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Address correspondence to William A. Catterall, Department of Pharmacology R-189, University of Washington, HSC, Box 357280, F-427, Seattle, WA 98195-7280. Tel.: (206) 543-1925. Fax: (206) 543-3882. E-mail: wcatt{at}u.washington.edu
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Abstract |
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Key Words: sodium channel; neurofascin; neural cell adhesion molecules; nodes of Ranvier; protein binding
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Introduction |
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Neurofascin belongs to the L1 family of neuronal CAMs containing extracellular Ig- and fibronectin (FN) type IIIlike domains, as well as ankyrin binding activity in their cytoplasmic domains (see Fig. 1
A). FIGQY, a highly conserved sequence in L1 family intracellular domains, is required for ankyrin binding, and phosphorylation of the FIGQY tyrosine residue abolishes binding (Garver et al., 1997). Neurofascin and another L1 family member, NrCAM, become clustered along rat sciatic nerve axons early during postnatal development, thus defining the sites for assembly of nodes of Ranvier (Davis et al., 1996). AnkyrinG and sodium channels are subsequently recruited to these sites as nodes mature (Lambert et al., 1997). Sodium channel and ß subunits also assemble during this developmental period (Wollner et al., 1988). There are several splice variants of neurofascin, including a 155- and a 186-kD isoform (Davis et al., 1996). The 155-kD isoform contains six extracellular Ig domains followed by four FN domains, whereas the third FN domain is absent from the 186-kD isoform, and a mucin-like domain is inserted after FN domain 4. Only the 186-kD isoform, neurofascin 186, appears to be localized to nodes of Ranvier, whereas the 155-kD isoform is expressed only in unmyelinated fibers (Davis et al., 1996). Neurofascin 186 is also localized to Purkinje cell axon initial segments, whereas the 155-kD isoform is expressed in cell bodies and dendrites (Davis et al., 1996).
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Results and discussion |
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ß1 and ß3 mRNAs are both found in central and peripheral neurons, with overlapping as well as distinct patterns of expression (Curtis, R.A., D. Lawson, P. Ge, P.S. DiStefano, and I. Solis-Santiago. 2000. Cloning and localization of a novel Na+/- channel ß3 subunit. Society of Neuroscience Annual Meeting. 418.22 [Abstr.]; Morgan et al., 2000; unpublished data), suggesting that ß1 and ß3 play similar roles. This concept is supported by the ability of both subunits to associate with neurofascin, whereas ß2, which shares less homology with ß1 and ß3, does not interact with neurofascin. ß2 subunits share homology with contactin/F3/F11 (Isom et al., 1995), a glycophosphatidylinositol (GPI)-anchored CAM that interacts with NrCAM (Morales et al., 1993; Sakurai et al., 1997). It will be interesting to investigate whether ß2 subunits are able to interact with NrCAM at nodes of Ranvier, thus providing a further mechanism by which sodium channels are targeted to these specialized regions.
Neurofascin and ß1 localize to nodes of Ranvier and associate in rat brain
Sodium channel subunits have been shown to localize to nodes of Ranvier (Ellisman and Levinson, 1982; Vabnick et al., 1996; Rasband et al., 1999), but concentration of ß1 subunits at nodes has not been confirmed. The localization of both neurofascin and ß1 subunits in sciatic nerve was determined by probing tissue from adult rats with polyclonal antiFN domain antibody and anti-ß1CT, respectively. Neurofascin has previously been localized to nodes of Ranvier in adult rats (Davis et al., 1996), and we confirmed these data for the sciatic nerve (Fig. 2
A) while demonstrating that ß1 subunits also localize to highly concentrated sites that appear identical to those stained by anti-FN antibodies (Fig. 2 B). This result shows that both ß1 subunits and neurofascin are highly concentrated at nodes of Ranvier.
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Neurofascin and NrCAM cluster at nodes of Ranvier in rat sciatic nerve between postnatal days 2 and 5, followed by the recruitment of ankyrinG and sodium channels to these sites (Lambert et al., 1997). To examine whether ß1 subunits form a complex with neurofascin from early postnatal development through to adulthood, both P5 and adult rat brain lysates were immunoprecipitated with polyclonal ß1 antibody, anti-ß1CT, or control rabbit IgG. After SDS-PAGE of immunoprecipitated proteins and immunoblotting, polyclonal antimucin domain antibody detected the 186-kD neurofascin isoform coimmunoprecipitating with ß1 subunits in both P5 and adult brain lysates (Fig. 2 C). The antimucin domain antibody was raised against the mucin domain present only in the neurofascin isoform detected at nodes. P5 and adult rat brain lysates were probed with anti-ß1CT to confirm that ß1 is expressed during early postnatal development (Fig. 2 D).
These results show that ß1 subunits interact with neurofascin in developing rat brain, and we propose that this association is involved in targeting sodium channels to specialized regions of the neuron such as nodes of Ranvier and axon initial segments. Previous reports suggest that mature nodes of Ranvier contain sodium channels, neurofascin, and NrCAM, bound in a complex by ankyrinG (Davis et al., 1996; Volkmer et al., 1996; Zhou et al., 1998) that is able to interact with several transmembrane molecules. AnkyrinG is likely to be present in our immunoprecipitates as well, but the experiments presented below show that sodium channels and neurofascin interact through their extracellular domains, so it is unlikely that ankyrinG is required for formation of the complex. Our data show that sodium channel ß1 subunits can interact directly with neurofascin in transfected cells and are associated with neurofascin in postnatal and adult rat brain, indicating that this interaction may be involved in both forming the nascent node of Ranvier and stabilizing the mature node.
Neurofascin and ß1 interact in cis in transfected tsA-201 cells
Both neurofascin and NrCAM form trans-interactions with molecules on adjacent cells (Volkmer et al., 1996). To investigate whether ß1 interacts with neurofascin in cis or in trans, tsA-201 cells were transfected together or separately with neurofascin 186 and ß1. 20 h after transfection cells were removed from culture dishes by treatment with EDTA, and the separately transfected ß1-expressing cells were mixed thoroughly with neurofascin-expressing cells and cultured for a further 24 h to 80% confluency. Cells were lysed and their proteins were immunoprecipitated with anti-ß1CT. SDS-PAGE and immunoblotting with monoclonal antibody anti-HA.11 detected interaction of ß1 and neurofascin in cis (Fig. 3
A), but were unable to detect any neurofascin 186 coimmunoprecipitating with ß1 subunits in trans following separate transfection (Fig. 3 B). Immunoblotting of cell lysates showed that both ß1 and neurofascin 186 were well expressed (Fig. 3 B). As a control, transfected cells were fixed and stained with anti-ß1CT (Fig. 3 C) or anti-HA.11 antibody to tagged neurofascin (Fig. 3 D). Both proteins localized to the plasma membrane, which would allow trans-heterophilic interactions to occur between them. Thus, our results in tsA-201 cells demonstrate that interaction of ß1 and neurofascin only occurs in cis within the same cell membrane in this experimental system. This type of interaction would be important in the formation of sodium channel clusters and targeting to nodes of Ranvier in the axonal membrane. However, it is possible that weaker trans-interactions occur between ß1 and neurofascin expressed on opposing cells such as the axon and perinodal astrocyte or Schwann cell microvilli. Such trans-interactions may be easily disrupted during cell lysis, and therefore are not detected in our immunoprecipitation experiments.
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Determination of the neurofascin binding site
The extracellular region of neurofascin 186 consists of multiple domains. To investigate which extracellular domains of neurofascin were able to interact with ß1, the Ig domains Ig16, the FN domains FN1, 2, and 4, and the mucin-like domain were expressed separately, fused at the NH2 terminus to the HA.11 tag, and at the COOH terminus to the GPI anchor sequence from human placental alkaline phosphatase. These constructs were coexpressed with sodium channel and ß1 subunits in tsA-201 cells. After immunoprecipitation with anti-SP20, only Ig1GPI and FN2GPI were able to associate with
/ß1 complexes in tsA-201 cells, suggesting that the neurofascin binding site is assembled from amino acids in both of these domains (Fig. 5
A). It was necessary to express ß1 complexed with
subunits in these experiments, as the GPI constructs were expressed at high levels and all of them bound nonspecifically to ß1 in the absence of
subunits. When
subunits were expressed alone with Ig1GPI or FN2GPI no interaction was observed, confirming that this association is ß1 dependent (Fig. 5 C). Both the 155- and 186-kD isoforms of neurofascin contain Ig1 and FN2, so ß1 should interact with both isoforms. However, ß1 is localized to nodes of Ranvier in sciatic nerve and is most likely to interact with neurofascin 186, which also concentrates at nodes. The interaction of Ig1 with ß1 in cis suggests that the neurofascin molecule folds back on itself to make this domain accessible to ß1, as drawn in Fig. 3 A; thus, binding of amino acids on Ig1 and FN2 with ß1 could then occur. It is also possible that the FN2 domain of neurofascin could interact with ß1 subunits in a cis configuration, whereas Ig1 interacts with ß1 subunits at lower affinity in trans on an opposing membrane. Development of new methods to measure lower-affinity trans-interactions of ß1 will be required to test this idea. Similar interactions are made by axonin 1/TAG-1like glycoproteins, a family of neural CAMs containing six Ig domains and four FN domains. Homophilic trans-interactions occur between Ig domains 2 and 3 of axonin 1 (Freigang et al., 2000), whereas the FN domains of TAX-1, the human homologue of axonin 1, are thought to form cis-homophilic interactions (Tsiotra et al., 1996).
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Neurofascin and NrCAM are only able to associate with ankyrinG when the conserved tyrosine residue within the FIGQY sequence is dephosphorylated (Garver et al., 1997). We reported recently that sodium channels associate with receptor protein tyrosine phosphatase ß (RPTPß) in developing rat brain (Ratcliffe et al., 2000). This interaction is mediated through the extracellular domain of the subunit, and the intracellular domains of both the
and ß1 subunit; it is observed in neonatal tissue, but is absent at postnatal day 16. RPTPß is expressed predominantly in glia, but RPTPß mRNAs have also been detected in neurons (Snyder et al., 1996). The close localization of neurofascin and NrCAM to RPTPß suggests that they could be substrates, with dephosphorylation occurring early in postnatal development, thus allowing interactions with ankyrinG to occur. As expression of RPTPß is restricted to the brain (Levy et al., 1993), a related tyrosine phosphatase may perform a similar function in the peripheral nervous system.
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Materials and methods |
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Sodium channel subunit mammalian expression plasmids pCDM8 (encoding Nav1.2a), pCDM8ß1, pCDM8ß2, and chimera pCDM8ß1ß2ß2 have been described previously (Auld et al., 1990; Ratcliffe et al., 2000). ß3 cDNA was amplified and subcloned into pcDNA3.1myc-his (Invitrogen) for expression of tagged ß3. ß1ecGPI was subcloned from pSP64T (McCormick et al., 1999) into pCDM8. cDNA encoding neurofascin 186 tagged at the NH2 terminus with HA.11 was provided by Dr. Vann Bennett. This cDNA was subcloned into the EcoRI site of pcDNA3.1 (Invitrogen). pcDNA3.1NF186
ic was constructed by introducing a stop codon after residue E1129. Neurofascin 186 GPI-tagged constructs were made as follows. A Cla1 restriction site was introduced into pcDNA3.1NF186 through a silent change of the codon for S8 from AGC to TCG. The ClaI-EcoRV fragment of pcDNA3.1NF186, which includes all but the first eight amino acids of neurofascin 186 and the HA.11 tag, was removed and replaced with a PCR product encompassing the mucin domain, amino acids P897A1084. The GPI anchor recognition sequence from human placental alkaline phosphatase (McCormick et al., 1999) was then cloned in frame into the EcoRV-XhoI sites of this construct to produce pcDNA3.1mucin-GPI. For construction of remaining GPI-tagged domain expression vectors, the ClaI-EcoRV fragment of pcDNA3.1mucin-GPI was replaced with PCR products coding for Ig1 (P16-Q11), Ig2 (V112-T212), Ig3 (R213-P313), Ig4 (Y314-P406), Ig5 (R407-T498), Ig6 (R499-L586), FN1 (A587-P703), FN2 (E704-L801), and FN4 (P802-A905).
Coimmunoprecipitation experiments
Membrane fractions were prepared from P5 and adult rat brains as described previously (Nishiwaki et al., 1998). For immunoprecipitation reactions, 500 µg of total protein was added to 20 µg of anti-ß1CT or control IgG antibody, and incubated for 2 h at 4°C. Protein Aagarose was added, and the incubation was continued overnight. The complex bound to the agarose beads, was washed extensively, and bound proteins were heat eluted in SDS loading buffer at 100°C for 10 min. Proteins were separated by SDS-PAGE and transferred onto nitrocellulose, and neurofascin 186 was detected using polyclonal antimucin domain antibody. For coimmunoprecipitation assays performed using tsA-201 cell lysates, 50 µg of an equimolar ratio of expression plasmids was transfected into cells in DMEM F12 supplemented with 10% FBS, 100 U/ml penicillin, and streptomycin plated on 150-mm dishes using the calcium phosphate method. At 40 h after transfection, cell monolayers were washed in PBS, lysed in 1% Nonidet P-40, 0.25% sodium deoxycholate, 15 mM NaCl, 1 mM EGTA, 50 mM Tris-Cl, pH 7.4, 1 mM PMSF, 10 µg/ml leupeptin, and 10 µg/ml aprotinin, and incubated at 4°C for 30 min. Lysates were centrifuged and the supernatant was used in immunoprecipitation experiments essentially as described above. HA-tagged neurofascin 186 was detected with monoclonal antibody anti-HA.11 (Covance Research Products, Inc.).
Immunocytochemistry
Transfected tsA-201 cells were plated on poly-L-lysinecoated glass coverslips for 24 h and fixed in 4% paraformaldehyde. Coverslips were blocked in 5% avidin, followed by 5% biotin, and then 10% milk solution in TBS. Primary antibody was added at a 1:100 (anti-ß1extra) or 1:1,000 dilution (anti-HA.11) in TBS containing 10% milk and 0.025% Triton X-100, and coverslips were incubated overnight at 4°C. Coverslips were then incubated with a 1:300 dilution of biotin-conjugated secondary antibody for 1 h at 37°C, followed by a 1-h incubation with fluorescein-conjugated avidin (Vector Laboratories). Coverslips were washed, dried, and mounted on glass slides in Vectashield (Vector Laboratories), and visualized under a confocal laser scanning microscope (model MRC 600; Bio-Rad Laboratories).
For staining of intact sciatic nerve, adult rats were anesthetized with nembutal and intracardially perfused with a solution of 4% paraformaldehyde in PB (0.1 M sodium phosphate, pH 7.4). The sciatic nerves were removed, postfixed for 2 h, and sunk in successive solutions of 10 and 30% (wt/vol) sucrose in PB at 4°C over a period of 72 h. Sciatic nerves were embedded in OCT compound, and 20 µm sections were cut and thaw mounted onto Superfrost Plus slides (Fisher Scientific). Sciatic sections were fixed in 4% paraformaldehyde, rinsed, and then blocked using 5% normal goat serum and 5% nonfat milk in 0.1 M TBS for 1 h. The sections were then incubated in anti-ß1extra antibody (diluted 1:15) overnight at room temperature, rinsed in TBS for 30 min, incubated in biotinylated goat antirabbit IgG (diluted 1:300; Vector Laboratories), rinsed in TBS for 30 min, and finally incubated in avidin D-fluorescein (diluted 1:300; Vector Laboratories) for 1 h. All antibodies were diluted in TBS containing 5% milk, 5% normal goat serum, and 0.05% Triton X-100. The slides were then rinsed in TBS for 5 min, in TB for 20 min, and in distilled water for 2 min, coverslipped with Vectashield, sealed with nail polish, and viewed using an MRC 600 confocal microscope. Control sections were incubated in normal rabbit serum, or the primary antibody was omitted. In both instances, no specific staining was observed.
For staining of teased sciatic nerves, fresh sciatic nerve removed from 3- and 10-d-old rats was rinsed in PB, treated with collagenase (3.5 mg/ml of PB) for 15 min, rinsed in PB, teased apart on a slide coated with cell tak, rinsed with PB, fixed in 4% paraformaldehyde, rinsed, and then blocked using 10% normal goat serum in TBS for 1 h. The sections were then incubated in anti-ß1extra (diluted 1:15) overnight at room temperature, and processed for immunocytochemistry.
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Footnotes |
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Acknowledgments |
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This research was supported by the Wellcome Trust (C.F. Ratcliffe) and National Institutes of Health Research Grant NS25704 (W.A. Catterall).
Submitted: 15 February 2001
Revised: 7 June 2001
Accepted: 13 June 2001
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References |
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