An ankyring-binding motif is necessary and sufficient for targeting Na _v1.6 sodium channels to axon initial segments and nodes of ranvier

Neurons are highly polarized cells with functionally distinct axonal and somatodendritic compartments. Voltage-gated sodium channels Nav1.2 and Nav1.6 are highly enriched at axon initial segments (AISs) and nodes of Ranvier, where they are necessary for generation and propagation of action potentials. Previous studies using reporter proteins in unmyelinated cultured neurons suggest that an ankyrinGbinding motif within intracellular loop 2 (L2) of sodium channels is sufficient for targeting these channels to the AIS, but mechanisms of channel targeting to nodes remain poorly understood. Using a CD4-Na _v1.2/L2 reporter protein in rat dorsal root ganglion neuron-Schwann cell myelinating cocultures, we show that the ankyrinG-binding motif is sufficient for protein targeting to nodes of Ranvier. However, reporter proteins cannot capture the complexity of full-length channels. To determine how native, full-length sodium channels are clustered in axons, and to show the feasibility of studying these channels in vivo, we constructed fluorescently tagged and functional mouse Nav1.6 channels for in vivo analysis using in utero brain electroporation. We show here that wild-type tagged-Nav1.6 channels are efficiently clustered at nodes and AISs in vivo. Furthermore, we show that mutation of a single invariant glutamic acid residue (E1100) within the ankyrinG-binding motif blocked Na _v1.6 targeting in neurons both in vitro and in vivo. Additionally, we show that caseine kinase phosphorylation sites within this motif, while not essential for targeting, can modulate clustering at the AIS. Thus, the ankyrinGbinding motif is both necessary and sufficient for the clustering of sodium channels at nodes of Ranvier and the AIS.