Regulation of the dopamine transporter - Addiction Research ...

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Schmitt & Reith DAT Regulation around 1 min, before quickly dropping back to baseline levels within 3 min. In accordance with most substrate-induced trafficking studies, DAT surface expression began to drop below vehicle levels after 20 min of amphetamine exposure. Curiously, dopamine itself was not found to induce the same immediate upregulation of surface DAT expression as amphetamine, indicating that this biphasic trafficking pattern might not extend to all DAT substrates and may contribute to amphetamine’s ability to stimulate dopamine efflux via the DAT. 31 However, a more recent study from this group— using total internal reflection fluorescent microscopy to visualize hDAT trafficking dynamics in N2Aneuroblastomacellswithreal-timetemporal resolution—shows evidence that both dopamine (10 �M) and d-amphetamine (5 �M) induce changes in surface DAT expression within seconds, but the effect of amphetamine persisted longer after substrate washout. 32 Effects of DAT inhibitors Compared with the robust literature on DAT regulation by phenethylamine substrates, there is a paucity of studies investigating the acute regulatory effects of DAT inhibitors. The few in vitro regulatory studies using heterologous cell systems, as well as the bulk of in vivo and clinical studies, have focused solely on the classical DAT inhibitor cocaine, doubtless a result of its notorious reputation as one of the most addictive compounds known to humans. 33 Atypical DAT inhibitors lacking cocaine-like abuse potential 34,35 remain almost entirely uninvestigated; thus, it is not yet possible to make global statements regarding the regulatory effects of inhibitors other than cocaine. However, at least for cocaine, it appears that nontranslocated DAT inhibitors not only block the acute regulatory effects of substrates, they may exert the opposite effect: insertion of DATs from the endosomic recycling pool into the plasma membrane. 16,36 Chronic administration of cocaine upregulates striatal DAT expression in rhesus monkeys, an effect that persists for more than 30 days after cocaine withdrawal. 37 Increased DAT expression has also been shown in postmortem analyses of brain tissue from human cocaine addicts 38 and synaptosomes prepared from this tissue exhibit greater [ 3 H]dopamine uptake than synaptosomes from age-matched cocaine-naïve individuals. 39 In addition, cyclic voltammetry studies of rats that exhibit a self-administration preference for high doses of cocaine reveal a specific increase in the V max of dopamine uptake with no effect on the Km for dopamine, suggesting an upregulation of DAT expression. 40 Interestingly, these results are exactly inverted compared with the findings for d-methamphetamine, indicating that the two psychostimulants possess opposing effects on DAT trafficking, despite their comparably high abuse potentials. Different effects on plasmalemmal DAT expression may play a role in the observed efficacy of d-amphetamine for the treatment of cocaine dependence in preclinical and early clinical trials. 41–44 In studies of hDAT-expressing cultured cells, preincubation with cocaine has been routinely demonstrated to prevent the DAT-internalizing effects of substrates (e.g., Refs. 23, 24, and 45), as well as the putative amphetamine-induced brief, transient upregulation of surface DAT levels. 31,32 The acute effects of cocaine on DAT trafficking in the absence of substrates are less consistent: whereas some research groups have shown that cocaine elicits a rapid increase in plasmalemmal DAT expression, 46,47 others have found no effect of cocaine pretreatment on DAT activity or surface expression. 24,48 In the study by Little et al., treatment of N2A-hDAT cells with 1 �M cocaine for 24 h increased cell surface DAT expression by approximately 30%. 47 Remarkably, this same magnitude of increase was detected using a wide array of techniques, including biotinylation, intact-cell [ 3 H]CFT binding, and visualization of plasma membrane anti-DAT immunofluorescence with confocal microscopy. 47 The long incubation time used in this study (24 h) may underlie some of the discrepancy with other reports showing no effect of cocaine, because most studies have used incubation times on the order of 30 min to 1 h—curiously, however, Daws et al.demonstrateda cocaine-mediated increase in surface expressed DAT in HEK-hDAT cells after only 1 h of treatment. 46 Cellular signaling cascades, phosphorylation, and DAT regulation DAT phosphorylation and trafficking Members of the neurotransmitter sodium symporter superfamily are large proteins—the hDAT possesses 620 amino acid residues—containing Ann. N.Y. Acad. Sci. 1187 (2010) 316–340 c○ 2010 New York Academy of Sciences. 321
DAT Regulation Schmitt & Reith many cytoplasmic-facing consensus sites for phosphorylation, many of which lie on the N terminus of the DAT protein (for a review of functional moieties in the DAT amino acid sequence, see Volz and Schenk 2 ). Protein phosphorylation is a de facto posttranslational strategy for regulating protein function, altering protein–protein interactions and transducing exogenous stimuli. Because phosphorylation of a particular residue in a protein can both drastically change its three-dimensional shape and serve as a signaling marker, is not surprising that the phosphorylation state of the DAT has a profound influence on both the intrinsic activity (i.e., affinity for and responsiveness to various ligands) and membrane distribution of the transporter. 4 Several protein kinases and phosphatases involved in key signaling cascades can affect DAT function, including members of the protein kinase C (PKC), protein kinase A, phosphatidylinositol-3-kinase (PI3K), protein tyrosine kinase, Ca 2+ /calmodulin kinase, protein phosphatase 1, and mitogen-activated protein kinase (MAPK) families. 49–52 Of these, PKC has been the most thoroughly investigated; however, a direct phosphotransferase reaction between PKC and the DAT has not been demonstrated, suggesting that other (currently undefined) downstream kinases may be responsible for the direct phosphorylation of the DAT. 4,51 In addition to possible direct effects on the DAT and on DAT membrane trafficking, activation (or, by the same token, inhibition) of these phosphorylation pathways can affect the regulatory and transmitter-releasing action of substrates, such as amphetamine. As we discuss in the following, both substrate exposure and PKC activation can result in DAT downregulation; however, despite a myriad of studies demonstrating the importance of protein kinases and phosphatases in substratemediated regulation of plasmalemmal DAT expression, explicit details regarding the mechanistic link between the two processes have been elusive. 50 In contrast with substrates, classical DAT inhibitors, such as cocaine, �-CFT, and methylphenidate, have little effect on DAT phosphorylation. 48 Protein kinase C Functional regulation of the DAT in response to PKC activation was first demonstrated in vitro by using phorbol-ester kinase modulators, such as 4�phorbol-12-myristate-13-acetate (�-PMA). Incubation with �-PMA (or other nonphorbol PKC activators) dramatically increases levels of 32 PO4labeled immunoprecipitated DAT in both synaptosomes 53 and cells heterologously expressing DAT. 54 Increased phosphorylation was not observed in the presence of the inactive phorbol analogue 4�phorbol-12,13-didecanoate, and all effects of �- PMA were abolished by cotreatment with the PKC inhibitor bisindoylmaleimide I, highlighting the involvement of PKC. 53 Analogous to the findings with DAT substrates, pretreatment with PKC activators acutely and rapidly reduces [ 3 H]dopamine uptake by lowering the V max for uptake without affecting the Km valueandreducestheBmax of [ 3 H]mazindol binding to intact Xenopus oocytes, 55 suggesting a role for phosphorylation in promoting the internalization of plasmalemmal DAT. A follow-up study by Melikian and Buckley substantiated this hypothesis: the authors found that induction of PKC with �-PMA results in intracellular redistribution of the DAT from the plasma membrane to recycling endosomal compartments in hDAT-expressing PC12 cells. 56 Endocytic trafficking of the DAT to early and recycling endosomes in response to PKC activation has since been demonstrated in many different cell models and has been visualized in live cells, using various fluorescent-tagged DATs. 25,57 PKCmediated loss of plasmalemmal DAT is probably due to a combination of increased clathrin-dependent endocytosis and decreased recycling from endosomal compartments. 58 Proteolytic digestion studies of 32 PO4-labeled DAT indicate that serine residues in the N terminus are major target sites of phosphorylation after exogenous pharmacological PKC activation. 59 In both constitutive and PKC-mediated DAT internalization, an endocytic motif has been shown to be required in the DAT C terminus, that is, residues 587–596; the results do not rule out the possibility that other DAT sequences play a role in the interaction between the transporter and the endocytic machinery, with such sequences being sensitive to the local environment within the 587–596 motif. 60 Essential elements of the DAT C-terminal endocytic motif are conserved within the SLC6 transporter family but not in any other protein, indicating that DAT endocytosis may involve a clathrinindependent component. 60 Recent studies by the group of Vaughan 61 does not indicate clathrinindependent endocytosis of DAT, but they do suggest a role for cholesterol-sensitive raftassociated DAT that responds to PKC with reduced 322 Ann. N.Y. Acad. Sci. 1187 (2010) 316–340 c○ 2010 New York Academy of Sciences.
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