Abstract
The mechanism by which voltage-gated sodium channels are trafficked to the surface of neurons is not well understood. Our previous work implicated the cytoplasmic N terminus of the sodium channel Na(v)1.6 in this process. We report that the N terminus plus the first transmembrane segment (residues 1-153) is sufficient to direct a reporter to the cell surface. To identify proteins that interact with the 117-residue N-terminal domain, we carried out a yeast two-hybrid screen of a mouse brain cDNA library. Three clones containing overlapping portions of the light chain of microtubule-associated protein Map1b (Mtap1b) were recovered from the screen. Interaction between endogenous Na(v)1.6 channels and Map1b in mouse brain was confirmed by co-immunoprecipitation. Map1b did not interact with the N terminus of the related channel Na(v)1.1. Alanine-scanning mutagenesis of the Na(v)1.6 N terminus demonstrated that residues 77-80 (VAVP) contribute to interaction with Map1b. Co-expression of Na(v)1.6 with Map1b in neuronal cell line ND7/23 resulted in a 50% increase in current density, demonstrating a functional role for this interaction. Mutation of the Map1b binding site of Na(v)1.6 prevented generation of sodium current in transfected cells. The data indicate that Map1b facilitates trafficking of Na(v)1.6 to the neuronal cell surface.
Highlights
Specific sodium channels have unique subcellular localizations within neurons
Cell Membrane Localization of CD74 Reporter—To determine whether the N terminus of Nav1.6 is sufficient to direct protein localization to the cell membrane, we used the extracellular domain of CD74 as a cell surface reporter [19]
We demonstrate here that the N terminus of Nav1.6 interacts with the adaptor protein Map1b, resulting in an increase in current density without a change in activation or fast inactivation of the channel
Summary
Specific sodium channels have unique subcellular localizations within neurons. Results: We identified a novel interaction of sodium channel Nav1.6 with a microtubule-associated protein. We recently characterized the ethylnitrosourea-induced mouse mutant Scn8aataxia, in which the amino acid substitution S21P results in trapping of the Nav1.6 channel protein in the Golgi [14] The location of this mutation in the N terminus suggested that this region might be involved in protein-protein interactions required for trafficking of the channel protein to the cell surface. To test this prediction, we carried out a yeast two-hybrid screen of a mouse brain cDNA library to identify proteins that interact with the 117-residue N terminus of the channel. The work reported here provides evidence that Nav1.6 is subject to microtubular transport to the cell membrane mediated by interaction with the light chain of Map1b
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