Identification of the cysteine residue responsible for disulfide linkage of Na+ channel alpha and beta2 subunits.
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Chen C, Calhoun JD, Zhang Y, Lopez-Santiago L, Zhou N, Davis TH, Salzer JL, Isom LL
Identification of the cysteine residue responsible for disulfide linkage of Na+ channel alpha and beta2 subunits.
J Biol Chem. 2012 Nov 9;287(46):39061-9. doi: 10.1074/jbc.M112.397646. Epub 2012 Sep 19.
- PubMed ID
- 22992729 [ View in PubMed]
- Abstract
Voltage-gated Na(+) channels in the brain are composed of a single pore-forming alpha subunit, one non-covalently linked beta subunit (beta1 or beta3), and one disulfide-linked beta subunit (beta2 or beta4). The final step in Na(+) channel biosynthesis in central neurons is concomitant alpha-beta2 disulfide linkage and insertion into the plasma membrane. Consistent with this, Scn2b (encoding beta2) null mice have reduced Na(+) channel cell surface expression in neurons, and action potential conduction is compromised. Here we generated a series of mutant beta2 cDNA constructs to investigate the cysteine residue(s) responsible for alpha-beta2 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 beta2 subunits. Loss of alpha-beta2 covalent complex formation disrupts the targeting of beta2 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 beta2 subcellular localization in vivo. WT beta2 subunits are resistant to live cell Triton X-100 detergent extraction from the hippocampal axon initial segment, whereas mutant beta2 subunits, which cannot form disulfide bonds with alpha, are removed by detergent. Taken together, our results demonstrate that alpha-beta2 covalent association via a single, extracellular disulfide bond is required for beta2 targeting to specialized neuronal subcellular domains and for beta2 association with the neuronal cytoskeleton within those domains.