Regulation of the dopamine transporter - Addiction Research ...

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Schmitt & Reith DAT Regulation DAT regulation have yet to be elucidated, we will attempt to highlight instances where DATaffecting drugs may directly influence intracellular signaling cascades (which can, in turn, affect DAT regulation). We will also briefly discuss the involvement of presynaptic GPCRs and transporterassociated scaffolding proteins in DAT regulation. Finally, the possible implications of transporter protein regulation to the putative toxicity of several substituted amphetamine derivatives will be discussed. Direct regulation of transporter function by DAT ligands Effects of transporter substrates Substrates that are actively translocated by the DAT (other than dopamine itself) include endogenous trace amines, such as tyramine and �phenethylamine, the neurotoxic mitochondrial poison 1-methyl-4-phenylpyridinium (MPP + ), and the amphetamines, such as the clinically used stimulant d-amphetamine and its more potent congener d-methamphetamine, a highly-abused addictive drug. 20 Extracellular substrates can produce either transient upregulation or significant downregulation of DAT activity, depending on the duration of substrate exposure. Evidence that substrates for the DAT can induce rapid alterations in transporter activity was first obtained in striatal synaptosomes prepared from rats 1 h after treatment with high-dose d-methamphetamine (15 mg/kg of body weight). At this dose, acute methamphetamine administration resulted in a 65% decrease in synaptosomal [ 3H]dopamine uptake compared with vehicle. 21 Further analysis showed that this downregulation is transient—it was observed in synaptosomes prepared 1 h after, but not 24 h after, methamphetamine treatment—and is due to a treatment-associated drop in transport velocity (V max), not an alteration in the transporter’s affinity (Km) for dopamine and is not associated with changes in overall DAT protein concentration. 22 These data are consistent with the hypothesis that methamphetamine rapidly and transiently diminishes the activity of the DAT at the cell surface; however, they do not distinguish between a trafficking-dependent mechanism (internalization of plasmalemmal transporters) and a posttranslational regulatory effect at the individual transporter level. Support for a trafficking-dependent mechanism has come from further research employing fluorescent microscopy to visualize tagged human DAT (hDAT) fusion proteins expressed in cultured cells. For example, Saunders et al. demonstrated that exposing HEK293 cells transfected with fluorescenttagged hDAT to d-amphetamine triggers migration of the fluorescent DAT from the cell surface to the intracellular compartment, with visible DAT accumulation in punctate intracellular vesicles. 23 In line with the previous findings for methamphetamine, preincubation of the HEK-hDAT cells with amphetamine for 1 h significantly reduced cellular [ 3 H]dopamine uptake, affecting the V max transport parameter but not the Km for dopamine. Amphetamine-induced transporter internalization was apparent after as little as 20 min (maximal at 1-h exposure time) and was found to be dependent on internalization of the DAT by clathrin-coated vesicles, as coexpression of a dominant-negative form of dynamin I—a GTPase responsible for cleaving nascent clathrin-coated vesicles budding from the plasma membrane—in the HEK-hDAT cells prevented the internalizing effect of amphetamine. 23 The authors also noted that dopamine itself causes a similar redistribution of plasmalemmal DAT to intracellular vesicles (albeit at a higher dosage than amphetamine), implying that regulation of surface DAT expression is a general effect of substratelike compounds. Extending this qualitative observation, Chi and Reith found that pretreatment with dopamine for 1 h decreases surface DAT expression by approximately 30% in both HEK-hDAT cells and rat striatal synaptosomes, an effect that cleavable biotinylation assays suggest is due to enhanced endocytosis of the transporter protein. 24 Further corroboration of increased internalization and sequestration of surface-localized transporters in response to substrates is provided by the fluorescent microscopy studies of Sorkina et al., who visualized amphetamine-mediated DAT endocytosis in living cells and later demonstrated that internalized DATs colocalize with well-characterized markers of early and recycling endosomes, such as Rab5 and EEA1. 25,26 In what quaternary form is DAT internalized by the action of amphetamine? Is there a relationship between the oligomerization of DAT and its internalization? We found that in hDAT-expressing HEK- 293 cells, d-amphetamine shifted the distribution of Ann. N.Y. Acad. Sci. 1187 (2010) 316–340 c○ 2010 New York Academy of Sciences. 319
DAT Regulation Schmitt & Reith surface DAT toward a smaller ratio of oligomers to monomers in what appears to reflect a dissociation of DAT oligomers. 27 Along with the reduction of the fraction of oligomerized DAT, the amount of surface DAT was decreased, and blocking endocytosis with phenylarsine oxide or sucrose counteracted these effects of amphetamine. 27 We speculated that DAT at the cell surface is distributed between oligomers and monomers and that monomers are internalized; d-amphetamine could promote the formation of monomers, which then become internalized, reducing the DAT’s presence at the surface. When endocytosisispreventedbyanexogenouslyaddedblocker, monomeric DAT formed by amphetamine remains at the surface and reassociates to form oligomers. In this scenario, oligomerization and internalization are linked in the effects of amphetamine. One can grasp the functional relevance of a changed distribution between oligomerized and monomeric DAT at the surface only once the functional properties of these forms of the transporter are known. Substrate-mediated downregulation of surface DAT expression has also been demonstrated with electrophysiological techniques. In heterologous expression systems, DAT function is assessed by measuring transporter-associated currents with twoelectrode voltage clamp recording, whereas in vivo, microamperometry is used to electrochemically monitor dopamine clearance in real-time. In hDATexpressing Xenopus oocytes, brief repeated bath perfusion with the substrates dopamine, amphetamine and tyramine (1 min of substrate exposure every 5 min, for 1 h in total) gradually reduces DAT function, as shown by progressive decline of substrate translocation-associated currents. 28 Of the three substrates, amphetamine was found to be the most efficacious, producing a significant drop in current magnitude after the fewest repeat perfusions. This gradual decline in DAT function is accompanied by a decrease in specific [ 3 H]CFT binding (Bmax) to intact oocytes, indicating that the reduction in transporter currents originates from loss of surface-localized DATs rather than modification of the electrophysiological parameters of individual transporters. Kahlig et al. provided further substantiation of this finding—using a combination of fluorescence microscopy and patch-clamp recording, the authors demonstrated that amphetamine-induced internalization of the DAT is not paralleled by alteration of single- transporter current dynamics, reinforcing the idea that substrate-mediated DAT regulation is the result of endocytosis of plasmalemmal DAT. 29 In line with the findings in heterologous cells, recurrent infusion of dopamine in vivo by reverse microdialysis (one infusion every 2 min) results in a robust reduction of dopamine clearance in rat striatum; however, no significant change was observed in the nucleus accumbens. 28 This observation implies that DAT regulation might be an anatomically specific phenomenon, differing between brain regions. A similar anatomical discrepancy has been shown for d-methamphetamine, which decreases [ 3 H]dopamine uptake in striatal synaptosomes but not in those prepared from the nucleus accumbens. 22 A recent study by Richards and Zahniser also highlights the difference between the striatum and the nucleus accumbens in terms of substrateinduced DAT regulation: brief (15 min) preincubation with 20 �M amphetamine reduces the V max of [ 3 H]dopamine uptake in rat striatal synaptosomes but does not significantly alter the V max in synaptosomes from the nucleus accumbens. 30 Curiously, however, the authors found that synaptosomes from both regions were significantly affected if prepared from rats treated systemically with 2-mg/kg amphetamine for 45 min. Further inquiry is needed to delineate potential region-specific differences in DAT regulation and between in vivo and in vitro approaches. Many studies have now conclusively shown that several substrates, such as dopamine, damphetamine, and d-methamphetamine, can trigger cytosolic redistribution of plasmalemmal DAT. Nearly all these studies used relatively long substrate exposure times—anywhere from 15 min to a few hours. Far less is known about the immediate—on a scale of seconds to minutes—effects of substrates on DAT trafficking. A study from the Gnegy lab 31 suggests that substrates, such as amphetamine, may have time-dependent biphasic effects on DAT membrane trafficking. Using confocal microscopy, reversible biotinylation, and intact-cell [ 3 H]CFT (2�-carbomethoxy-3�-(4-fluorophenyl)tropane) binding, the authors demonstrated that acute exposure to 3 �M d-amphetamine causes an extremely rapid upregulation in plasmalemmal DAT expression: surface DAT levels were significantly increased (on the order of 60–70%) within 30 s of amphetamine treatment and remained elevated for 320 Ann. N.Y. Acad. Sci. 1187 (2010) 316–340 c○ 2010 New York Academy of Sciences.
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