title: Dopamine and amphetamine rapidly increase dopamine transporter trafficking to the surface: live-cell imaging using total internal reflection fluorescence microscopy.
abstract: Rapid treatment (1 min) of rat striatal synaptosomes with low-dose amphetamine increases surface expression of the dopamine transporter (DAT). Using mouse neuroblastoma N2A cells, stably transfected with green fluorescent protein-DAT, we demonstrate the real-time substrate-induced rapid trafficking of DAT to the plasma membrane using total internal reflection fluorescence microscopy (TIRFM). Both the physiological substrate, dopamine, and amphetamine began to increase surface DAT within 10 s of drug addition and steadily increased surface DAT until removal 2 min later. The substrate-induced rise in surface DAT was dose-dependent, was blocked by cocaine, and abated after drug removal. Although individual vesicle fusion was not visually detectable, exocytosis of DAT was blocked using both tetanus neurotoxin and botulinum neurotoxin C to cleave soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Notably, the dopamine-induced increase in surface DAT was cocaine-sensitive but D(2)-receptor independent. TIRFM data were confirmed in human DAT-N2A cells using biotinylation, and similar effects were detected in rat striatal synaptosomes. A specific inhibitor of protein kinase C-beta blocked the substrate-mediated increase in surface DAT in both DAT-N2A cells and rat striatal synaptosomes. These data demonstrate that the physiological substrate, dopamine, and amphetamine rapidly increase the trafficking of DAT to the surface by a mechanism dependent on SNARE proteins and protein kinase C-beta but independent of dopamine D(2) receptor activation. Importantly, this study suggests that the reuptake system is poised to rapidly increase its function during dopamine secretion to tightly regulate dopaminergic neurotransmission.
authors.affiliation: Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109, USA.
authors.forename: Cheryse A
authors.initials: CA
authors.lastname: Furman
authors.forename: Rong
authors.initials: R
authors.lastname: Chen
authors.forename: Bipasha
authors.initials: B
authors.lastname: Guptaroy
authors.forename: Minjia
authors.initials: M
authors.lastname: Zhang
authors.forename: Ronald W
authors.initials: RW
authors.lastname: Holz
authors.forename: Margaret
authors.initials: M
authors.lastname: Gnegy
mesh_terms: D000661:Amphetamine / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D004298:Dopamine / Q000378:metabolism* / Q000494:pharmacology; D050483:Dopamine Plasma Membrane Transport Proteins / Q000819:agonists / Q000378:metabolism*; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D008857:Microscopy, Interference / Q000379:methods*; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D051381:Rats
chemical_list: D050483:Dopamine Plasma Membrane Transport Proteins; D000661:Amphetamine; D004298:Dopamine