Dependence of axon initial segment formation on Na+ channel expression.
Spinal motor neurons were isolated from embryonic rats, and grown in culture. By 2 days in vitro, the axon initial segment was characterized by colocalization and clustering of Na+ channels and ankyrinG. By 5 days, NrCAM, and neurofascin could also be detected at most initial segments. We sought to determine, as one important aim, whether Na+ channels themselves played an essential role in establishing this specialized axonal region. Small hairpin RNAs (shRNAs) were used to target multiple subtypes of Na+ channels for reduced expression by RNA interference. Transfection resulted in substantial knockdown of these channels within the cell body and also as clusters at initial segments. Furthermore, Na+ currents originating at the initial segment, and recorded under patch clamp, were strongly reduced by shRNA. Control shRNA against a nonmammalian protein was without effect. Most interestingly, targeting Na+ channels also blocked clustering of ankyrinG, NrCAM, and neurofascin at the initial segment, although these proteins were seen in the soma. Thus, both Na+ channels and ankyrinG are required for formation of this essential axonal domain. Knockdown of Na+ channels was somewhat less effective when introduced after the initial segments had formed. Disruption of actin polymerization by cytochalasin D resulted in multiple initial segments, each with clusters of both Na+ channels and ankyrinG. The results indicate that initial segment formation occurs as Na+ channels are transported into the nascent axon membrane, diffuse distally, and link to the cytoskeleton by ankyrinG. Subsequently, other components are added, and stability is increased. A computational model closely reproduced the experimental results.
Xu
Xiaorong
X
Department of Neurobiology and Anatomy, University of Rochester Medical Center, Rochester, New York 14642, USA.
Shrager
Peter
P
D000818:Animals; D017487:Ankyrins / Q000378:metabolism; D001369:Axons / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002452:Cell Count / Q000379:methods; D002478:Cells, Cultured; D003198:Computer Simulation; D015638:Cytochalasin D / Q000494:pharmacology; D004622:Embryo, Mammalian; D015870:Gene Expression / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009046:Motor Neurons / Q000378:metabolism*; D009414:Nerve Growth Factors / Q000378:metabolism; D019006:Neural Cell Adhesion Molecules / Q000378:metabolism; D016900:Neurofilament Proteins / Q000378:metabolism; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D018408:Patch-Clamp Techniques / Q000379:methods; D034622:RNA Interference / Q000502:physiology; D016337:RNA, Catalytic / Q000378:metabolism; D051381:Rats; D015222:Sodium Channels / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D013116:Spinal Cord / Q000166:cytology*; D013997:Time Factors; D014162:Transfection
C490614:Ank3 protein, rat; D017487:Ankyrins; D015815:Cell Adhesion Molecules; D009414:Nerve Growth Factors; D019006:Neural Cell Adhesion Molecules; D016900:Neurofilament Proteins; C504594:Nfasc protein, rat; D019384:Nucleic Acid Synthesis Inhibitors; D016337:RNA, Catalytic; D015222:Sodium Channels; C418481:hairpin ribozyme; D049452:Green Fluorescent Proteins; D015638:Cytochalasin D