Structural Requirements for Interaction of Sodium Channel β1 Subunits with Ankyrin

Abstract Sodium channel β subunits modulate channel kinetic properties and cell surface expression levels and function as cell adhesion molecules. β1 and β2 participate in homophilic cell adhesion resulting in ankyrin recruitment to cell contact sites. We hypothesized that a tyrosine residue in the cytoplasmic domain of β1 may be important for ankyrin recruitment and tested our hypothesis using β1 mutants replacing Tyr181 with alanine (β1Y181A), phenylalanine (β1Y181F), or glutamate (β1Y181E), or a truncated construct deleting all residues beyond Tyr181(β1L182STOP). Ankyrin recruitment was observed in β1L182STOP, showing that residues Ile166-Tyr181 contain the major ankyrin recruiting activity of β1. Ankyrin recruitment was abolished in β1Y181E, suggesting that tyrosine phosphorylation of β1 may inhibit β1-ankyrin interactions. AnkyrinG and β1 associate in rat brain membranes and in transfected cells expressing β1 and ankyrinG in the absence of sodium channel α subunits. β1 subunits are recognized by anti-phosphotyrosine antibodies following treatment of these cell lines with fibroblast growth factor. β1 and ankryinG association is not detectable in cells following treatment with fibroblast growth factor. AnkyrinG and β1Y181E do not associate even in the absence of fibroblast growth factor treatment. β1 subunit-mediated cell adhesion and ankyrin recruitment may contribute to sodium channel placement at nodes of Ranvier. The phosphorylation state of β1Y181 may be a critical regulatory step in these developmental processes.

Sodium channel ␤ subunits modulate channel kinetic properties and cell surface expression levels and function as cell adhesion molecules. ␤1 and ␤2 participate in homophilic cell adhesion resulting in ankyrin recruitment to cell contact sites. We hypothesized that a tyrosine residue in the cytoplasmic domain of ␤1 may be important for ankyrin recruitment and tested our hypothesis using ␤1 mutants replacing Tyr 181 with alanine (␤1Y181A), phenylalanine (␤1Y181F), or glutamate (␤1Y181E), or a truncated construct deleting all residues beyond Tyr 181 (␤1L182 STOP ). Ankyrin recruitment was observed in ␤1L182 STOP , showing that residues Ile 166 -Tyr 181 contain the major ankyrin recruiting activity of ␤1. Ankyrin recruitment was abolished in ␤1Y181E, suggesting that tyrosine phosphorylation of ␤1 may inhibit ␤1-ankyrin interactions. Ankyrin G and ␤1 associate in rat brain membranes and in transfected cells expressing ␤1 and ankyrin G in the absence of sodium channel ␣ subunits. ␤1 subunits are recognized by anti-phosphotyrosine antibodies following treatment of these cell lines with fibroblast growth factor. ␤1 and ankryin G association is not detectable in cells following treatment with fibroblast growth factor. Ankyrin G and ␤1Y181E do not associate even in the absence of fibroblast growth factor treatment. ␤1 subunit-mediated cell adhesion and ankyrin recruitment may contribute to sodium channel placement at nodes of Ranvier. The phosphorylation state of ␤1Y181 may be a critical regulatory step in these developmental processes.
Sodium channels are unique among voltage-and ligandgated ion channels in that they contain auxiliary subunits that not only modulate channel kinetics, but also function as cell adhesion molecules (CAMs) 1 that direct channel insertion into the plasma membrane and channel interaction with other signaling proteins. We are exploring the novel idea that the cell adhesive functions of sodium channel ␤ subunits may be as important or even more important than modulation of channel gating in excitable cells. We propose that, as CAMs, sodium channel ␤ subunits act as critical communication links between extra-and intracellular signaling molecules.
Sodium channel auxiliary ␤ subunits are multifunctional CAMs of the Ig superfamily (1). ␤ subunits participate in homophilic cell adhesion (2), heterophilic adhesion with contactin and neurofascin (3,4), interactions with extracellular matrix molecules (5,6), and recruitment of ankyrin to the plasma membrane at sites of cell-cell contact in response to homophilic cell adhesion (2). Interestingly, ␤ subunit cell adhesive interactions are independent of participation in the ion conduction complex, suggesting that the ␤ subunits may be bifunctional molecules (2,3,6). We have proposed that the cell adhesive functions of ␤ subunits play critical roles in the regulation of sodium channel density and localization.
What is the molecular basis for the interaction of ␤1 with the cytoskeleton? We showed previously (2) that ␤1-mediated homophilic cell adhesion results in recruitment of ankyrin to sites of cell-cell contact in Drosophila S2 cells. Removal of the intracellular cytoplasmic domain of ␤1 had no obvious effects on homophilic adhesion but completely abolished ankyrin recruitment, demonstrating that the intracellular domain is required for cytoskeletal interactions. The purpose of the present study was to identify residues in the cytoplasmic domain of ␤1 that are required for ankyrin recruitment. Previous experiments have shown that deletion of a 5-amino acid sequence from the intracellular carboxyl-terminal domain of the L1-CAM family member neurofascin (FIGQY) abolished ankyrin binding activity (7,8). Phosphorylation or deletion of just the tyrosine residue in this sequence abolished ankyrin binding and significantly reduced neurofascin-mediated cell adhesion. Similar experiments were performed to investigate the structural basis for signaling by Drosophila neuroglian, another member of the L1-CAM family (9). The results of this study identified a conserved intracellular 36-amino acid sequence that is responsible for ankyrin binding. Mutation of the conserved FIGQY tyrosine residue in this region reduced the extent of ankyrin recruitment as well as cell adhesion. A missense mutation of the FIGQY tyrosine in human L1 (Y1229H) results in clinical disease and confirms this residue as critical for the proper function of L1 in neuronal development (10).
We hypothesized that an intracellular tyrosine residue in ␤1 may play a role in ankyrin recruitment similar to that observed for the conserved FIGQY tyrosine residue in members of the L1-CAM family. Interestingly, abolishment of the entire cytoplasmic domain of sodium channel ␤1 or ␤2 subunits does not have any significant effects on homophilic cell adhesion (2). Like the ␤ subunits, the intracellular domains of L1-CAMs, such as neurofascin and neuroglian, are not essential for cellular aggregation (11,12). However, unlike the ␤ subunits, * This work was supported in part by National Institutes of Health Grants R01MH59980 and R01HD29388 (to L. L. I. and M. H.). 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  mutations reducing ankyrin binding result in a reduction in both the rate and extent of cellular aggregation (7)(8)(9). The extracellular Ig domain of ␤1 is homologous to the Ig loop of the CAM myelin P o (13,14). Unlike ␤1, however, deletion of the intracellular domain of myelin P o results in abolishment of homophilic adhesion (15). Thus, the sodium channel ␤ subunits appear to diverge from L1-CAMs and myelin P o in this respect.
We tested our hypothesis that an intracellular tyrosine residue is important for ankyrin recruitment by expressing a series of truncated and site-directed ␤1 subunit mutant constructs in Drosophila S2 cells. Our results show that the sequence Ile 166 -Tyr 181 contains the major ankyrin recruiting activity of ␤1. Ankyrin recruitment was retained in ␤1Y181F, but was abolished in ␤1Y181E and impaired in ␤1Y181A. The data suggest the possibility that tyrosine phosphorylation, mimicked by introduction of a negative charge in ␤1Y181E, may regulate ankyrin-␤1 interactions. ␤1 subunits are recognized by two different anti-phosphotyrosine antibodies, PY20 and PY100, in transfected mammalian cells following treatment with fibroblast growth factor (FGF). Tyr 181 mutant ␤1 subunits are not recognized by anti-phosphotyrosine antibodies, indicating that Tyr 181 is the only intracellular tyrosine residue that is phosphorylated, even though two additional tyrosine residues (Tyr 161 and Tyr 163 ) are present near the intracellular end of the transmembrane segment. Tyr 161 and Tyr 163 are present in ␤1 STOP , a truncation mutant ending with Lys 165 that was previously shown to be deficient in ankyrin recruiting activity (2). Ankyrin G and ␤1 are co-immunoprecipitated from rat brain membranes and from transfected cells expressing ␤1 and GFPtagged ankyrin G in the absence of sodium channel ␣ subunits. Treatment of this cell line with FGF abolishes ␤1-ankyrin G interactions. As predicted by the S2 cell experiments, GFPankyrin G and ␤1Y181E do not associate in transfected cells. Our results suggest that reversible, receptor-mediated changes in tyrosine phosphorylation modulate the association of sodium channels with ankyrin during axonal fasciculation or at specialized domains such as nodes of Ranvier. We conclude that the association of ankyrin and sodium channel ␤1 subunits is mediated through a 16-amino acid segment of the intracellular domain of ␤1 containing a tyrosine residue and that this event occurs independently of ␤1 subunit association with the ionconducting pore.

EXPERIMENTAL PROCEDURES
Materials-Drosophila S2 cells were obtained from the American Type Culture Collection. Rabbit polyclonal antisera to an extracellular domain of ␤1 (KRRSETTAETFTEWTFR), ␤1 EX , was described previously (2). Polyclonal antiserum to an intracellular domain of ␤1 (LAIT-SESKENCTGVQVAE), ␤1 IN , was generated and affinity purified by Research Genetics (Huntsville, AL). Mouse anti-Drosophila ankyrin antibodies were generated as previously described (16). Anti-ankyrin G , anti-ankyrin B , and anti-PY20 antibodies were obtained from Zymed Laboratories Inc. (South San Francisco, CA). Anti-GFP antibody was obtained from Molecular Probes (Eugene, OR). ␣-Amanitin and FGF were obtained from Sigma. Anti-PY100 was obtained from Cell Signaling Technology (Beverly, MA). A GFP-tagged rat ankyrin G construct in the vector pEGFP-N1 (CLONTECH) was a gift from Dr. V. Bennett, Duke University. Chinese hamster lung 1610 and 1610␤1 cells have been described previously (6,17).
All final products were cloned into pCR2.1 (Invitrogen). ␤1Y181F was then liberated with EcoRV and XhoI for subcloning into the SmaI and XhoI sites, respectively, of pRmHa3, containing the Drosophila metallothionein promoter (18). ␤1Y181A and ␤1Y181E were liberated with EcoRI and subcloned into the EcoRI site of pRmHa3, respectively. ␤1L182 STOP was liberated with XhoI and SacI and subcloned into the SalI and SacI sites, respectively, of pRmHa3. Each construct was sequenced completely (Thermosequenase (Amersham Biosciences)) prior to use.
Transfection of S2 Cells-Drosophila Schneiders' line 2 (S2) cells were grown and maintained as previously described (11,19). S2 cells were transfected with FuGENE according to the manufacturer's instructions (Roche Molecular Biochemicals). The cells were co-transfected with pPC4 to confer ␣-amanitin resistance as a selectable marker as previously described (20) and cloned using the soft agar method (19). Cells were then induced overnight in the presence of 0.7 mM CuSO 4 and mechanical shaking, as previously described (11), and analyzed by Western blot for protein expression of the transfected cDNA. For Western blot analysis, ␤1-transfected S2 cells were solubilized in 5% SDS and boiled in SDS-PAGE sample buffer containing 5% ␤-mercaptoethanol. Samples were separated on 10% acrylamide SDS-PAGE gels and transferred to nitrocellulose. Western blots were probed with anti-␤1 EX antibody, as indicated, at 1:500 dilution, and then with horseradish peroxidase-conjugated goat anti-rabbit antibody at 1:100,000 dilution. Immunoreactive bands were visualized with WestDura chemiluminescent reagent (Pierce). These and subsequent digital images were prepared using Adobe Photoshop 6.0.
Aggregation Assays-Equal aliquots of wild type or cloned transfected S2 cells were induced in the presence of 0.7 mM CuSO 4 followed by mixing at room temperature on a rotary shaker, as previously described (11). 10-l aliquots of each cell line were removed after 20, 40, 60, and 80 min and analyzed by counting cells with a hemocytometer to quantitate the extent of aggregation by counting non-aggregated cells. The data were analyzed using Prism software.
Immunocytochemical Detection of Ankyrin in S2 Cells-Immunocytochemical analysis of Drosophila ankyrin distribution in S2 cells was performed following the aggregation experiments described above. Cells were fixed with 2% paraformaldehyde and permeabilized with 0.5% Triton X-100. Mouse anti-Drosophila ankyrin was used as the primary antibody followed by incubation with fluorescein isothiocyanate-conjugated anti-mouse IgG antibody. Slides were then viewed with a Bio-Rad MRC 600 confocal scanning laser microscope in the Microscopy and Image Analysis Laboratory Core Facility at the University of Michigan.
Transfection of Mammalian Cells with ␤1Y181 Mutants and/or Ankyrin G -Chinese hamster lung 1610 and 1610␤1 (6) cell lines were transfected with GFP-tagged ankyrin G and/or ␤1Y181(A, E, or F) using FuGENE as the transfection reagent. G418 and hygromycin were used to select for ankyrin G and ␤1Y181 mutant expression, respectively. Expression of ␤1, ␤1Y181 mutants, and/or GFP-ankyrin G in each line was monitored by Western blot analysis.
Co-immunoprecipitation of Wild Type and Mutant ␤1 Subunits with Ankyrin G -Immunoprecipitations were performed from rat brain membranes prepared as previously described (21) or from transfected cells. Membranes or transfected cells were solubilized in 1.25% Triton X-100 and the soluble fraction was incubated overnight at 4°C with anti-␤1 EX antibody or anti-GFP antibody, as indicated in the figure legends. Protein A-Sepharose (50 l of a 1:1 suspension) was then added and the incubation was continued for 2 h at 4°C. The Protein A-Sepharose beads were precipitated in a microcentrifuge and washed with 50 mM Tris-HCl, pH 7.5, containing 0.1% Triton X-100. Immunoprecipitates were eluted from the Protein A-Sepharose with SDS-PAGE sample buffer and separated on 5 or 10% acrylamide SDS-PAGE gels as indicated in the figure legends. Proteins were then transferred to nitrocellulose and probed with anti-␤1 EX or anti-GFP antibodies, as indicated in the figure legend. Chemiluminescent detection of immunoreactive bands was accomplished with WestDura reagent.
Immunoprecipitation of ␤1 Subunits with Anti-phosphotyrosine Antibodies-Immunoprecipitations were performed from solubilized transfected cell pellets following stimulation of cell monolayers with FGF or vehicle (50 ng/ml for 30 min at 37°C). Membranes or cells were solubilized in 1.25% Triton X-100 containing 1 mM sodium orthovanadate and the soluble fraction was incubated overnight at 4°C with anti-␤1 IN antibody in the presence of 1 mM sodium orthovanadate. Protein A-Sepharose (50 l of a 1:1 suspension) was then added and the incubation was continued for 2 h at 4°C. The Protein A-Sepharose beads were precipitated in a microcentrifuge and washed with 50 mM Tris-HCl, pH 7.5, containing 0.1% Triton X-100. Immunoprecipitates were eluted from the Protein A-Sepharose with SDS-PAGE sample buffer and separated on 10% acrylamide SDS-PAGE gels. Proteins were then transferred to nitrocellulose and probed with anti-phosphotyrosine antibody, as indicated in the figure legends. Whereas both anti-PY20 and anti-PY100 recognized ␤1 subunits equally well, only the experiments with anti-PY20 are shown. Chemiluminescent detection of immunoreactive bands was accomplished with WestDura reagent.
Immunocytochemcial Localization of Ankyrin in 1610 Fibroblasts-Chinese hamster lung 1610 and 1610␤1 (6) cell lines were used to analyze ankyrin B localization. Cells were fixed with 2% paraformaldehyde and permeabilized with 0.5% Triton X-100. Mouse anti-ankyrin B (Zymed Laboratories Inc.) was used as the primary antibody (1:200), followed by incubation with fluorescein isothiocyanate-conjugated antimouse IgG antibody (1:200). Slides were then viewed with a Zeiss LSM 510 confocal microscope mounted on a Zeiss Axiovert 100M inverted microscope in the Microscopy and Image Analysis Laboratory Core Facility at the University of Michigan.

An intracellular tyrosine residue (Tyr 181 ) is essential for ␤1ankyrin interactions but is not required for cell adhesion-To
test our hypothesis that the single intracellular tyrosine residue downstream of Lys 165 in ␤1, Tyr 181 , is critical for ankyrin recruitment, we constructed a number of mutant ␤1 cDNAs in pRmHa3, including ␤1L182 STOP , ␤1Y181F, ␤1Y181E, and ␤1Y181A (Fig. 1A) and co-transfected them with a plasmid conferring ␣-amanitin resistance into Drosophila S2 cells. Each cell line was then cloned using the previously described soft agar method (19). Fig. 1B demonstrates that each of these mutant constructs was efficiently expressed. S2 cells transfected with each of the ␤1Y181 mutants formed aggregates following induction with CuSO 4 and mechanical shaking (Fig.  2). Untransfected S2 cells treated similarly did not aggregate. The percent of non-aggregated cells was quantified for each cell line following 20, 40, 60, and 80 min of induction. In contrast to studies with L1 family CAMs, the extent of cellular aggregation after 80 min was similar for wild type ␤1 and the ␤1 mutants. These results are consistent with our previous studies showing that the intracellular domain of ␤1 beyond Ile 166 is not necessary for cell adhesion (2). Interestingly, there appears to be subtle differences in the rate of aggregation in the Tyr 181 mutant constructs compared with wild type.
We also showed previously that the intracellular domain of ␤1 from Ile 166 to the carboxyl terminus is required for homophilic adhesion-induced ankyrin recruitment (2). Immunocytochemical analysis of ␤1L182 STOP -induced cell aggregates using an antibody to Drosophila ankyrin showed ankyrin recruitment to sites of cell-cell contact similar to wild type ␤1 (72 versus 80% recruitment, Table I), indicating that residues Ile 166 -Tyr 181 contain the major ankyrin recruiting activity for this molecule (Fig. 3, A and B). We next tested a series of point mutations to evaluate the importance of Tyr 181 in ␤1-mediated ankyrin recruitment. ␤1Y181F-induced cell adhesion resulted in ankyrin recruitment to an extent similar to wild type ␤1 (75 versus 80% recruitment, Table I) (Fig.  3, C and D). In contrast, ankyrin recruitment induced by ␤1Y181A was significantly impaired (15 versus 80% recruitment, Table I) (Fig. 3, E and F). These results are similar to those reported for neurofascin (22), and suggest that the aromatic ring of Tyr 181 may be critical for ␤1-ankyrin inter- Positions of tyrosine residues are indicated by the filled circles. B, equal aliquots of wild type ␤1or ␤1 mutant-transfected S2 cells, as indicated, were solubilized in 5% SDS and boiled in SDS-PAGE sample buffer containing 5% ␤-mercaptoethanol. Samples were separated by 10% acrylamide SDS-PAGE and transferred to nitrocellulose. Western blots were probed with anti-␤1 EX antibody at 1:500 dilution and then with horseradish peroxidaseconjugated goat anti-rabbit antibody at 1:100,000 dilution. Immunoreactive bands were visualized with WestDura chemiluminescent substrate. Molecular mass markers are given in kDa. Previous studies with neurofascin have also shown that phosphorylation of an intracellular tyrosine residue in the conserved FIGQY domain (Tyr 1229 in human neurofascin) resulted in the abolishment of ankyrin recruiting activity (8). Mutation of this tyrosine residue to glutamic acid mimicked phosphorylation by introducing negative charges and greatly impaired both cell adhesion and ankyrin recruitment (22). We performed a similar experiment using the ␤1Y181E mutant construct. This substitution completely abolished ankyrin recruitment in S2 cells (0% recruitment, Table I) (Fig. 3, G and H). In contrast to studies using neurofascin or neuroglian, the Y181E mutation had little effect on ␤1 subunit-induced cell adhesion fol-lowing 80 min of induction compared with wild type ␤1 (Fig. 2). Under bright field illumination, cells in Fig. 3, panels G and H, were tightly aggregated (data not shown), yet ankyrin staining was absent from cell-cell contact sites and appeared to be completely cytoplasmic. We conclude that the major ankyrin recruiting activity of ␤1 is contained in residues Ile 166 -Tyr 181 in the intracellular domain and that Tyr 181 is critical for ␤1ankyrin interactions. These results are summarized in Table I.
Sodium Channel ␤1 Subunits Are Tyrosine-phosphorylated-The results shown in Fig. 3 suggest that tyrosine phosphorylation may be a key regulatory event in ␤1-mediated ankyrin recruitment in Drosophila S2 cells. Substitution of ␤1Y181 with glutamic acid to mimic phosphorylation by introducing negative charges resulted in the abolishment of ankyrin recruitment. Are ␤1 subunits phosphorylated in mammalian cells? Fig. 4 shows that ␤1 subunits expressed in 1610␤1 fibroblasts are recognized by anti-PY20 antibodies following treatment of the cells with FGF in the presence of orthovanadate. Prior to Western blot analysis, ␤1 subunits were immunoprecipitated with anti-␤1 IN antibody. Both panels represent the identical blot probed with 2 different antibodies following immunopreciptation with anti-␤1 antibodies. In Fig. 4, left panel, the blot was probed with anti-␤1 antibody to show approximately equal expression of ␤1 subunits in 1610␤1 cells with and without FGF treatment. The blot was then stripped and reprobed with anti-PY20 (Fig. 4, right panel). Arrows indicate the position of the immunoreactive ␤1 band in both panels. It is clear from this experiment that ␤1 subunits are directly phosphorylated on tyrosine residues following FGF stimulation. Longer exposures of co-immunoprecipitation experiments in the absence of FGF treatment revealed a low level of basal phosphorylation of ␤1 subunits in the presence of orthovanadate alone (data not shown). We were also able to detect low levels of basal phosphorylation of ␤1 subunits in rat brain membranes in the presence of orthovanadate (data not shown). Two different anti-phosphotyrosine antibodies, anti-PY20 and anti-PY100, recognized ␤1 subunits equally well. However, only the experiments with anti-PY20 are shown. Tyr 181 mutant ␤1 subunits expressed in FGF-treated 1610 cells are not recognized by anti-phosphotyrosine antibodies, indicating that Tyr 181 is the only intracellular tyrosine residue that is phos-  FIG. 4. ␤1 subunits contain phosphotyrosine residues. Confluent 100-mm dishes of 1610␤1 cells were stimulated with 50 ng/ml FGF (ϩ) or vehicle (Ϫ) for 30 min at 37°C, solubilized in 1.25% Triton X-100 containing 1 mM sodium orthovanadate and the soluble fraction was incubated overnight at 4°C with 5 g of anti-␤1 IN antibody, also containing 1 mM sodium orthovanadate. Protein A-Sepharose was added and the incubation was continued for 2 h at 4°C. The Protein A-Sepharose beads were precipitated in a microcentrifuge and washed with 50 mM Tris-HCl, pH 7.5, 0.1% Triton X-100. Immunoprecipitates were eluted from the Protein A-Sepharose with SDS-PAGE sample buffer and separated on 10% acrylamide SDS-PAGE gels. Proteins were then transferred to nitrocellulose and probed with anti-␤1 IN antibody (1:500 dilution, left panel) followed by horseradish peroxidase-conjugated goat anti-rabbit IgG (1:100,000 dilution). Right panel, the blot used in the left panel was stripped and reprobed with anti-PY20 antibody (1:2000 dilution) followed by horseradish peroxidase-conjugated goat anti-rabbit IgG (1:100,000 dilution). Chemiluminescent detection of immunoreactive bands was accomplished with WestDura reagent. PY20, anti-PY20 antibody; ␤1, anti-␤1 IN antibody. Molecular mass markers are indicated in kDa. Arrows indicate the positions of ␤1 immunoreactive bands. phorylated, even though two additional tyrosine residues (Tyr 161 and Tyr 163 , Fig. 1A) are present near the intracellular end of the transmembrane segment (Fig. 5). We have shown previously, using biochemical as well as functional assays, that 1610 cells do not contain endogenous sodium channel ␣ or ␤ subunits (21) and reconfirmed this for the present study by reverse transcriptase-PCR (data not shown). Thus, the immune complex recognized by anti-phosphotyrosine in our experiments does not include sodium channel ␣ subunits, strengthening the argument that it is the ␤1 subunit that is phosphorylated.
Co-immunoprecipitation of ␤1 Subunits and Ankyrin G -Our experiments in S2 cells strongly suggest that sodium channel ␤1 subunits interact directly with Drosophila ankyrin. How does this relate to mammalian physiology? Do sodium channel ␤1 subunits interact with ankyrin in mammalian cells? To answer these questions, we first performed co-immunoprecipitation experiments to demonstrate the association of ␤1 and ankyrin G in solubilized rat brain membranes (Fig. 6A). This result, whereas positive, could have been indirect because of direct interaction of ankyrin G with sodium channel ␣ subunits, as has been suggested previously (23,24). To investigate this question in greater detail, we transfected Chinese hamster lung 1610␤1 fibroblasts with a GFP-tagged ankyrin G cDNA construct and then tested for ␤1 subunit-ankyrin G interactions. Immunoprecipitation of solubilized 1610␤1 cells with anti-␤1 antibody followed by Western blot analysis with anti-GFP showed that ␤1 and ankyrin G interact in these cells in the absence of ␣ subunits (Fig. 6B). Immunocytochemistry experiments demonstrate that endogenous ankyrin B is localized diffusely throughout the cytoplasm in untransfected 1610 cells and becomes concentrated at the plasma membrane at sites of cell-cell contact in cells stably transfected with ␤1 (Fig. 7).
␤1Y181A and ␤1Y181F also associate with GFP-ankyrin G in 1610 cells (Fig. 6C). In this co-immunoprecipitation experiment, ␤1Y181A appears to associate less efficiently with ankyrin compared with wild type, although the difference is clearly not as large as that seen in the ankyrin recruitment assays (Fig. 3, Table I). We attribute this apparent discrepancy to the non-quantitative nature of chemiluminescent detection of Western blots as well as the possibility of signal saturation in this assay. As predicted from the ankyrin recruitment experiments, we were not able to co-immunoprecipitate ␤1Y181E subunits and GFP-ankyrin G from co-transfected 1610 cells (Fig. 6C). Western blot analysis of cell lysates showed that ␤1Y181E was efficiently expressed in this cell line (Fig. 6C). Finally, treatment of 1610␤1 cells with FGF abolished ␤1ankyrin B interactions (Fig. 6D). Thus, ␤1 subunits appear to bind ankyrin G and ankyrin B directly. The sodium channel signaling complex at nodes of Ranvier may include both ␣-ankyrin and ␤1-ankyrin linkages. The present data suggest that ␤1ankyrin interactions are negatively regulated by phosphorylation of ␤1 subunits at residue Tyr 181 . We propose that this regulatory process plays a critical role in sodium channel localization and clustering at mammalian nodes of Ranvier. DISCUSSION We demonstrated previously that sodium channel ␤1 and ␤2 subunits are members of the Ig superfamily and mediate homophilic cell adhesion leading to recruitment of ankyrin to points of cell-cell contact in transfected Drosophila S2 cells (2). Deletion of the intracellular carboxyl-terminal domain of ␤1 or ␤2 abolished ankyrin binding but retained homophilic cell adhesion, indicating that the intracellular domains of ␤ subunits are critical for signal transduction events leading to cytoskeletal communication (2). In the present study we focused on the mechanism of ␤1 subunit interactions with ankyrin. ␤1 subunits contain a single intracellular tyrosine residue downstream of Lys 165 , identified as Tyr 181 (25). We hypothesized that this tyrosine may play a role in signal transduction leading to cytoskeletal communication following cell adhesion. To test our hypothesis, we generated mutant ␤1 subunit constructs, focusing on Tyr 181 . We found that ␤1 subunit-mediated cellular aggregation was retained in all of the mutant constructs. Although subtle differences in the relative rates of aggregation were observed, the extent of aggregation following 80 min of induction was unchanged. Ankyrin recruitment was retained in ␤1L182 STOP , indicating that the sequence Ile 166 -Tyr 181 contains the major ankyrin recruiting activity of ␤1. Ankyrin recruitment was retained in ␤1Y181F, impaired in ␤1Y181A, and abolished in ␤1Y181E, denoting the importance of an aromatic residue at this position and suggesting the FIG. 5. Tyr 181 mutant ␤1 subunits are not recognized by antiphosphotyrosine antibodies. A, confluent 100-mm dishes of 1610 cells transfected with ␤1, ␤1Y181A, or ␤1Y181F were stimulated with 50 ng/ml FGF for 30 min at 37°C, solubilized in 1.25% Triton X-100 containing 1 mM sodium orthovanadate and the soluble fraction was incubated overnight at 4°C with 5 g of anti-PY20 antibody, also containing 1 mM sodium orthovanadate. Protein A-Sepharose was added and the incubation was continued for 2 h at 4°C. The Protein A-Sepharose beads were precipitated in a microcentrifuge and washed with 50 mM Tris-HCl, pH 7.5, 0.1% Triton X-100. Immunoprecipitates were eluted from the Protein A-Sepharose with SDS-PAGE sample buffer and separated on 10% acrylamide SDS-PAGE gels. Proteins were then transferred to nitrocellulose and probed with anti-␤1 IN antibody (1:500 dilution) followed by horseradish peroxidase-conjugated goat anti-rabbit IgG (1:100,000 dilution). B, confluent 100-mm dishes of 1610 cells transfected with ␤1, ␤1Y181A, or ␤1Y181F were stimulated with 50 ng/ml FGF for 30 min at 37°C, solubilized in SDS-PAGE sample buffer, separated on 10% acrylamide SDS-PAGE gels, transferred to nitrocellulose, and probed with anti-␤1 EX antibody followed by horseradish peroxidase-conjugated goat anti-rabbit IgG (1:100,000 dilution). Brain, 50 g of rat brain membrane served as a positive control for ␤1 expression. Chemiluminescent detection of immunoreactive bands was accomplished with WestDura reagent. Molecular mass markers are indicated in kDa. Arrows indicate the positions of ␤1 immunoreactive or IgG bands. possibility that tyrosine phosphorylation, mimicked by introduction of a negative charge in ␤1Y181E, may negatively regulate ankyrin-␤1 interactions. ␤1 subunits transfected into 1610 fibroblasts recruit endogenously expressed ankyrin B to the plasma membrane. ␤1 subunits in these cells are efficiently recognized by anti-phosphotyrosine antibodies following FGF stimulation, with a low level of phosphorylation detected under basal conditions, suggesting that ␤1 subunit phosphorylation FIG. 6. Co-immunoprecipitation of ␤1 subunits and ankyrin G . A, ␤1 and ankyrin G are associated in rat brain membranes. Rat brain membranes were solubilized in 1.25% Triton X-100, and the soluble fraction was incubated overnight at 4°C with 2.5 g of anti-ankyrin G antibody. Protein A-Sepharose was added and the incubation was continued for 2 h at 4°C. The Protein A-Sepharose beads were precipitated in a microcentrifuge and washed with 50 mM Tris-HCl, pH 7.5, 0.1% Triton X-100. Immunoprecipitates were eluted from the Protein A-Sepharose with SDS-PAGE sample buffer and separated on 10% acrylamide SDS-PAGE gels. Proteins were then transferred to nitrocellulose and probed with anti-␤1 EX antibody (1:500 dilution) followed by horseradish peroxidase-conjugated goat anti-rabbit IgG (1:100,000 dilution). Chemiluminescent detection of immunoreactive bands was accomplished with WestDura reagent. IgG, nonimmune serum; Ank, antiankyrin G antibody; ␤1, anti-␤1 EX antibody. The arrow indicates the position of the ␤1 immunoreactive band. Molecular mass markers are indicated in kDa. B, ␤1 and ankyrin G associate in the absence of sodium channel ␣ subunits. 1610␤1 cells were transfected with GFP-ankyrin G , solubilized in 1.25% Triton X-100, and the soluble fraction was incubated overnight at 4°C with 5 g of anti-␤1 EX antibody. Protein A-Sepharose was added and the incubation was continued for 2 h at 4°C. The Protein A-Sepharose beads were precipitated in a microcentrifuge and washed with 50 mM Tris-HCl, pH 7.5, 0.1% Triton X-100. Immunoprecipitates were eluted from the Protein A-Sepharose beads with SDS-PAGE sample buffer and separated on 5% acrylamide SDS-PAGE gels. Proteins were then transferred to nitrocellulose and probed with anti-GFP antibody (1:500 dilution) followed by horseradish peroxidase-conjugated goat anti-rabbit IgG (1:100,000 dilution). Chemiluminescent detection of immunoreactive bands was accomplished with WestDura reagent. IgG, nonimmune serum; GFP, anti-GFP antibody; ␤1, anti-␤1 EX antibody. The arrow indicates the position of the GFPankyrin G immunoreactive band. Molecular mass markers are indicated in kDa. C, ␤1Y181A and ␤1Y181F associate with ankyrin G , but ␤1Y181E does not. Left panel, 1610 cells were co-transfected with ␤1Y181A, ␤1Y181E, or ␤1Y181F and GFP-ankyrin G , solubilized in 1.25% Triton X-100 and the soluble fraction was incubated overnight at 4°C with 5 g of anti-␤1 EX antibody. Protein A-Sepharose was added and the incubation was continued for 2 h at 4°C. The Protein A-Sepharose beads were precipitated in a microcentrifuge and washed with 50 mM Tris-HCl, pH 7.5, 0.1% Triton X-100. Immunoprecipitates were eluted from the Protein A-Sepharose with SDS-PAGE sample buffer and separated on 5% acrylamide SDS-PAGE gels. Proteins were then transferred to nitrocellulose and probed with anti-GFP antibody (1:500 dilution) followed by horseradish peroxidase-conjugated goat anti-rabbit IgG (1:100,000 dilution). Right panel, Western blot analysis of ␤1Y181E-transfected cells to demonstrate that the ␤1 subunit mutant was efficiently expressed. Cells were solubilized in SDS-PAGE sample buffer and separated on 10% acrylamide SDS-PAGE gels. Proteins were then transferred to nitrocellulose and probed with anti-␤1 EX antibody (1:500 dilution) followed by horseradish peroxidase-conjugated goat anti-rabbit IgG (1:100,000 dilution). Chemiluminescent detection of immunoreactive bands in both panels was accomplished with WestDura reagent. IgG, nonimmune serum; GFP, anti-GFP antibody; ␤1, anti-␤1 EX antibody. The arrow indicates the position of the ␤1 immunoreactive band. Molecular mass markers are indicated in kDa. D, FGF treatment abolishes ␤1-ankyrin interactions. 1610␤1 cells were stimulated with FGF (ϩ) or vehicle (Ϫ) as described in the legend to Fig.  4, solubilized in 1.25% Triton X-100 and the soluble fraction was incubated overnight at 4°C with 5 g of anti-␤1 EX antibody. Protein A-Sepharose was added and the incubation was continued for 2 h at 4°C. The Protein A-Sepharose beads were precipitated in a microcentrifuge and washed with 50 mM Tris-HCl, pH 7.5, 0.1% Triton X-100. Immunoprecipitates were eluted from the Protein A-Sepharose with SDS-PAGE sample buffer and separated on 5% acrylamide SDS-PAGE gels. Proteins were then transferred to nitrocellulose and probed with antiankyrin B antibody (1:500 dilution) followed by horseradish peroxidaseconjugated goat anti-rabbit IgG (1:100,000 dilution). Chemiluminescent detection of immunoreactive bands was accomplished with WestDura reagent. IgG, nonimmune serum. The arrow indicates the position of the ankyrin B immunoreactive band. Molecular mass markers are indicated in kDa. may be a regulated event. ␤1 and ankyrin G , the ankyrin isoform present at axon initial segments and nodes of Ranvier, associate in transfected 1610 cells in the absence of ␣ subunits, suggesting that these two proteins may interact in mammalian neurons in vivo. In contrast, ␤1Y181E and ankyrin G do not associate, and FGF treatment of cells results in inhibition of wild type ␤1 interactions with ankyrin, supporting our conclusion that tyrosine phosphorylation inhibits ␤1-mediated ankyrin recruitment.
Previous studies identified a highly conserved FIGQY sequence in the cytoplasmic domain of neurofascin as the principal site of regulation of ankyrin binding (7,8). Phosphorylation of this tyrosine residue abolished cell adhesion and ankyrin recruitment in cells expressing this CAM (7,8). Structural requirements for the association of neurofascin with ankyrin were found to be very similar to the present results (22). Briefly, substitution of the critical tyrosine residue (Tyr 1229 ) in neurofascin with phenylalanine retained ankyrin binding, whereas substitution with alanine or glutamic acid greatly impaired this interaction. Interestingly, manipulation of the cytoplasmic domain of neurofascin had dramatic effects on the rate and extent of cell adhesion, suggesting an additional inside-out signaling mechanism. In contrast, the cytoplasmic domain of ␤1 appears to have little influence on homophilic cell adhesion, indicating that whereas ␤1-mediated extracellular adhesion events influence cytoskeletal communications, cytoskeletal interactions have little effect on ␤1-mediated cell adhesion.
Neuronal sodium channel ␣ and ␤1 subunits interact with receptor protein-tyrosine phosphatase ␤ during neonatal development (26). A consequence of formation of this signaling complex is to reduce tyrosine phosphorylation of ␣ subunits. Coexpression of receptor protein-tyrosine phosphatase ␤ and Nav1.2 ␣ subunits in tsA-201 cells resulted in a depolarizing shift in the voltage dependence of sodium channel inactivation and an increase in whole cell current. The present data introduce the possibility that receptor protein-tyrosine phosphatase ␤ may also function to regulate tyrosine phosphorylation of sodium channel ␤1 subunits resulting in modulation of ankyrin recruitment to the plasma membrane. These events would be predicted to influence sodium channel localization and density via CAM-cytoskeletal interactions during formation of the node of Ranvier or during axonal fasiculation. In glia, where receptor protein-tyrosine phosphatase ␤ and sodium channel ␤1 subunits are also expressed (27)(28)(29)(30)(31), modulation of ␤1 subunit tyrosine phosphorylation may be critical to axoglial communication during myelination and nodal formation.
Mutations in voltage-gated sodium channel ␣ subunit genes have been shown to be linked to inherited human diseases causing paroxysmal events such as long QT syndrome in the heart and hyperkalemic periodic paralysis in skeletal muscle (32). Until recently there were no identified neuronal sodium channel mutations associated with human disease. Mutations in the human SCN1A ␣ subunit gene have been linked to two families with idiopathic generalized epilepsy with febrile seizures plus type 2 (GEFSϩ2) (33)(34)(35). A mutation in SCN1B has been shown to be linked to a large family with GEFSϩ1 (36). This mutation, C121W (C102W using the original numbering presented in Isom et al. (25)), changes a critical cysteine residue defining the carboxyl-terminal region of the Ig loop in ␤1 to a tryptophan, most likely destabilizing this critical cell adhesion and ␣ subunit association domain. Coexpression of the GEFSϩ1 mutant ␤1 subunit with rat brain Nav1.2 sodium channels in Xenopus oocytes revealed a lack of the classic ␤1-mediated acceleration of the inactivation rate (36). It is not yet known whether GEFSϩ1 mutant ␤1 subunits are ex-pressed at the plasma membrane or, if so, whether they are associated with ␣ subunits. It will be important in future studies to examine the coexpression of these mutant ␤1 subunits with ␣ subunits in transfected mammalian cells and transgenic animals as well as their cell adhesive properties in S2 cells. Nevertheless, this important study showed for the first time that mutations in sodium channel ␤ subunits result in pathophysiology of the human nervous system and predicted that cell adhesion may play a role in the epileptic phenotype.
Sodium channel ␤ subunits play important roles in channel modulation and regulation of channel density at the plasma membrane. We have shown that ␤ subunits also function as cell adhesion molecules in heterologous expression systems. What are the potential implications of these findings? It has been shown previously that ankyrin G , the CAMs neurofascin and NrCAM, and voltage-gated sodium channels co-localize at the axonal membrane of the adult node of Ranvier in specialized membrane domains (37)(38)(39). Early clusters of neurofascin and NrCAM are joined later by ankyrin G and sodium channels during differentiation of myelinated axons. Formation of the node of Ranvier may then result from the fusion of two cluster intermediates. An ankyrin G -mediated link between neurofascin, NrCAM, and ion channels may allow these CAMs to cluster sodium channels in the axonal membrane. It was proposed that sodium channel ␤2 subunits, because of their homology to F3/contactin, may interact in a lateral or cis fashion with NrCAM and thus contribute to sodium channel localization (38). Recent studies have shown that ␤1 also interacts with neurofascin-186 directly (4). Our present data propose a direct link between sodium channel ␤1 subunits and ankyrin G that is modulated by tyrosine phosphorylation of ␤1 subunits. The multivalent membrane-binding domain of ankyrin G (40) may allow interaction with multiple CAMs, including neurofascin, NrCAM, as well as sodium channel ␤1 subunits, forming a dynamic sodium channel signaling complex at the node of Ranvier that may also include the cell adhesion molecule contactin, as well as receptor protein-tyrosine phosphatase ␤ and tyrosine kinases. As has been proposed for neurofascin (7), tyrosine phosphorylation of ␤1 may be receptor-mediated and reversible, resulting in strong versus weak cytoskeletal connections depending on cellular signaling. ␤1 subunit mRNA expression has been described in sciatic nerve Schwann cells, astrocytes from spinal cord, optic nerve, and sciatic nerve, oligodendrocytes, and B104 oligodendrocyte precursor cells in culture (27)(28)(29)31). trans-Homophilic cell adhesion may occur between axonal and glial cell sodium channel ␤ subunits in addition to the proposed cis interactions. This putative adhesion may also contribute to sodium channel clustering at nodes of Ranvier during the process of myelination. Our challenge now is to relate these exciting observations in heterologous expression systems to physiological events.