Single-Molecule Imaging of Nav1.6 on the Surface of Hippocampal Neurons Reveals Somatic Nanoclusters

Voltage-gated sodium (Na-v) channels are responsible for the depolarizing phase of the action potential in most nerve cells, and Na-v channel localization to the axon initial segment is vital to action potential initiation. Na-v channels in the soma play a role in the transfer of axonal output information to the rest of the neuron and in synaptic plasticity, although little is known about Na-v channel localization and dynamics within this neuronal compartment. This study uses single-particle tracking and photoactivation localization microscopy to analyze cell-surface Na(v)1.6 within the soma of cultured hippocampal neurons. Mean-square displacement analysis of individual trajectories indicated that half of the somatic Na(v)1.6 channels localized to stable nanoclusters similar to 230 nm in diameter. Strikingly, these domains were stabilized at specific sites on the cell membrane for >30 min, notably via an ankyrin-independent mechanism, indicating that the means by which Na(v)1.6 nanoclusters are maintained in the soma is biologically different from axonal localization. Nonclustered Na(v)1.6 channels showed anomalous diffusion, as determined by mean-square-displacement analysis. High-density single-particle tracking of Na-v channels labeled with photoactivatable fluorophores in combination with Bayesian inference analysis was employed to characterize the surface nanoclusters. A subpopulation of mobile Na(v)1.6 was observed to be transiently trapped in the nanoclusters. Somatic Na(v)1.6 nanoclusters represent a new, to our knowledge, type of Na-v channel localization, and are hypothesized to be sites of localized channel regulation.