Substrate-induced internalization of the high-affinity choline transporter.
Cholinergic neurons are endowed with a high-affinity choline uptake system for efficient synthesis of acetylcholine at the presynaptic terminals. The high-affinity choline transporter CHT1 is responsible for choline uptake, the rate-limiting step in acetylcholine synthesis. However, endogenous physiological factors that affect CHT1 expression or function and consequently regulate the acetylcholine synthesis rate are essentially unknown. Here we demonstrate that extracellular substrate decreases the cell-surface expression of CHT1 in rat brain synaptosomes, primary cultures from the basal forebrain, and mammalian cell lines transfected with CHT1. Extracellular choline rapidly decreases cell-surface CHT1 expression by accelerating its internalization, a process that is mediated by a dynamin-dependent endocytosis pathway in HEK293 cells. Specific inhibitor hemicholinium-3 decreases the constitutive internalization rate and thereby increases cell-surface CHT1 expression. We also demonstrate that the constitutive internalization of CHT1 depends on extracellular pH in cultured cells. Our results collectively suggest that the internalization of CHT1 is induced by extracellular substrate, providing a novel feedback mechanism for the regulation of acetylcholine synthesis at the cholinergic presynaptic terminals.
Department of Pharmacology, Faculty of Pharmacy, Keio University, Minato-ku, Tokyo 105-8512, Japan. okuda-tk@pha.keio.ac.jp
Takashi
T
Okuda
Asami
A
Konishi
Hidemi
H
Misawa
Tatsuya
T
Haga
D000818:Animals; D000831:Animals, Newborn; D019921:Biotinylation / Q000379:methods; D002478:Cells, Cultured; D002794:Choline / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian; D005260:Female; D006426:Hemicholinium 3 / Q000493:pharmacokinetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D021381:Protein Transport / Q000187:drug effects / Q000235:genetics; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D020665:Septum of Brain / Q000166:cytology; D027981:Symporters / Q000235:genetics / Q000378:metabolism*; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism; D014162:Transfection; D014316:Tritium / Q000378:metabolism / Q000493:pharmacokinetics
D008024:Ligands; D034741:RNA, Small Interfering; C510731:SLC5A7 protein, human; D027981:Symporters; D014316:Tritium; D006426:Hemicholinium 3; D011189:Potassium Chloride; D002794:Choline