Visualization of dopamine transporter trafficking in live neurons by use of fluorescent cocaine analogs.
The dopamine transporter (DAT) mediates reuptake of dopamine from the synaptic cleft and is a target for widely abused psychostimulants such as cocaine and amphetamine. Nonetheless, little is known about the cellular distribution and trafficking of natively expressed DAT. Here we use novel fluorescently tagged cocaine analogs to visualize DAT and DAT trafficking in cultured live midbrain dopaminergic neurons. The fluorescent tags were extended from the tropane N-position of 2beta-carbomethoxy-3beta-(3,4-dichlorophenyl)tropane using an ethylamino-linker. The rhodamine-, OR Green-, or Cy3-labeled ligands had high binding affinity for DAT and enabled specific labeling of DAT in live neurons and visualization by confocal imaging. In the dopaminergic neurons, DAT was uniformly distributed in the plasma membrane of the soma, the neuronal extensions, and varicosities along these extensions. FRAP (fluorescence recovery after photobleaching) experiments demonstrated bidirectional movement of DAT in the extensions and indicated that DAT is highly mobile both in the extensions and in the varicosities (immobile fraction less than approximately 30%). DAT was constitutively internalized into vesicular structures likely representing intracellular transporter pools. The internalization was blocked by lentiviral-mediated expression of dominant-negative dynamin and internalized DAT displayed partial colocalization with the early endosomal marker EGFP-Rab5 and with the transferrin receptor. DAT internalization and function was not affected by activation of protein kinase C (PKC) with phorbol-12-myristate-13-acetate (PMA) or by inhibition with staurosporine or GF109203X. These data are in contrast to findings for DAT in transfected heterologous cells and challenge the paradigm that trafficking and cellular distribution of endogenous DAT is subject to regulation by PKC.
Eriksen
Jacob
J
Molecular Neuropharmacology Group and Center for Pharmacogenomics, Department of Neuroscience and Pharmacology, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark.
Rasmussen
Søren G F
SG
Rasmussen
Trine Nygaard
TN
Vaegter
Christian Bjerggaard
CB
Cha
Joo Hwan
JH
Zou
Mu-Fa
MF
Newman
Amy Hauck
AH
Gether
Ulrik
U
D000409:Alanine / Q000235:genetics; D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D003042:Cocaine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D004298:Dopamine / Q000494:pharmacology; D050483:Dopamine Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D018765:Dopamine Uptake Inhibitors / Q000737:chemistry / Q000378:metabolism*; D034281:Dynamins / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008239:Lysine / Q000235:genetics; D008301:Maleimides / Q000494:pharmacology; D008636:Mesencephalon / Q000166:cytology; D009154:Mutation / Q000502:physiology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D010703:Phorbol Esters / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D051381:Rats; D011990:Receptors, Transferrin / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014162:Transfection / Q000379:methods; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism; D050493:Vesicular Monoamine Transport Proteins / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism
D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors; D004791:Enzyme Inhibitors; D007211:Indoles; D008301:Maleimides; D010703:Phorbol Esters; D011990:Receptors, Transferrin; C493452:SLC18A2 protein, human; D050493:Vesicular Monoamine Transport Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C021279:(1R-(exo,exo))-3-(4-fluorophenyl)-8-methyl-8- azabicyclo(3.2.1)octane-2-carboxylic acid, methyl ester; C032974:12-O-retinoylphorbol-13-acetate; D014446:Tyrosine 3-Monooxygenase; D020696:rab5 GTP-Binding Proteins; D034281:Dynamins; D003042:Cocaine; D008239:Lysine; C070515:bisindolylmaleimide I; D000409:Alanine; D004298:Dopamine