High resolution optical analysis of Nav1.6 localization and trafficking

Abstract

Voltage-gated sodium (Naᵥ) channels are responsible for the depolarizing phase of the action potential in most nerve cell membranes. As such, these proteins are essential for nearly all functions of the nervous system including thought, movement, sensation, and many other basic physiological processes. Neurons precisely control the number, type, and location of these important ion channels. The density of Naᵥ channels within the axon initial segment (AIS) of neurons can be more than 35-fold greater than that in the somatodendritic region and this localization is vital to action potential initiation. Dysfunction or mislocalization of Naᵥ channels is linked to many diseases including epilepsy, cardiac arrhythmias, and pain disorders. Despite the importance of Naᵥ channels, knowledge of their trafficking and cell-surface dynamics is severely limited. Research in this area has been hampered by the lack of modified Naᵥ constructs suitable for investigations into neuronal Naᵥ cell biology. This dissertation demonstrates the successful creation of modified Naᵥ1.6 cDNAs that retain wild-type function and trafficking following expression in cultured rat hippocampal neurons. The Naᵥ1.6 isoform is emphasized because it 1) is the most abundant Naᵥ channel in the mammalian brain, 2) is involved in setting the action potential threshold, 3) controls repetitive firing in Purkinje neurons and retinal ganglion cells, 4) and can contain mutations causing epilepsy, ataxia, or mental retardation. Using single-molecule microscopy techniques, the trafficking and cell-surface dynamics of Naᵥ1.6 were investigated. In contrast to the current dogma that Naᵥ channels are localized to the AIS of neurons through diffusion trapping and selective endocytosis, the experiments presented here demonstrate that Naᵥ1.6 is directly delivered to the AIS via a vesicular delivery mechanism. The modified Naᵥ1.6 constructs were also used to investigate the distribution and cell-surface dynamics of Naᵥ1.6. Somatic Naᵥ1.6 channels were observed to localize to small membrane regions, or nanoclusters, and this localization is ankyrinG independent. These sites, which could represent sites of localized channel regulation, represent a new Naᵥ localization mechanism. Channels within the nanoclusters appear to be stably bound on the order of minutes to hours, while non-clustered Naᵥ1.6 channels are mobile. Novel single-particle tracking photoactivation localization microscopy (spt-PALM) analysis of Naᵥ1.6-Dendra2 demonstrated that the nanoclusters can be modeled as energy wells and the depth of these interactions increase with neuronal age. The research presented in this dissertation represents the first single-molecule approaches to any Naᵥ channel isoform. The approaches developed during the course of this dissertation research will further our understanding of Naᵥ1.6 cell biology under both normal and pathological conditions.