Identification of the Cysteine Residue Responsible for Disulfide Linkage of Na+ Channel α and β2 Subunits

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