title: 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.
authors: Cell and Molecular Biology Graduate Program, Colorado State University, Fort Collins, Colorado, United States of America; Molecular, Cellular and Integrative Neuroscience Program, Colorado State University, Fort Collins, Colorado, United States of America.
authors: Elizabeth J
authors: EJ
authors: Akin
authors: Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado, United States of America.
authors: Laura
authors: L
authors: Solé
authors: Rehabilitation Research Center, Veterans Administration Connecticut Healthcare System, West Haven, Connecticut, United States of America; Department of Neurology, Yale University School of Medicine, New Haven, Connecticut, United States of America.
authors: Sulayman D
authors: SD
authors: Dib-Hajj
authors: Rehabilitation Research Center, Veterans Administration Connecticut Healthcare System, West Haven, Connecticut, United States of America; Department of Neurology, Yale University School of Medicine, New Haven, Connecticut, United States of America.
authors: Stephen G
authors: SG
authors: Waxman
authors: Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado, United States of America; Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado, United States of America; Molecular, Cellular and Integrative Neuroscience Program, Colorado State University, Fort Collins, Colorado, United States of America.
authors: Michael M
authors: MM
authors: Tamkun
mesh_terms: D000200:Action Potentials / Q000502:physiology; D000818:Animals; D017487:Ankyrins / Q000235:genetics* / Q000378:metabolism; D001369:Axons / Q000378:metabolism* / Q000648:ultrastructure; D045744:Cell Line, Tumor; D004622:Embryo, Mammalian; D036681:Fluorescence Recovery After Photobleaching; D018507:Gene Expression Regulation, Developmental; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D051379:Mice; D062557:NAV1.6 Voltage-Gated Sodium Channel / Q000235:genetics* / Q000378:metabolism; D055495:Neurogenesis / Q000235:genetics*; D061251:Primary Cell Culture; D011485:Protein Binding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011984:Sensory Receptor Cells / Q000378:metabolism* / Q000648:ultrastructure; D015398:Signal Transduction; D014162:Transfection; D022161:Transport Vesicles / Q000378:metabolism
chemical_list: C094681:Ank3 protein, mouse; D017487:Ankyrins; D062557:NAV1.6 Voltage-Gated Sodium Channel; D011993:Recombinant Fusion Proteins; C491046:Scn8a protein, mouse; D049452:Green Fluorescent Proteins