Identification of the cysteine residue responsible for disulfide linkage of Na+ channel α and ß2 subunits.

Voltage-gated Na(+) channels in the brain are composed of a single pore-forming α subunit, one non-covalently linked ß subunit (ß1 or ß3), and one disulfide-linked ß subunit (ß2 or ß4). The final step in Na(+) channel biosynthesis in central neurons is concomitant α-ß2 disulfide linkage and insertion into the plasma membrane. Consistent with this, Scn2b (encoding ß2) null mice have reduced Na(+) channel cell surface expression in neurons, and action potential conduction is compromised. Here we generated a series of mutant ß2 cDNA constructs to investigate the cysteine residue(s) responsible for α-ß2 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 ß2 subunits. Loss of α-ß2 covalent complex formation disrupts the targeting of ß2 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 ß2 subcellular localization in vivo. WT ß2 subunits are resistant to live cell Triton X-100 detergent extraction from the hippocampal axon initial segment, whereas mutant ß2 subunits, which cannot form disulfide bonds with α, are removed by detergent. Taken together, our results demonstrate that α-ß2 covalent association via a single, extracellular disulfide bond is required for ß2 targeting to specialized neuronal subcellular domains and for ß2 association with the neuronal cytoskeleton within those domains.