Abstract
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. DAT and its trafficking was visualized in cultured live midbrain dopaminergic (DA) neurons using novel fluorescently tagged cocaine analogs. The ligands enabled specific labelling and visualization of DAT in live neurons. In the DA neurons, DAT was uniformly distributed in the plasma membrane of the soma, the neuronal extensions, and varicosities along these extensions. Moreover, DAT was constitutively internalized into vesicular structures and the internalization was blocked by lentiviralmediated expression of dominant-negative dynamin. 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. To further characterize the intracellular pool of constitutively endocytosed DAT several strategies were employed in both cell lines and cultured DA neurons. First, to investigate the constitutive trafficking of heterologously expressed DAT we fused the N-terminus of DAT to the intracellular tail of the single-membrane spanning protein Tac, thereby creating an extracellular antibody epitope. Upon expression in HEK293 cells this TacDAT fusion protein displayed functional properties similar to the wild type transporter. In an ELISA based internalization assay, TacDAT intracellular accumulation was increased by inhibitors of lysosomal degradation and moreover TacDAT colocalized with the late endosomal marker Rab7. In the DA cell line 1Rb3An27 TacDAT also co-localized with EGFP-Rab7 and not with the recycling endosomal marker EGFP-Rab11. To assess whether sorting to late endosomes/lysosomes was a property also inherent to natively expressed transporter, DAT was visualized directly in cultured DA neurons using the fluorescent cocaine analog JHC 1-64. These data showed pronounced colocalization upon constitutive internalization with Lysotracker, a late endosomal/lysosomal marker; however only little cololization was observed with Alexa488-conjugated transferrin. Additionally, when expressing EGFP-Rab7 with lentivirus in cultured DA neurons a marked co-localization between JHC 1-64 and EGFP-Rab7 was observed. The data suggest that constitutively internalized DAT primarily is targeted to late endosomes and lysosomes both in heterologous cells and in cultured DA neurons.
DAT has been shown to be regulated by the dopamine D2 receptor (D2R), the primary target foranti-psychotics, through a direct interaction. D2R is among other places expressed as an autoreceptor in DA neurons. Transient over-expression of DAT with D2R in HEK293 cells reduced surface expression of D2R while DAT surface levels remained unaffected. Furthermore ß-Arrestin2 recruitment to D2R was compromised and D2R-mediated inhibition of cAMP accumulation was reduced as a consequence of the reduced surface level expression of D2R. The effects of DAT on D2R surface expression and signalling was not unique for DAT and D2R. Thus, transient coexpression of ß2AR with DAT resulted in reduced surface levels of ß2AR. In addition, coexpression of the serotonin transporter with D2R appeared to modulate the ß-Arrestin2 recruitment to D2R and the D2R-mediated inhibition of cAMP accumulation in a manner similar to coexpression of DAT. The data suggest that DAT affect D2R function and expression with no apparent specificity.
| Original language | English |
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| Number of pages | 196 |
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| Publication status | Published - 2010 |