title: Identification of the cysteine residue responsible for disulfide linkage of Na+ channel α and β2 subunits.
abstract: Voltage-gated Na(+) channels in the brain are composed of a single pore-forming α subunit, one non-covalently linked β subunit (β1 or β3), and one disulfide-linked β subunit (β2 or β4). The final step in Na(+) channel biosynthesis in central neurons is concomitant α-β2 disulfide linkage and insertion into the plasma membrane. Consistent with this, Scn2b (encoding β2) null mice have reduced Na(+) channel cell surface expression in neurons, and action potential conduction is compromised. Here we generated a series of mutant β2 cDNA constructs to investigate the cysteine residue(s) responsible for α-β2 subunit covalent linkage. We demonstrate that a single cysteine-to-alanine substitution at extracellular residue Cys-26, located within the immunoglobulin (Ig) domain, abolishes the covalent linkage between α and β2 subunits. Loss of α-β2 covalent complex formation disrupts the targeting of β2 to nodes of Ranvier in a myelinating co-culture system and to the axon initial segment in primary hippocampal neurons, suggesting that linkage with α is required for normal β2 subcellular localization in vivo. WT β2 subunits are resistant to live cell Triton X-100 detergent extraction from the hippocampal axon initial segment, whereas mutant β2 subunits, which cannot form disulfide bonds with α, are removed by detergent. Taken together, our results demonstrate that α-β2 covalent association via a single, extracellular disulfide bond is required for β2 targeting to specialized neuronal subcellular domains and for β2 association with the neuronal cytoskeleton within those domains.
authors.affiliation: Department of Pharmacology and Cellular and Molecular Biology Program, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
authors.forename: Chunling
authors.initials: C
authors.lastname: Chen
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Calhoun
authors.forename: Yanqing
authors.initials: Y
authors.lastname: Zhang
authors.forename: Luis
authors.initials: L
authors.lastname: Lopez-Santiago
authors.forename: Ningna
authors.initials: N
authors.lastname: Zhou
authors.forename: Tigwa H
authors.initials: TH
authors.lastname: Davis
authors.forename: James L
authors.initials: JL
authors.lastname: Salzer
authors.forename: Lori L
authors.initials: LL
authors.lastname: Isom
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism; D002448:Cell Adhesion; D002462:Cell Membrane / Q000378:metabolism; D018920:Coculture Techniques; D003545:Cysteine / Q000737:chemistry*; D003599:Cytoskeleton / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D000939:Epitopes / Q000737:chemistry; D057809:HEK293 Cells; D006624:Hippocampus / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D009154:Mutation; D062550:NAV1.1 Voltage-Gated Sodium Channel / Q000737:chemistry* / Q000235:genetics; D009474:Neurons / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods; D051381:Rats; D012583:Schwann Cells; D015222:Sodium Channels / Q000737:chemistry
chemical_list: D004220:Disulfides; D000939:Epitopes; D062550:NAV1.1 Voltage-Gated Sodium Channel; D015222:Sodium Channels; D003545:Cysteine