Identification of the cysteine residue responsible for disulfide linkage of Na+ channel ? and ?2 subunits.

ABSTRACT: Voltage-gated Na(+) channels in the brain are composed of a single pore-forming ? subunit, one non-covalently linked ? subunit (?1 or ?3), and one disulfide-linked ? subunit (?2 or ?4). The final step in Na(+) channel biosynthesis in central neurons is concomitant ?-?2 disulfide linkage and insertion into the plasma membrane. Consistent with this, Scn2b (encoding ?2) null mice have reduced Na(+) channel cell surface expression in neurons, and action potential conduction is compromised. Here we generated a series of mutant ?2 cDNA constructs to investigate the cysteine residue(s) responsible for ?-?2 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 ? and ?2 subunits. Loss of ?-?2 covalent complex formation disrupts the targeting of ?2 to nodes of Ranvier in a myelinating co-culture system and to the axon initial segment in primary hippocampal neurons, suggesting that linkage with ? is required for normal ?2 subcellular localization in vivo. WT ?2 subunits are resistant to live cell Triton X-100 detergent extraction from the hippocampal axon initial segment, whereas mutant ?2 subunits, which cannot form disulfide bonds with ?, are removed by detergent. Taken together, our results demonstrate that ?-?2 covalent association via a single, extracellular disulfide bond is required for ?2 targeting to specialized neuronal subcellular domains and for ?2 association with the neuronal cytoskeleton within those domains.

SUBMITTER: Chen C 

PROVIDER: S-EPMC3493947 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature