Brain enriched voltage-gated sodium channel (VGSC) Na_v1.2 and Na_v1.6 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_v1.2 and Na_v1.6 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_v1.2 and Na_v1.6 in cultured mouse and rat neurons and in male and female mouse brain. We discovered a previously uncharacterized trafficking pathway that targets Na_v1.2 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_v1.2 in the axon initial segment and facilitate its membrane loading at the distal axon. As mouse pyramidal neurons undergo myelination, Na_v1.2 is gradually excluded from the distal axon as Na_v1.6 becomes the dominant VGSC in the axon initial segment and nodes of Ranvier. In addition, we revealed exquisite developmental regulation of Na_v1.2 and Na_v1.6 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.

脑富集电压门控钠通道 (VGSC) Na _v 1.2 和 Na _v 1.6 对于 CNS 中的电信号传导至关重要。先前的研究广泛描述了这两种 VGSC 的细胞类型特异性表达和电生理特性，以及它们的差异如何有助于神经元兴奋性的微调。然而，由于缺乏可靠的标记和成像方法，这些同源 Na _v 1.2 和 Na _v 1.6 通道的亚细胞定位和动力学仍未得到充分研究。为了克服这一挑战，我们结合了基因组编辑、超分辨率和活细胞单分子成像来探测培养的小鼠和大鼠神经元以及雄性神经元中 Na _v 1.2 和 Na _v 1.6 的亚细胞组成、相对丰度和运输动态。和雌性小鼠的大脑。我们发现了一种以前未表征的运输途径，该途径将 Na _v 1.2 靶向无髓鞘神经元的远端轴突。该途径利用跨膜结构域 I 和 II 之间的细胞内环 1 中的不同信号来抑制 Na _v 1.2 在轴突初始段中的保留，并促进其在远端轴突的膜负载。当小鼠锥体神经元经历髓鞘形成时，Na _v 1.2 逐渐从远端轴突中排除，因为 Na _v 1.6 成为轴突初始段和 Ranvier 节点中的主要 VGSC。此外，我们还揭示了轴突初始段和树突中 Na _v 1.2 和 Na _v 1.6 定位的精细发育调控，阐明了这些亚细胞区室中钠通道的分子身份。 总之，这些结果揭示了 VGSC 的区室特异性定位和运输机制，可以单独调节这些机制以调节大脑中的膜兴奋性。

意义声明直接观察内源性电压门控钠通道揭示了以前未表征的远端轴突靶向机制以及不同亚细胞区室中钠通道的分子特性。