Direct Observation of Compartment-Specific Localization and Dynamics of Voltage-Gated Sodium Channels

Brain enriched voltage-gated sodium channel (VGSC) Na(v)1.2 and Na(v)1.6 are critical for electrical signaling in the CNS. Previous studies have extensively characterized cell-type-specific expression and electrophysiological properties of these two VGSCs and how their differences contribute to fine-tuning of neuronal excitability. However, because of a lack of reliable labeling and imaging methods, the subcellular localization and dynamics of these homologous Na(v)1.2 and Na(v)1.6 channels remain understudied. To overcome this challenge, we combined genome editing, super-resolution, and live-cell single-molecule imaging to probe subcellular composition, relative abundances, and trafficking dynamics of Na(v)1.2 and Na(v)1.6 in cultured mouse and rat neurons and in male and female mouse brain. We discovered a previously uncharacterized trafficking pathway that targets Na(v)1.2 to the distal axon of unmyelinated neurons. This pathway uses distinct signals residing in the intracellular loop 1 between transmembrane domain I and II to suppress the retention of Na(v)1.2 in the axon initial segment and facilitate its membrane loading at the distal axon. As mouse pyramidal neurons undergo myelination, Na(v)1.2 is gradually excluded from the distal axon as Na(v)1.6 becomes the dominant VGSC in the axon initial segment and nodes of Ranvier. In addition, we revealed exquisite developmental regulation of Na(v)1.2 and Na(v)1.6 localizations in the axon initial segment and dendrites, clarifying the molecular identity of sodium channels in these subcellular compartments. Together, these results unveiled compartment-specific localizations and trafficking mechanisms for VGSCs, which could be regulated separately to modulate membrane excitability in the brain. SIGNIFICANCE STATEMENT Direct observation of endogenous voltage-gated sodium channels reveals a previously uncharacterized distal axon targeting mechanism and the molecular identity of sodium channels in distinct subcellular compartments.