Preferential Targeting of Nav1.6 Voltage-Gated Na+ Channels to the Axon Initial Segment during Development

Abstract

During axonal maturation, voltage-gated sodium (Nav) channels accumulate at the axon initial segment (AIS) at high concentrations. This localization is necessary for the efficient initiation of action potentials. The mechanisms underlying channel trafficking to the AIS during axonal development have remained elusive due to a lack of Nav reagents suitable for high resolution imaging of channels located specifically on the cell surface. Using an optical pulse-chase approach in combination with a novel Nav1.6 construct containing an extracellular biotinylation domain we demonstrate that Nav1.6 channels are preferentially inserted into the AIS membrane during neuronal development via direct vesicular trafficking. Single-molecule tracking illustrates that axonal channels are immediately immobilized following delivery, while channels delivered to the soma are often mobile. Neither a Nav1.6 channel lacking the ankyrin-binding motif nor a chimeric Kv2.1 channel containing the Nav ankyrinG-binding domain show preferential AIS insertion. Together these data support a model where ankyrinG-binding is required for preferential Nav1.6 insertion into the AIS plasma membrane. In contrast, ankyrinG-binding alone does not confer the preferential delivery of proteins to the AIS.

Polarized localization of voltage-gated Na + channels is regulated by concerted FGF13 and FGF14 action

“…Previous studies have defined specific intracellular domains within VGSCs that bind directly with the cytoskeletal adaptor molecule ankyrin-G and promote the concentration of VGSCs in the AIS (7,10,11). The localization of ankyrin-G is not significantly changed by either knockdown treatment (Fig.…”

“…In most primary neurons, efficient action potential initiation depends on the clustering of VGSCs at the axon initial segment (AIS) (2)(3)(4). VGSC density within the AIS is ∼10-to 80-fold higher than in the somatodendritic compartment (5,6), even though VGSCs are initially inserted into both somatodendritic and axonal membranes (7). Several mechanisms contribute to VGSC concentration in the AIS.…”

Another piece to the intracellular FGF/Na + channel puzzle

“…Studies using chimeric reporter proteins containing the ankyrinbinding motif from the Na v 1.2 channel implicate selective endocytosis from the somatic membrane combined with stable tethering to ankyrin within the AIS as a central mechanism in the establishment of Na v channel polarization (13). Experiments using the full-length Na v 1.6 channel and the single-molecule detection sensitivity of total internal reflection fluorescence microscopy demonstrate a direct, ankyrin-dependent vesicular delivery to the AIS where they are immediately immobilized, and ankyrinindependent delivery of mobile channels to the soma (14).…”

“…Thus far, only indirect evidence suggests that FGF13 is involved in steady-state clathrin-mediated endocytosis. Direct evidence would include measures of Na v channel membrane surface stability using single-molecule imaging techniques of tagged channels (14) and observed recruitment of Na v channels to clathrin-coated pits. Such experiments have been done for other ion channels, such as K v 2.1 (19).…”

Surface dynamics of voltage-gated ion channels

“…84,109 Apart from this neuron-specific structure, most of the surface expressed voltage-gated ion channels are mobile within cellular membranes. 3,32,40,108,119,124 The existence of such surface mobility produces the potential to characterize molecular interactions of the observed molecule based merely on changes in its diffusion behavior. Given that kinetic properties of most ion channels depend not only on the pore-forming subunit, but to a large extent on the assembly and interaction with accessory subunits, one can assume that channel composition, and hence function, might be tuned by a dynamic assembly over time.…”