Title: Heterophilic Interactions of Sodium Channel β1 Subunits with Axonal and Glial Cell Adhesion Molecules
Abstract: 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 α subunit and at least one β subunit. β1 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 β1 interactions with contactin and Nf186 in greater detail and investigated interactions of β1 with NrCAM, Nf155, and sodium channel β2 and β3 subunits. Using Fc fusion proteins and immunocytochemical techniques, we show that β1 interacts with the fibronectin-like domains of contactin. β1 also interacts with NrCAM, Nf155, sodium channel β2, and Nf186 but not with sodium channel β3. The interaction of the extracellular domains of β1 and β2 requires the region 169TEEEGKTDGEGNA181 located in the intracellular domain of β2. Interaction of β1 with Nf186 results in increased Nav1.2 cell surface density over α alone, similar to that shown previously for contactin and β2. We propose that β1 is the critical communication link between sodium channels, nodal cell adhesion molecules, and ankyrinG. 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 α subunit and at least one β subunit. β1 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 β1 interactions with contactin and Nf186 in greater detail and investigated interactions of β1 with NrCAM, Nf155, and sodium channel β2 and β3 subunits. Using Fc fusion proteins and immunocytochemical techniques, we show that β1 interacts with the fibronectin-like domains of contactin. β1 also interacts with NrCAM, Nf155, sodium channel β2, and Nf186 but not with sodium channel β3. The interaction of the extracellular domains of β1 and β2 requires the region 169TEEEGKTDGEGNA181 located in the intracellular domain of β2. Interaction of β1 with Nf186 results in increased Nav1.2 cell surface density over α alone, similar to that shown previously for contactin and β2. We propose that β1 is the critical communication link between sodium channels, nodal cell adhesion molecules, and ankyrinG. 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 (1Isom L.L. Front. Biosci. 2002; 7: 12-23Crossref PubMed Google Scholar, 2Custer A.W. Kazarinova-Noyes K. Sakurai T. Xu X. Simon W. 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The nodal gap and the juxtaparanode are separated by the paranode, a region containing septate-like junctions composed of cell adhesion molecules (CAMs) 1The 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. that act as diffusion barriers to ion channel movement (2Custer A.W. Kazarinova-Noyes K. Sakurai T. Xu X. Simon W. Grumet M. Shrager P. J. Neurosci. 2003; 23: 10032-10039Crossref PubMed Google Scholar, 6Poliak S. Peles E. Nat. Rev. Neurosci. 2003; 4: 968-980Crossref PubMed Scopus (502) Google Scholar, 7Boyle M.E. Berglund E.O. Murai K.K. Weber L. Peles E. Ranscht B. Neuron. 2001; 30: 385-397Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar, 8Bhat M.A. Rios J.C. Lu Y. Garcia-Fresco G.P. Ching W. St Martin M. Li J. Einheber S. Chesler M. Rosenbluth J. Salzer J.L. Bellen H.J. Neuron. 2001; 30: 369-383Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar). This specific arrangement of voltage-gated sodium and potassium channels results in rapid and efficient saltatory conduction of action potentials (9Chen C. Westenbroek R.E. Xu X. Edwards C.A. Sorenson D.R. Chen Y. McEwen D.P. O'Malley H.A. Bharucha V. Meadows L.S. Knudsen G.A. Vilaythong A. Noebels J.L. Saunders T.L. Scheuer T. Shrager P. Catterall W.A. Isom L.L. J. Neurosci. 2004; 24: 4030-4042Crossref PubMed Scopus (217) Google Scholar, 10Catterall W. Neuron. 2000; 26: 13-25Abstract Full Text Full Text PDF PubMed Scopus (1760) Google Scholar, 11Salzer J.L. Neuron. 2003; 40: 297-318Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 12Scherer S.S. Ann. N. Y. Acad. Sci. 1999; 883: 131-142Crossref PubMed Scopus (28) Google Scholar). Voltage-gated sodium channels are composed of a pore-forming α subunit, from the gene family containing Nav1.1 through Nav1.9 and at least one β subunit from the gene family containing β1, β1A, β2, β3, and β4 (10Catterall W. Neuron. 2000; 26: 13-25Abstract Full Text Full Text PDF PubMed Scopus (1760) Google Scholar, 13Yu F.H. Westenbroek R.E. Silos-Santiago I. McCormick K.A. Lawson D. Ge P. Ferriera H. Lilly J. DiStefano P.S. Catterall W.A. Scheuer T. Curtis R. J. Neurosci. 2003; 23: 7577-7585Crossref PubMed Google Scholar, 14Isom L.L. Neuroscientist. 2001; 7: 42-54Crossref PubMed Scopus (292) Google Scholar, 15Meadows L. Malhotra J.D. Stetzer A. Isom L.L. Ragsdale D.S. J. Neurochem. 2001; 76: 1871-1878Crossref PubMed Scopus (55) Google Scholar, 16Kazen-Gillespie K.A. Ragsdale D.S. D'Andrea M.R. Mattei L.N. Rogers K.E. Isom L.L. J. Biol. Chem. 2000; 275: 1079-1088Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). 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) (2Custer A.W. Kazarinova-Noyes K. Sakurai T. Xu X. Simon W. Grumet M. Shrager P. J. Neurosci. 2003; 23: 10032-10039Crossref PubMed Google Scholar, 17Kazarinova-Noyes K. Malhotra J.D. McEwen D.P. Mattei L.N. Berglund E.O. Ranscht B. Levinson S.R. Schachner M. Shrager P. Isom L.L. Xiao Z.C. J. Neurosci. 2001; 21: 7517-7525Crossref PubMed Google Scholar, 18Lustig M. Zanazzi G. Sakurai T. Blanco C. Levinson S.R. Lambert S. Grumet M. Salzer J.L. Curr. Biol. 2001; 11: 1864-1869Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Sodium channel β1 and β2 subunits are also members of the Ig superfamily of CAMs. β1 and β2 colocalize with sodium channel α subunits at nodes of Ranvier, and β1 interacts with contactin and with Nf186 in vitro (9Chen C. Westenbroek R.E. Xu X. Edwards C.A. Sorenson D.R. Chen Y. McEwen D.P. O'Malley H.A. Bharucha V. Meadows L.S. Knudsen G.A. Vilaythong A. Noebels J.L. Saunders T.L. Scheuer T. Shrager P. Catterall W.A. Isom L.L. J. Neurosci. 2004; 24: 4030-4042Crossref PubMed Scopus (217) Google Scholar, 17Kazarinova-Noyes K. Malhotra J.D. McEwen D.P. Mattei L.N. Berglund E.O. Ranscht B. Levinson S.R. Schachner M. Shrager P. Isom L.L. Xiao Z.C. J. Neurosci. 2001; 21: 7517-7525Crossref PubMed Google Scholar, 19Chen C. Bharucha V. Chen Y. Westenbroek R.E. Brown A. Malhotra J.D. Jones D. Avery C. Gillespie P.J. II I Kazen-Gillespie K.A. Kazarinova-Noyes K. Shrager P. Saunders T.L. Macdonald R.L. Ransom B.R. Scheuer T. Catterall W.A. Isom L.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 17072-17077Crossref PubMed Scopus (159) Google Scholar, 20Ratcliffe C.F. Westenbroek R.E. Curtis R. Catterall W.A. J. Cell Biol. 2001; 154: 427-434Crossref PubMed Scopus (155) Google Scholar). Nf186, NrCAM, β1, and β2 each interact in vitro with a key cytoskeletal anchoring protein, ankyrinG, that is also localized to nodes of Ranvier (21Davis J.Q. Bennett V. J. Biol. Chem. 1994; 269: 27163-27166Abstract Full Text PDF PubMed Google Scholar, 22Malhotra J.D. Kazen-Gillespie K. Hortsch M. Isom L.L. J. Biol. Chem. 2000; 275: 11383-11388Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 23Malhotra J.D. Koopmann M.C. Kazen-Gillespie K.A. Fettman N. Hortsch M. Isom L.L. J. Biol. Chem. 2002; 277: 26681-26688Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). β1- or β2-mediated homophilic cell-adhesive interactions result in ankyrin recruitment in Drosophila S2 cells, and the interaction of sodium channel α subunits with ankyrinG is greatly enhanced in the presence of β1 subunits in vitro (22Malhotra J.D. Kazen-Gillespie K. Hortsch M. Isom L.L. J. Biol. Chem. 2000; 275: 11383-11388Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 24McEwen D.P. Meadows L.S. Chen C. Thyagarajan V. Isom L.L. J. Biol. Chem. 2004; 279: 16044-16049Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). 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 β1 subunits. In sciatic nerve, Nf186 and NrCAM are early markers of nodal formation that cluster at presumptive nodes prior to sodium channels and ankyrinG (2Custer A.W. Kazarinova-Noyes K. Sakurai T. Xu X. Simon W. Grumet M. Shrager P. J. Neurosci. 2003; 23: 10032-10039Crossref PubMed Google Scholar, 25Lambert S. Davis J. Bennett V. J. Neurosci. 1997; 17: 7025-7036Crossref PubMed Google Scholar). In the peripheral nervous system (PNS), Schwann cell contact at the beginning stages of myelin ensheathement is required for sodium channel clustering (26Dugandzija-Novakovic S. Koszowski A.G. Levinson S.R. Shrager P. J. Neurosci. 1995; 15: 492-503Crossref PubMed Google Scholar, 27Vabnick I. Novakovic S.D. Levinson S.R. Schachner M. Shrager P. J. Neurosci. 1996; 16: 4914-4922Crossref PubMed Google Scholar). In optic nerve, developing nodes of Ranvier are defined by clustering of ankyinG prior to the arrival of CAMs and sodium channels (28Jenkins S.M. Bennett V. J. Cell Biol. 2001; 155: 739-746Crossref PubMed Scopus (363) Google Scholar, 29Jenkins S.M. Bennett V. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2303-2308Crossref PubMed Scopus (102) Google Scholar), and this process is independent of paranodal axoglial cell adhesion (29Jenkins S.M. Bennett V. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2303-2308Crossref PubMed Scopus (102) Google Scholar). In the central nervous system (CNS), conditioned medium from optic nerve glia induces clustering of sodium channels in retinal ganglion cell axons in vitro (30Kaplan M.R. Meyer-Franke A. Lambert S. Bennett V. Duncan I.D. Levinson S.R. Barres B.A. Nature. 1997; 386: 724-728Crossref PubMed Scopus (286) Google Scholar). In vivo, the presence of oligodendrocytes is necessary for sodium channel clustering and nodal formation in the CNS (30Kaplan M.R. Meyer-Franke A. Lambert S. Bennett V. Duncan I.D. Levinson S.R. Barres B.A. Nature. 1997; 386: 724-728Crossref PubMed Scopus (286) Google Scholar). 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 (31Peles E. Nativ M. Lustig M. Grumet M. Schilling J. Martinez R. Plowman G.D. Schlessinger J. EMBO J. 1997; 16: 978-988Crossref PubMed Scopus (361) Google Scholar, 32Rios J.C. Melendez-Vasquez C.V. Einheber S. Lustig M. Grumet M. Hemperly J. Peles E. Salzer J.L. J. Neurosci. 2000; 20: 8354-8364Crossref PubMed Google Scholar, 33Tait S. Gunn-Moore F. Collinson J.M. Huang J. Lubetzki C. Pedraza L. Sherman D.L. Colman D.R. Brophy P.J. J. Cell Biol. 2000; 150: 657-666Crossref PubMed Scopus (272) Google Scholar). In the CNS, contactin is expressed in the nodal gap, where it interacts with sodium channels (17Kazarinova-Noyes K. Malhotra J.D. McEwen D.P. Mattei L.N. Berglund E.O. Ranscht B. Levinson S.R. Schachner M. Shrager P. Isom L.L. Xiao Z.C. J. Neurosci. 2001; 21: 7517-7525Crossref PubMed Google Scholar, 32Rios J.C. Melendez-Vasquez C.V. Einheber S. Lustig M. Grumet M. Hemperly J. Peles E. Salzer J.L. J. Neurosci. 2000; 20: 8354-8364Crossref PubMed Google Scholar), 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 (17Kazarinova-Noyes K. Malhotra J.D. McEwen D.P. Mattei L.N. Berglund E.O. Ranscht B. Levinson S.R. Schachner M. Shrager P. Isom L.L. Xiao Z.C. J. Neurosci. 2001; 21: 7517-7525Crossref PubMed Google Scholar), and this interaction is dependent on presence of β1 subunits (9Chen C. Westenbroek R.E. Xu X. Edwards C.A. Sorenson D.R. Chen Y. McEwen D.P. O'Malley H.A. Bharucha V. Meadows L.S. Knudsen G.A. Vilaythong A. Noebels J.L. Saunders T.L. Scheuer T. Shrager P. Catterall W.A. Isom L.L. J. Neurosci. 2004; 24: 4030-4042Crossref PubMed Scopus (217) Google Scholar, 17Kazarinova-Noyes K. Malhotra J.D. McEwen D.P. Mattei L.N. Berglund E.O. Ranscht B. Levinson S.R. Schachner M. Shrager P. Isom L.L. Xiao Z.C. J. Neurosci. 2001; 21: 7517-7525Crossref PubMed Google Scholar). In β1 (-/-) 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 β1 (9Chen C. Westenbroek R.E. Xu X. Edwards C.A. Sorenson D.R. Chen Y. McEwen D.P. O'Malley H.A. Bharucha V. Meadows L.S. Knudsen G.A. Vilaythong A. Noebels J.L. Saunders T.L. Scheuer T. Shrager P. Catterall W.A. Isom L.L. J. Neurosci. 2004; 24: 4030-4042Crossref PubMed Scopus (217) Google Scholar). The number of mature nodes of Ranvier is significantly reduced in β1 (-/-) optic nerves, and we have proposed that the loss of sodium channel-contactin interactions at the node may be responsible for this effect (9Chen C. Westenbroek R.E. Xu X. Edwards C.A. Sorenson D.R. Chen Y. McEwen D.P. O'Malley H.A. Bharucha V. Meadows L.S. Knudsen G.A. Vilaythong A. Noebels J.L. Saunders T.L. Scheuer T. Shrager P. Catterall W.A. Isom L.L. J. Neurosci. 2004; 24: 4030-4042Crossref PubMed Scopus (217) Google Scholar). The purpose of the present study was to test the hypothesis that sodium channel β1 subunits participate in heterophilic cell-adhesive interactions with CAMs found in the axo-glial complex. We show that β1, expressed in the absence of sodium channel α subunits, interacts with NrCAM, Nf186, contactin, and Nf155. We observed an interaction of the β1 and β2 subunit extracellular domains only in the presence of a region of the β2 intracellular domain, suggesting that cytoskeletal interactions may affect β2-mediated heterophilic cell adhesion. We did not detect β1 interactions with sodium channel β3 subunits in our system. β1-Nf186 interactions result in increased sodium channel α subunit cell surface expression in vitro, similar to what we showed previously for contactin and β2. In contrast, although β1 is required for NrCAM interactions with Nav1.2, coexpression of β1 and NrCAM did not increase α subunit cell surface expression. These results suggest that β1 subunits interact with multiple CAMs to form the basis of the sodium channel complex at nodes of Ranvier. In addition, the present results showing β1 interaction with Nf155, coupled with our previous observation that β1 (-/-) mice lack septate-like junctions at the periphery of the nodal gap in a subset of CNS and PNS axons (9Chen C. Westenbroek R.E. Xu X. Edwards C.A. Sorenson D.R. Chen Y. McEwen D.P. O'Malley H.A. Bharucha V. Meadows L.S. Knudsen G.A. Vilaythong A. Noebels J.L. Saunders T.L. Scheuer T. Shrager P. Catterall W.A. Isom L.L. J. Neurosci. 2004; 24: 4030-4042Crossref PubMed Scopus (217) Google Scholar), support our hypothesis that β1 subunits may also participate in axo-glial communication at the nodal-paranodal boundary. Antibodies and Constructs—Rabbit polyclonal antisera to sodium channel β1 and β2 subunits were described previously (19Chen C. Bharucha V. Chen Y. Westenbroek R.E. Brown A. Malhotra J.D. Jones D. Avery C. Gillespie P.J. II I Kazen-Gillespie K.A. Kazarinova-Noyes K. Shrager P. Saunders T.L. Macdonald R.L. Ransom B.R. Scheuer T. Catterall W.A. Isom L.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 17072-17077Crossref PubMed Scopus (159) Google Scholar, 22Malhotra J.D. Kazen-Gillespie K. Hortsch M. Isom L.L. J. Biol. Chem. 2000; 275: 11383-11388Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 24McEwen D.P. Meadows L.S. Chen C. Thyagarajan V. Isom L.L. J. Biol. Chem. 2004; 279: 16044-16049Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). A polyclonal anti-β3 antibody was a gift from Dr. William A. Catterall (University of Washington, Seattle, WA) and has been described previously (20Ratcliffe C.F. Westenbroek R.E. Curtis R. Catterall W.A. J. Cell Biol. 2001; 154: 427-434Crossref PubMed Scopus (155) Google Scholar). 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) (33Tait S. Gunn-Moore F. Collinson J.M. Huang J. Lubetzki C. Pedraza L. Sherman D.L. Colman D.R. Brophy P.J. J. Cell Biol. 2000; 150: 657-666Crossref PubMed Scopus (272) Google Scholar). 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 (18Lustig M. Zanazzi G. Sakurai T. Blanco C. Levinson S.R. Lambert S. Grumet M. Salzer J.L. Curr. Biol. 2001; 11: 1864-1869Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The Nf186 cDNA construct was a gift from Dr. Vann Bennett (Duke University, Durham, NC) (34Zhang X. Davis J.Q. Carpenter S. Bennett V. J. Biol. Chem. 1998; 273: 30785-30794Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Nf155-Fc and Cn-Fc cDNA constructs were provided by Dr. Elior Peles (Weizmann Institute, Rehovot, Israel) (35Gollan L. Sabanay H. Poliak S. Berglund E.O. Ranscht B. Peles E. J. Cell Biol. 2002; 157: 1247-1256Crossref PubMed Scopus (92) Google Scholar, 36Gollan L. Salomon D. Salzer J.L. Peles E. J. Cell Biol. 2003; 163: 1213-1218Crossref PubMed Scopus (119) Google Scholar). The Cn and CnIg-Fc constructs were gifts from Dr. Genviève Rougon (Laboratoire de Génétique et Physiologie du Développement, Marseille, France) (37Revest J.M. Faivre-Sarrailh C. Maeda N. Noda M. Schachner M. Rougon G. Eur. J. Neurosci. 1999; 11: 1134-1147Crossref PubMed Scopus (54) Google Scholar). cDNAs encoding β1 and β2 were described previously (24McEwen D.P. Meadows L.S. Chen C. Thyagarajan V. Isom L.L. J. Biol. Chem. 2004; 279: 16044-16049Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). 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 β Subunit Fc Fusion Proteins—β1, β2, or β3 cDNAs (24McEwen D.P. Meadows L.S. Chen C. Thyagarajan V. Isom L.L. J. Biol. Chem. 2004; 279: 16044-16049Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 38Isom L.L. De Jongh K.S. Patton D.E. Reber B.F. Offord J. Charbonneau H. Walsh K. Goldin A.L. Catterall W.A. Science. 1992; 256: 839-842Crossref PubMed Scopus (615) Google Scholar, 39Isom L.L. Ragsdale D.S. De Jongh K.S. Westenbroek R.E. Reber B.F. Scheuer T. Catterall W.A. 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Briefly, the 3′ end of each β 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 β subunits and the hinge, CH2, and CH3 regions of the human IgG1. The resulting chimeras are called β1-Fc, β2-Fc, and β3-Fc, respectively. Generation of β2 Truncation Mutants—Truncation mutants of the β2 intracellular domain were generated using polymerase chain reaction. β2Δ181–186 lacks the terminal six amino acids in the carboxyl domain, β2Δ174–186 lacks the terminal 13 amino acids, β2Δ169–186 lacks the terminal 18 amino acids, and β2Δ164–186 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 (35Gollan L. Sabanay H. Poliak S. Berglund E.O. Ranscht B. Peles E. J. Cell Biol. 2002; 157: 1247-1256Crossref PubMed Scopus (92) Google Scholar, 36Gollan L. Salomon D. Salzer J.L. Peles E. J. Cell Biol. 2003; 163: 1213-1218Crossref PubMed Scopus (119) Google Scholar). 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 (35Gollan L. Sabanay H. Poliak S. Berglund E.O. Ranscht B. Peles E. J. Cell Biol. 2002; 157: 1247-1256Crossref PubMed Scopus (92) Google Scholar). 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 40× 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 and DP Manager version 1.1.1.71 software. Stable Transfection and Characterization of Cell Lines—CHL cells stably expressing Nav1.2, Nav1.2/β1, or Nav1.2/Cn/β1 have been described previously (17Kazarinova-Noyes K. Malhotra J.D. McEwen D.P. Mattei L.N. Berglund E.O. Ranscht B. Levinson S.R. Schachner M. Shrager P. Isom L.L. Xiao Z.C. J. Neurosci. 2001; 21: 7517-7525Crossref PubMed Google Scholar, 42Isom L.L. Scheuer T. Brownstein A.B. Ragsdale D.S. Murphy B.J. Catterall W.A. J. Biol. Chem. 1995; 270: 3306-3312Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 43West J.W. Patton D.E. Scheuer T. Wang Y. Goldin A.L. Catterall W.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10910-10914Crossref PubMed Scopus (676) Google Scholar). 1 μg of cDNA encoding NrCAM, Nf155, or Nf186 was transfected into Nav1.2, Nav1.2/β1, and Nav1.2/Cn/β1 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 (42Isom L.L. Scheuer T. Brownstein A.B. Ragsdale D.S. Murphy B.J. Catterall W.A. J. Biol. Chem. 1995; 270: 3306-3312Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar) 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). Production of Fc Fusion Proteins and Binding Assays—To investigate potential β1-interacting CAMs, we used soluble Fc fusion proteins encoding the extracellular domains of contactin, Nf155, NrCAM, sodium channel β2, or sodium channel β3 (Fig. 1A) (18Lustig M. Zanazzi G. Sakurai T. Blanco C. Levinson S.R. Lambert S. Grumet M. Salzer J.L. Curr. Biol. 2001; 11: 1864-1869Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 36Gollan L. Salomon D. Salzer J.L. Peles E. J. Cell Biol. 2003; 163: 1213-1218Crossref PubMed Scopus (119) Google Scholar, 37Revest J.M. Faivre-Sarrailh C. Maeda N. Noda M. Schachner M. Rougon G. Eur. J. Neurosci. 1999; 11: 1134-1147Crossref PubMed Scopus (54) Google Scholar, 44Lustig M. Erskine L. Mason C.A. Grumet M. Sakurai T. J. Comp. Neurol. 2001; 434: 13-28Crossref PubMed Scopus (75) Google Scholar) as well as full-length Nf186, full-length β2, and the truncation mutant β2STOP (22Malhotra J.D. Kazen-Gillespie K. Hortsch M. Isom L.L. J. Biol. Chem. 2000; 275: 11383-11388Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 34Zhang X. Davis J.Q. Carpenter S. Bennett V. J. Biol. Chem. 1998; 273: 30785-30794Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Immunoblots of cell supernatants from transiently transfected CHL fibroblasts indicated expression of all of the soluble Fc fusion proteins used (Fig. 1B). 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 β1 only did not bind the Fc protein backbone (Fig. 1C, c). Thus, any interaction that was observed between CHL cells transfected with β1 and incubated with the Fc fusion proteins was interpreted as a specific interaction between β1 and the Fc fusion protein. β1 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 (17Kazarinova-Noyes K. Malhotra J.D. McEwen D.P. Mattei L.N. Berglund E.O. Ranscht B. Levinson S.R. Schachner M. Shrager P. Isom L.L. Xiao Z.C. J. Neurosci. 2001; 21: 7517-7525Crossref PubMed Google Scholar). The association of contactin with sodium channels in vitro is dependent on the presence of β1, and this interaction results in increased sodium channel α subunit expression at the cell surface (17Kazarinova-Noyes K. Malhotra J.D. McEwen D.P. Mattei L.N. Berglund E.O. Ranscht B. Levinson S.R. Schachner M. Shrager P. Isom L.L. Xiao Z.C. J. Neurosci. 2001; 21: 7517-7525Crossref PubMed Google Scholar). The interaction between β1 and contactin requires the Ig l