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.
Highlights
Voltage-gated sodium (Nav) channels are responsible for the initiation and conduction of action potentials in neurons and are densely accumulated at the axon initial segment (AIS) [1]
GFP and biotinylation domain-tagged Nav1.6 channels localize to the axon initial segment in cultured hippocampal neurons
To enable real-time imaging of Nav1.6 dynamics in neurons, GFP was fused to the C-terminus of the mouse Nav1.6 channel and a biotin acceptor domain (BAD) (Tamkun et al, 2007) was inserted into the S1-S2 extracellular loop of domainIV (Fig 1A)
Summary
Voltage-gated sodium (Nav) channels are responsible for the initiation and conduction of action potentials in neurons and are densely accumulated at the axon initial segment (AIS) [1]. Nav channels are composed of a highly post-translationally modified pore-forming α-subunit and auxiliary β-subunits [2]. Of the nine Nav alpha subunits (Nav1.1–1.9), Nav1.1, Nav1.2, Nav1.3. Nav1.6 Delivery to the Developing Axon Initial Segment
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