PT  - JOURNAL ARTICLE
AU  - Jacob Eriksen
AU  - Søren G. F. Rasmussen
AU  - Trine Nygaard Rasmussen
AU  - Christian Bjerggaard Vaegter
AU  - Joo Hwan Cha
AU  - Mu-Fa Zou
AU  - Amy Hauck Newman
AU  - Ulrik Gether
TI  - Visualization of Dopamine Transporter Trafficking in Live Neurons by Use of Fluorescent Cocaine Analogs
AID  - 10.1523/JNEUROSCI.4177-08.2009
DP  - 2009 May 27
TA  - The Journal of Neuroscience
PG  - 6794--6808
VI  - 29
IP  - 21
4099  - http://www.jneurosci.org/content/29/21/6794.short
4100  - http://www.jneurosci.org/content/29/21/6794.full
SO  - J. Neurosci.2009 May 27; 29
AB  - 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 2β-carbomethoxy-3β-(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 ∼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.