title: Single-Molecule Imaging of Nav1.6 on the Surface of Hippocampal Neurons Reveals Somatic Nanoclusters.
abstract: Voltage-gated sodium (Nav) channels are responsible for the depolarizing phase of the action potential in most nerve cells, and Nav channel localization to the axon initial segment is vital to action potential initiation. Nav 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 Nav channel localization and dynamics within this neuronal compartment. This study uses single-particle tracking and photoactivation localization microscopy to analyze cell-surface Nav1.6 within the soma of cultured hippocampal neurons. Mean-square displacement analysis of individual trajectories indicated that half of the somatic Nav1.6 channels localized to stable nanoclusters ∼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 Nav1.6 nanoclusters are maintained in the soma is biologically different from axonal localization. Nonclustered Nav1.6 channels showed anomalous diffusion, as determined by mean-square-displacement analysis. High-density single-particle tracking of Nav channels labeled with photoactivatable fluorophores in combination with Bayesian inference analysis was employed to characterize the surface nanoclusters. A subpopulation of mobile Nav1.6 was observed to be transiently trapped in the nanoclusters. Somatic Nav1.6 nanoclusters represent a new, to our knowledge, type of Nav channel localization, and are hypothesized to be sites of localized channel regulation.
authors.affiliation: Cell and Molecular Biology Graduate Program, Colorado State University, Fort Collins, Colorado; Molecular, Cellular and Integrative Neuroscience Program, Colorado State University, Fort Collins, Colorado; Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado.
authors.forename: Elizabeth J
authors.initials: EJ
authors.lastname: Akin
authors.affiliation: Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado.
authors.forename: Laura
authors.initials: L
authors.lastname: Solé
authors.affiliation: Molecular, Cellular and Integrative Neuroscience Program, Colorado State University, Fort Collins, Colorado; Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado.
authors.forename: Ben
authors.initials: B
authors.lastname: Johnson
authors.affiliation: Physico-Chimie Curie, Institut Curie, Paris Sciences Lettres, CNRS UMR 168, Université Pierre et Marie Curie, Paris, France.
authors.forename: Mohamed El
authors.initials: ME
authors.lastname: Beheiry
authors.affiliation: Institut Pasteur, Decision and Bayesian Computation, Centre National de la Recherche Scientifique (CNRS) UMR 3525, Paris, France; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia.
authors.forename: Jean-Baptiste
authors.initials: JB
authors.lastname: Masson
authors.affiliation: School of Biomedical Engineering, Colorado State University, Fort Collins, Colorado; Department of Electrical and Computer Engineering, Colorado State University, Fort Collins, Colorado. Electronic address: krapf@engr.colostate.edu.
authors.forename: Diego
authors.initials: D
authors.lastname: Krapf
authors.affiliation: Cell and Molecular Biology Graduate Program, Colorado State University, Fort Collins, Colorado; Molecular, Cellular and Integrative Neuroscience Program, Colorado State University, Fort Collins, Colorado; Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado; Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado. Electronic address: michael.tamkun@colostate.edu.
authors.forename: Michael M
authors.initials: MM
authors.lastname: Tamkun
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D017487:Ankyrins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D002966:Clathrin / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology* / Q000378:metabolism*; D007091:Image Processing, Computer-Assisted; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D009038:Motion; D062557:NAV1.6 Voltage-Gated Sodium Channel / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000166:cytology* / Q000378:metabolism*; D051381:Rats; D051669:Shab Potassium Channels / Q000378:metabolism; D000072760:Single Molecule Imaging
chemical_list: D000199:Actins; C490614:Ank3 protein, rat; D017487:Ankyrins; D002966:Clathrin; C496991:Kcnb1 protein, rat; D062557:NAV1.6 Voltage-Gated Sodium Channel; C507811:Scn8a protein, rat; D051669:Shab Potassium Channels; D049452:Green Fluorescent Proteins