The MAP1B Binding Domain of Na_v1.6 Is Required for Stable Expression at the Axon Initial Segment

Abstract

Na v 1.6 (SCN8A) is a major voltage-gated sodium channel in the mammalian CNS, and is highly concentrated at the axon initial segment (AIS). As previously demonstrated, the microtubule associated protein MAP1B binds the cytoplasmic N terminus of Na v 1.6, and this interaction is disrupted by the mutation p.VAVP(77-80)AAAA. We now demonstrate that this mutation results in WT expression levels on the somatic surface but reduced surface expression at the AIS of cultured rat embryonic hippocampal neurons from both sexes. The mutation of the MAP1B binding domain did not impair vesicular trafficking and preferential delivery of Na v 1.6 to the AIS; nor was the diffusion of AIS inserted channels altered relative to WT. However, the reduced AIS surface expression of the MAP1B mutant was restored to WT levels by inhibiting endocytosis with Dynasore, indicating that compartment-specific endocytosis was responsible for the lack of AIS accumulation. Interestingly, the lack of AIS targeting resulted in an elevated percentage of persistent current, suggesting that this late current originates predominantly in the soma. No differences in the voltage dependence of activation or inactivation were detected in the MAP1B binding mutant relative to WT channel. We hypothesize that MAP1B binding to the WT Na v 1.6 masks an endocytic motif, thus allowing long-term stability on the AIS surface. This work identifies a critical and important new role for MAP1B in the regulation of neuronal excitability and adds to our understanding of AIS maintenance and plasticity, in addition to identifying new target residues for pathogenic mutations of SCN8A. Significance StatementNa v 1.6 is a major voltage-gated sodium channel in human brain, where it regulates neuronal activity due to its localization at the axon initial segment (AIS). Na v 1.6 mutations cause epilepsy, intellectual disability, and movement disorders. In the present work, we show that loss of interaction with MAP1B within the Na v 1.6 N terminus reduces the steady-state abundance of Na v 1.6 at the AIS. The effect is due to increased Na v 1.6 endocytosis at this neuronal compartment rather than a failure of forward trafficking to the AIS. This work confirms a new biological role of MAP1B in the regulation of sodium channel localization and will contribute to future analysis of patient mutations in the cytoplasmic N terminus of Na v 1.6.

Trafficking mechanisms underlying Nav channel subcellular localization in neurons

“…It was reasonable to assume that MAP1B binding was involved in the forward trafficking of Na v 1.6 since, in addition to its classical role in microtubule stabilization, MAP1B is also known to regulate the localization, function and trafficking of different channels and receptors [96]. However, as our recent work demonstrated, and to our surprise, the Na v 1.6 MAPM traffics to the somatic surface of hippocampal neurons but is not concentrated within the AIS (Figure 4(a)) [28]. While we expected that this distribution was due to a lack of trafficking to the AIS, the fluorescent biotinylation assay described above confirmed that this was not the case, with the MAPM being readily delivered to the AIS surface where it was immediately immobilized just like the wild-type channel.…”

“…While we expected that this distribution was due to a lack of trafficking to the AIS, the fluorescent biotinylation assay described above confirmed that this was not the case, with the MAPM being readily delivered to the AIS surface where it was immediately immobilized just like the wild-type channel. We found that MAPM is not concentrated at the AIS because, without MAP1B binding, it is internalized via compartment-specific Na v 1.6 endocytosis [28]. Although MAPM channels are still tethered at the AIS plasma membrane by AnkG binding (as demonstrated by their restricted diffusion, similar to wild-type Na v 1.6 channels), these channels are endocytosed from the axonal compartment when MAP1B is not able to bind to Na v 1.6 (as illustrated in Figure 4(b)).…”

“…However, biology is not always simple, as was reinforced when we applied our imaging technology to Na v 1.6 mutants previously reported to have forward trafficking defects. One such Na v 1.6 mutant (MAPM) carried amino acid substitutions in its MAP1B binding domain [28]. MAP1B light chain interaction with wild-type Na v 1.6 was demonstrated by both yeast two-hybrid experiments and co-immunoprecipitation from mouse brain membranes.…”

“…Progress in high resolution structure determination [25][26][27] added a physical context to much of the site-directed mutagenesis work that had occurred over the previous 20 years. Recent progress in Na v channel biology focuses on the mechanisms underlying channel trafficking and localization and how localization determines function [28,29], for proper nerve function requires the precise targeting of Na v isoforms to distinct subcellular domains. While this review will mainly focus on the localization and trafficking of Na v 1.6, other Na v channel isoforms will be discussed when appropriate.…”

Deletion of Class II ADP-Ribosylation Factors in Mice Causes Tremor by the Nav1.6 Loss in Cerebellar Purkinje Cell Axon Initial Segments

“…Therefore, ARF4 ϩ/Ϫ /ARF5 Ϫ/Ϫ mouse PCs may display a failure in trafficking Nav1.6 proteins to the AIS via an ankyrin-G-independent mechanism. However, it is also possible that the decreased localization of Nav1.6 to the AIS depends on the enhanced Nav1.6 endocytosis from the AIS, rather than a reduced forward trafficking to the AIS (Solé et al, 2019). The trafficking of Nav1.6 proteins to the AIS needs to be examined directly in real-time imaging to determine whether the maintenance of Nav1.6 proteins in the AIS is affected.…”