfected with full-length {beta}1 and incubated with {beta}2-Fc, we observed no interaction between these two sodium channel subunits (Fig. 6B). As a positive control, we showed that full-length {beta}2 binds {beta}2-Fc (Fig. 6C), consistent with our previous results showing {beta}2-{beta}2 interactions in Drosophila S2 cells (22). Interestingly, performing the reverse experiment, cells transfected with full-length {beta}2 bound {beta}1-Fc (Fig. 6D), suggesting that the {beta}2 intracellular domain may play a role in the interaction of the {beta}2 extracellular domain with the extracellular domain of {beta}1. To test this hypothesis, CHL cells were transfected with {beta}2STOP, a protein lacking the intracellular domain of {beta}2 (22). {beta}2STOP-transfected CHL cells incubated with {beta}1-Fc showed no binding above background (Fig. 6E), suggesting that the intracellular domain of {beta}2 is required for interaction with the {beta}1 extracellular domain. Alternatively, the inability of {beta}2STOP to interact with {beta}1-Fc may be due to a conformational change in the {beta}2 protein due to the lack of an intracellular domain. This possibility is unlikely, however, since {beta}2STOP expression in S2 cells results in cellular aggregation (22), indicating that this construct is able to function as a homophilic CAM (Fig. 6F). Our results suggested that {beta}2-mediated inside-out signaling and/or {beta}2-cytoskeletal interactions may be required for {beta}2-mediated heterophilic cell-adhesive interactions with {beta}1.



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  		FIG. 6.
Interaction between the extracellular domains of {beta}1 and {beta}2 requires a region of the {beta}2 intracellular domain. Binding was performed to detect interactions between the extracellular domains of {beta}1 and {beta}2 subunits. Homophilic {beta}2-{beta}2 binding interactions are shown as a positive control. A, schematic representation of wild type {beta}2. Sequences of the intracellular domains of {beta}2 and of the {beta}2 truncation mutants used in the binding studies are indicated below. The Ig loop and juxtamembrane segment constitute the extracellular domain (ED) of {beta}2, with the transmembrane-spanning segment (TM) and the intracellular domain (ID) forming the remainder of each construct. B, CHL cells transfected with {beta}1 (green, inset) and incubated with {beta}2-Fc (red). C, CHL cells transfected with {beta}2 (green, inset) and incubated with {beta}2-Fc (red). D, CHL cells transfected with full-length {beta}2(green, inset) and incubated with {beta}1-Fc (red). E, CHL cells transfected with {beta}2STOP (green, inset) and incubated with {beta}1-Fc (red). F, CHL cells transfected with {beta}2STOP (green, inset) and incubated with {beta}2-Fc (red). G, CHL cells transfected with {beta}2{Delta}181–186 (green, inset) and incubated with {beta}1-Fc (red). H and I, CHL cells transfected with {beta}2{Delta}174–186 (green, inset) and incubated with {beta}1-Fc (red) to show examples of positive and negative cells. J, CHL cells transfected with {beta}2{Delta}169–186 (green, inset) and incubated with {beta}1-Fc (red). K, CHL cells transfected with {beta}2{Delta}164–186 (green, inset) and incubated with {beta}1-Fc (red). No positive cells were observed in J or K. The arrows indicate examples of {beta}2-, {beta}2STOP-, {beta}2{Delta}181–186-, or {beta}2{Delta}174–186-expressing cells positive for {beta}2-Fc (C and F) or {beta}1-Fc (D, G, and I) binding. The arrowheads indicate examples of {beta}1-, {beta}2STOP-, {beta}2{Delta}174–186-, {beta}2{Delta}169–186-, or {beta}2{Delta}164–186-expressing cells negative for {beta}2-Fc (B) or {beta}1-Fc (E, H, J, and K) binding. Scale bars, 50 µm.

 
To further investigate the role of the {beta}2 intracellular domain in the extracellular interaction of {beta}2 with {beta}1, we created a series of {beta}2 truncation mutants in which increasing numbers of amino acids were deleted from the intracellular domain (Fig. 6A). These truncation mutants are termed {beta}2{Delta}181–186, {beta}2{Delta}174–186, {beta}2{Delta}169–186, and {beta}2{Delta}164–186, respectively. When {beta}2{Delta}181–186 was transfected into CHL cells and incubated with {beta}1-Fc, it interacted with {beta}1-Fc to a similar extent as wild type {beta}2 (Fig. 6G). {beta}2{Delta}174–186, however, showed an impaired ability to interact with {beta}1-Fc, since only a small portion of the transfected cells exhibited positive binding for {beta}1-Fc (Fig. 6, H and I). Further deletion of the {beta}2 intracellular domain, {beta}2{Delta}169–186, and {beta}2{Delta}164–186 resulted in the abolishment of {beta}2 binding to {beta}1-Fc despite robust expression of these mutant proteins at the plasma membrane, indicating that the region 169TEEEGKTDGEGNA181 in the intracellular domain of {beta}2 is critical for interaction of the {beta}2 extracellular domain with {beta}1.

{beta}1, Contactin, and Nf186, but Not Nf155 or NrCAM, Increase Cell Surface Sodium Channel Expression—{beta}1 increases Nav1.2 cell surface expression in transfected cells by ~2-fold over that observed with {alpha} alone (17, 42). Coexpression of {beta}2 or {beta}3 with Nav1.2 in the absence of {beta}1 resulted in no significant increases in sodium channel cell surface density over {alpha} alone, whereas the presence of both contactin and {beta}1 increased Nav1.2 at the cell surface by 6-fold (17, 24). We next investigated whether Nf186, Nf155, or NrCAM affected Nav1.2 cell surface expression levels. Stable CHL cell lines expressing Nav1.2 in combination with {beta}1, Nf186, Nf155, and/or NrCAM were generated by stably transfecting Nf186, Nf155, or NrCAM into the previously established Nav1.2 or Nav1.2/{beta}1 stable cell lines (17, 24, 42). Two independent stable clones showing robust expression of each respective CAM by Western blot were selected for the binding studies (Fig. 7A). Cell surface sodium channel expression was measured using whole cell [3H]STX binding as previously described (24). When expressed with Nav1.2 in the absence of {beta}1, neither Nf186, Nf155, nor NrCAM increased Nav1.2 cell surface levels over {alpha} alone (Fig. 7B). In fact, these CAMs produced statistically significant decreases in [3H]STX binding compared with {alpha} alone. We have observed similar results for {beta}2 and contactin (17) but do not as yet have an explanation for these effects. In the presence of {beta}1, neither Nf155 nor NrCAM increased cell surface Nav1.2 expression over that observed with the Nav1.2/{beta}1 cell line (Fig. 7B). In contrast, Nf186 did increase Nav1.2 density at the cell surface by ~2-fold in the presence of {beta}1 compared with Nav1.2/{beta}1. We next investigated whether NrCAM or Nf186 might increase the level of sodium channel cell surface expression observed in the presence of {beta}1 and contactin. The cell lines Nav1.2/{beta}1/Cn/Nf186 and Nav1.2/{beta}1/Cn/NrCAM were generated from the previously established Nav1.2/{beta}1/Cn line (17). Neither Nf186 nor NrCAM increased sodium channel cell surface expression over that observed in the presence of {beta}1 and contactin (Fig. 7B), suggesting that the level of Nav1.2 expressed at the cell surface in the presence of {beta}1 and contactin may be the maximal cell surface density of channels attainable in this heterologous system. [3H]STX binding to Nav1.2/{beta}1/Cn/Nf186 was actually decreased from that observed for Nav1.2/{beta}1/Cn; however, we suspect that this could be due to variations in individual cells in the original Nav1.2/{beta}1/Cn cell line.



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  		FIG. 7.
Coexpression of {beta}1 and Nf186 increases while coexpression of {beta}1 with Nf155 or NrCAM has no effect on Nav1.2 cell surface density. A, Western blot analysis of Nav1.2 or Nav1.2/{beta}1 or Nav1.2/{beta}1/Cn cell lines that were transfected with Nf155, Nf186, or NrCAM as indicated. Immunblots were probed with anti-Nf155 (1: 2000), anti-HA.11 (to detect Nf186, 1:1000), and anti-NrCAM (1:200), respectively. B, [3H]STX binding to intact CHL cells expressing either Nav1.2 alone or Nav1.2 with a combination of contactin, Nf155, Nf186, NrCAM, and/or {beta}1. The error bars indicate means ± S.E. for Nav1.2 (n = 6), Nav1.2/Nf155 (n = 3), Nav1.2/Nf186 (n = 3), Nav1.2/NrCAM (n = 3), Nav1.2/{beta}1 (n = 6), Nav1.2/{beta}1/Nf155 (n = 3), Nav1.2/{beta}1/Nf186 (n = 6), Nav1.2/{beta}1/NrCAM (n = 3), Nav1.2/{beta}1/Cn (n = 5), Nav1.2/{beta}1/Cn/Nf186 (n = 3), and Nav1.2/{beta}1/Cn/NrCAM (n = 3) cells. For all cell lines, binding was performed on at least two different stable clones. 10 µM tetrodotoxin was used to determine nonspecific binding as previously described (8). [3H]STX binding was normalized to protein using Advanced protein assay reagent (Cytoskeleton, Inc., Denver, CO). *, statistically significant compared with Nav1.2 (p < 0.05). **, statistically significant compared with Nav1.2/{beta}1 (p < 0.05). ***, statistically significant compared with Nav1.2/{beta}1/Cn (p < 0.05). C, immunoprecipitation (IP) of Nav1.2 with NrCAM from stably expressing Nav1.2/NrCAM or Nav1.2/{beta}1/NrCAM CHL cell lines. Equal amounts of cell lysates were immunoprecipitated with 5 µl of either nonimmune IgG or Sp11II for Nav1.2. Immunoblots were probed with anti-NrCAM antibodies (1:200). Cell lysates indicate equal expression of NrCAM in both cell lines (lanes 4 and 5).

 
We propose that {beta}1 plays a key role in the interaction of sodium channels with CAMs at nodes of Ranvier. Our previous results showed that contactin and sodium channels do not interact in the absence of {beta}1 in vitro or in vivo (9, 17). Ratcliffe et al. showed that the interaction of Nf186 with the sodium channel complex is dependent on the presence of the {beta}1 subunit in transfected cells (20). We were next interested in determining whether {beta}1 is required for the interaction of NrCAM with Nav1.2. Lysates from the Nav1.2/NrCAM and Nav1.2/{beta}1/NrCAM cell lines used in the binding studies were immunoprecipitated with either nonimmune IgG or the anti-Nav1.2 antibody, Sp11II. Immunoblotting with anti-NrCAM antibodies showed that Nav1.2 and NrCAM associate in the Nav1.2/{beta}1/NrCAM cell line but not in the absence of {beta}1 (Fig. 7C, lane 3). The control IgG and Nav1.2/NrCAM lanes (Fig. 7C, lanes 1 and 2) were negative for NrCAM staining despite similar NrCAM expression levels in the two cell lines (Fig. 7C, lanes 4 and 5). This result indicates that, similar to contactin and Nf186, {beta}1 is required for the interaction of NrCAM with the sodium channel {alpha} subunit.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Sodium channel {beta}1 subunits are multifunctional proteins that are involved in channel modulation as well as cell adhesion (17, 22–24, 42). {beta}1 subunits interact homophilically in vitro, resulting in cellular aggregation and ankyrin recruitment to points of cell-cell contact, as well as heterophilically in vitro with contactin and Nf186 (17, 20, 22, 24). The present data identify the CAMs Nf155, NrCAM, and sodium channel {beta}2 subunits as additional heterophilic binding partners of {beta}1 in vitro. In contrast, sodium channel {beta}3 subunits do not appear to interact with {beta}1. Nf155 and NrCAM are important for formation and maintenance of both CNS and PNS nodes of Ranvier (2, 3, 6). {beta}2 is critical for sodium channel cell surface expression in vitro and in vivo (19, 46, 47). We show that contactin, Nf186 and NrCAM increase channel cell surface expression in the presence but not in the absence of {beta}1, similar to that shown previously for contactin and {beta}2 (17, 24). Thus, we propose that {beta}1 functions as the critical communication link between sodium channels, ankyrinG, and other CAMs at the node of Ranvier and may participate in paranodal septate-like junction formation via interaction with Nf155.

We have previously described the interaction between sodium channel {beta}1 subunits and the CAM contactin (17, 24). This interaction depends on the presence of the {beta}1 Ig loop domain and results in a 4–6-fold increase in Nav1.2 expression at the cell surface in vitro (24). In the present study, we show that the fibronectin-like repeats of contactin, but not the Ig loop domains, are essential for {beta}1 interactions. In {beta}1 (-/-) mice, the association of sodium channels with contactin is disrupted, confirming the interpretation of our in vitro results that {beta}1 subunits are required for formation of the sodium channel-contactin complex (9). We have proposed that the loss of sodium channel-contactin interactions in vivo results in destabilization of sodium channel cell surface expression at nodes and the subsequent decrease in the number of mature CNS nodes of Ranvier observed in {beta}1 (-/-) mice (9). Our present results suggest that the "horseshoe" configuration of contactin (48–50) is not required for its interaction with {beta}1, leaving the Ig domains of contactin free to interact with other CAMs while simultaneously interacting with {beta}1. This proposed arrangement would thus increase the number and diversity of CAMs that can be involved in the sodium channel complex.

Recent evidence has shown a role for NrCAM and Nf186 in delineating presumptive PNS nodes of Ranvier prior to sodium channel and ankyrinG clustering (2, 18). NrCAM (-/-) mice exhibit a delay in nodal development as well as a delay in the localization of ankyrinG to PNS nodal areas (2). In CNS myelinated axons, ankyrinG has been shown to define nodal regions prior to CAM and sodium channel clustering (28, 29). AnkyrinG (-/-) mice exhibit disrupted localization of sodium channels, neurofascin, and NrCAM at initial segments of unmyelinated cerebellar granule cell axons and myelinated Purkinje neurons (28, 51). Thus, coordinated temporal and spatial adhesive events between NrCAM, Nf186, sodium channels, and ankyrinG are critical to sodium channel clustering and may exhibit different mechanisms in the PNS compared with the CNS. In the present study, we show that sodium channel {beta}1 subunits interact with the nodal, ankyrin-binding CAMs NrCAM, Nf186, and {beta}2, in addition to contactin. Furthermore, we show that, like contactin and Nf186, the interaction of NrCAM with the sodium channel complex is dependent on {beta}1. The interaction of {beta}1 with contactin, with Nf186, or with {beta}2 (17) results in significant increases in Nav1.2 cell surface expression in vitro. We showed previously that Nav1.2 channels interact weakly with ankyrinG in CHL cells, and coexpression of {beta}1 greatly enhances this interaction (24). In the present study, interaction of {beta}1 with the ankyrin-binding CAMs NrCAM, Nf186, and {beta}2 could result in increased interaction of sodium channels with the endogenous ankyrinB protein in CHL cells (23), stabilizing sodium channels at the cell surface. We propose that {beta}1 may act to stabilize sodium channels at the nodal plasma membrane in vivo through multiple extracellular interactions with ankyrin-binding CAMs as well as through its own intracellular ankyrin-binding domain. Furthermore, we propose that the reduction in action potential conduction velocity, reduction in the integral of the optic nerve compound action potential, loss of sodium channel cell surface expression, and reduction in the number of mature nodes of Ranvier observed in {beta}1(-/-) and/or {beta}2 (-/-) mice (9, 19) result from the loss of these critical interactions between nodal CAMs and sodium channels.

Our results show that sodium channel {beta}1 and {beta}2 subunits interact through heterophilic cell-adhesive interactions in addition to the interaction of each of these proteins with the pore-forming {alpha} subunit (10, 52). Our observation that a region of the {beta}2 intracellular domain is required for heterophilic cell adhesion between the {beta}2 and {beta}1 extracellular domains raises some interesting possibilities. {beta}2 subunits participate in homophilic cell adhesion, resulting in the recruitment of ankyrin to points of cell-cell contact in Drosophila S2 cells (22). {beta}2STOP, a {beta}2 truncation mutant lacking the intracellular domain, maintains homophilic adhesion yet does not recruit ankyrin (22). In the present study, when full-length {beta}1 was incubated with soluble {beta}2-Fc, no interaction was detected between these two subunits. However, when full-length {beta}2 was incubated with soluble {beta}1-Fc, interactions between the extracellular domains of {beta}1 and {beta}2 were observed. These results suggested that a region in the intracellular domain of {beta}2 must be present for heterophilic interaction with the extracellular domain of {beta}1 to occur. Using a series of {beta}2 truncation mutants, we found that the intracellular 169TEEEGKTDGEGNA181 region of {beta}2 is critical for extracellular, heterophilic adhesion with {beta}1. We propose that an interaction in the {beta}2 intracellular domain, possibly with ankyrin, stabilizes extracellular, heterophilic cell adhesive events. We showed previously that a single residue in the {beta}1 intracellular domain, Tyr181, is critical for ankyrin binding and that phosphorylation of this residue negatively regulates ankyrin interactions (22). Whereas we have not yet defined the ankyrin-binding domain of {beta}2, the region that we have identified as critical for {beta}2 heterophilic adhesion is located in a region of the protein similar in position to the ankyrin-binding domain of {beta}1. The {beta}2 intracellular domain does not contain tyrosine residues and is not similar to other ankyrin-binding domains reported in the literature (22). However, it does contain three possible casein kinase II phosphorylation sites according to the PhosphoBase program (available on the World Wide Web at www.cbs.dtu.dk/databases/PhosphoBase/) provided by the Technical University of Denmark. These putative sites include QKLS163TDD, DLKT169EEE, and EGKT175DGE. The residues Thr169 and Thr175 are located in the region determined to be required for heterophilic adhesion. It will be interesting in the future to investigate whether this is indeed the location of the ankyrin-binding domain of {beta}2 and, if so, whether it is regulated by phosphorylation.

{beta}1(-/-) mice exhibit disrupted axo-glial contacts in a subset of axons (9). In these axons, the septate-like junctions at the nodal-paranodal boundaries are everted or pulled away from the axon, leaving all other septate-like junctions, seen as transverse bands by transmission electron microscopy, intact. We proposed that axonal {beta}1 subunits located at the nodal-paranodal border may interact in cis with axonal contactin molecules in the paranode or in trans with glial Nf155 molecules at the nodal-paranodal border region. Alternatively, because we have also shown {beta}1 expression in oligodendrocytes (53), it is possible that glial {beta}1 subunits may interact in trans with axonal {beta}1 subunits, in cis with glial Nf155, or in trans with axonal contactin molecules. In the present study, we tested the hypothesis that {beta}1 interacts with Nf155 and showed that these molecules interact in our assay. Thus, we propose that {beta}1 is associated with sodium channels at the node of Ranvier, where it serves a dual function as a channel modulator and as a CAM. In addition, we propose that {beta}1 may be expressed in the absence of sodium channels in the nodal-paranodal border region of axons or in oligodendrocytes, where it functions as a CAM only. Interaction of {beta}1 with sodium channels at nodes of Ranvier results in modulation of channel function and stabilization of channel cell surface expression through interactions with {beta}2, contactin, Nf186, NrCAM, and ankyrinG. Axonal {beta}1 subunits located at the nodal-paranodal border may also interact in cis with paranodal contactin or in trans with glial Nf155. {beta}1 subunits expressed in oligodendrocytes may participate in the formation of septate-like junctions at the nodal-paranodal border through cell-adhesive interactions in cis with Nf155 and/or in trans with contactin. It will be necessary to perform in vivo experiments using {beta}1 mutant constructs to investigate the importance of the different functional domains of {beta}1 on the maintenance and stabilization of nodes of Ranvier.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health (NIH) Grant R01MH59980 (to L. L. I.) and by a subcontract from NIH Grant R01NS17965-17 from Dr. Peter Shrager at the University of Rochester (to L. L. I.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Supported by National Research Service Award Predoctoral Fellowship NS43067. Back

§ To whom correspondence should be addressed: Dept. of Pharmacology, University of Michigan, 1150 W. Medical Center Dr., 1301 MSRB III, Ann Arbor, MI 48109-0632. Tel.: 734-936-3050; Fax: 734-763-4450; E-mail: lisom{at}umich.edu.

1 The abbreviations used are: CAM, cell adhesion molecule; Nf186, neurofascin 186; PNS, peripheral nervous system; CNS, central nervous system; Nf155, neurofascin 155; CHL, Chinese hamster lung fibroblast(s); Cn, contactin; NrCAM, Nr-Fc, NrCAM-Fc; STX, saxitoxin; PBS, phosphate-buffered saline. Back


   ACKNOWLEDGMENTS
 
We acknowledge the technical expertise of Chunling Chen for cloning of {beta}3 cDNA. We also acknowledge Sam Holmstrom for the generation of the {beta}2 truncation mutants and Emily Slat for technical assistance. We thank Dr. William Catterall for the anti-{beta}3 antibody, Dr. Vann Bennett for the Nf186 construct, Dr. Peter Brophy for the Nf155 construct and anti-Nf155 antibodies, Dr. Elior Peles for the Nf155-Fc and Cn-Fc constructs, Dr. Martin Grumet for the NrCAM antibody and the Nr-Fc and NrCAM constructs, and Dr. Genviève Rougon for the Cn and CnIg-Fc constructs and the pIG1 vector. We also thank Dr. Elior Peles and Dr. Martin Grumet for advice in the preparation of this paper.



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 ABSTRACT
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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