Amphetamine (AMPH) elicits its behavioral effects by acting on the dopamine (DA) transporter (DAT) to induce DA efflux into the synaptic cleft. We previously demonstrated that a human DAT construct in which the first 22 amino acids were truncated was not phosphorylated by activation of protein kinase C, in contrast to wild-type (WT) DAT, which was phosphorylated. Nonetheless, in all functions tested to date, which include uptake, inhibitor binding, oligomerization, and redistribution away from the cell surface in response to protein kinase C activation, the truncated DAT was indistinguishable from the full-length WT DAT. Here, however, we show that in HEK-293 cells stably expressing an N-terminal-truncated DAT (del-22 DAT), AMPH-induced DA efflux is reduced by approximately 80%, whether measured by superfusion of a population of cells or by amperometry combined with the patch-clamp technique in the whole cell configuration. We further demonstrate in a full-length DAT construct that simultaneous mutation of the five N-terminal serine residues to alanine (S/A) produces the same phenotype as del-22—normal uptake but dramatically impaired efflux. In contrast, simultaneous mutation of these same five serines to aspartate (S/D) to simulate phosphorylation results in normal AMPH-induced DA efflux and uptake. In the S/A background, the single mutation to Asp of residue 7 or residue 12 restored a significant fraction of WT efflux, whereas mutation to Asp of residues 2, 4, or 13 was without significant effect on efflux. We propose that phosphorylation of one or more serines in the N-terminus of human DAT, most likely Ser7 or Ser12, is essential for AMPH-induced DAT-mediated DA efflux. Quite surprisingly, N-terminal phosphorylation shifts DAT from a “reluctant” state to a “willing” state for AMPH-induced DA efflux, without affecting inward transport. These data raise the therapeutic possibility of interfering selectively with AMPH-induced DA efflux without altering physiological DA uptake.

Evidence suggests that protein kinase C (PKC) and intracellular calcium are important for amphetaminestimulated outward transport of dopamine in rat striatum. In this study, we examined the effect of select PKC isoforms on amphetamine-stimulated dopamine efflux, focusing on Ca 2؉ -dependent forms of PKC. Efflux of endogenous dopamine was measured in superfused rat striatal slices; dopamine was measured by high performance liquid chromatography. The non-selective classical PKC inhibitor Gö 6976 inhibited amphetamine-stimulated dopamine efflux, whereas rottlerin, a specific inhibitor of PKC␦, had no effect. A highly specific PKC␤ inhibitor, LY379196, blocked dopamine efflux that was stimulated by either amphetamine or the PKC activator, 12-O-tetradecanoylphorbol-13-acetate. None of the PKC inhibitors significantly altered [3 H]dopamine uptake. PKC␤ I and PKC␤ II , but not PKC␣ or PKC␥, were coimmunoprecipitated from rat striatal membranes with the dopamine transporter (DAT). Conversely, antisera to PKC␤ I and PKC␤ II but not PKC␣ or PKC␥ were able to co-immunoprecipitate DAT. Amphetamine-stimulated dopamine efflux was significantly enhanced in hDAT-HEK 293 cells transfected with PKC␤ II as compared with hDAT-HEK 293 cells alone, or hDAT-HEK 293 cells transfected with PKC␣ or PKC␤ I . These results suggest that classical PKC␤ II is physically associated with DAT and is important in maintaining the amphetamine-stimulated outward transport of dopamine in rat striatum.

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Rapid treatment (1 min) of rat striatal synaptosomes with low-dose amphetamine increases surface expression of the dopamine transporter (DAT). Using mouse neuroblastoma N2A cells, stably transfected with green fluorescent protein-DAT, we demonstrate the realtime substrate-induced rapid trafficking of DAT to the plasma membrane using total internal reflection fluorescence microscopy (TIRFM). Both the physiological substrate, dopamine, and amphetamine began to increase surface DAT within 10 s of drug addition and steadily increased surface DAT until removal 2 min later. The substrate-induced rise in surface DAT was dose-dependent, was blocked by cocaine, and abated after drug removal. Although individual vesicle fusion was not visually detectable, exocytosis of DAT was blocked using both tetanus neurotoxin and botulinum neurotoxin C to cleave soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Notably, the dopamine-induced increase in surface DAT was cocaine-sensitive but D 2 -receptor independent. TIRFM data were confirmed in human DAT-N2A cells using biotinylation, and similar effects were detected in rat striatal synaptosomes. A specific inhibitor of protein kinase C-␤ blocked the substrate-mediated increase in surface DAT in both DAT-N2A cells and rat striatal synaptosomes. These data demonstrate that the physiological substrate, dopamine, and amphetamine rapidly increase the trafficking of DAT to the surface by a mechanism dependent on SNARE proteins and protein kinase C-␤ but independent of dopamine D 2 receptor activation. Importantly, this study suggests that the reuptake system is poised to rapidly increase its function during dopamine secretion to tightly regulate dopaminergic neurotransmission.

The human dopamine transporter (hDAT) regulates synaptic dopamine (DA) levels and is the site of action of abused and therapeutic drugs. Here we study the effect of a threonine residue (Thr62 in hDAT) that is highly conserved within a canonical phosphorylation site (RETW) in the juxtamembrane Nterminal region of monoamine transporters. In stably transfected human embryonic kidney 293T cells, expression of T62D-hDAT was reduced compared with hDAT or T62A-hDAT. T62D-hDAT displayed dramatically reduced [ 3 H]dopamine uptake but exhibited a higher basal dopamine efflux compared with hDAT or T62A-hDAT, as determined by measurements of can rescue reduced DA uptake in mutant transporters that are predominantly inward-facing, micromolar concentrations of Zn 2ϩ markedly potentiated [ 3 H]dopamine uptake in T62D-hDAT and permitted the measurement of amphetamine-stimulated dopamine efflux. These results suggest that T62D-hDAT prefers an inward-facing conformation in the transition between inward-and outward-facing conformations. For T62A-hDAT, however, the measured 50% reduction in both [ 3 H]dopamine uptake and [ 3 H]dopamine efflux was consistent with a slowed transition between inward-and outward-facing conformations. The mechanism underlying the important functional role of Thr62 in hDAT activity suggested by these findings is examined in a structural context using dynamic simulations of a threedimensional molecular model of DAT.The strength and duration of dopaminergic neurotransmission is tightly controlled by the dopamine transporter (DAT), which mediates reuptake of synaptic dopamine (DA) (Chen and Reith, 2000) and is a target for therapeutic drugs and abused psychostimulants (Sulzer et al., 2005). Amphetamine (AMPH), a substrate for DAT, competitively inhibits DA reuptake and elicits outward transport of DA by reversal of the transporter (Sulzer et al., 2005), purportedly by an exchange diffusion mechanism (Fischer and Cho, 1979;Sulzer et al., 2005). This model assumes that the transporter transitions between two primary conformational states-an "outwardfacing" conformation favoring substrate binding and influx, and an "inward-facing" conformation in which the binding sites are available to the intracellular milieu and which favors substrate efflux. In the absence of substrate, the transporter favors an outward-facing conformation ready to bind

Cues (conditioned stimuli; CSs) associated with rewards can come to motivate behavior, but there is considerable individual variation in their ability to do so. For example, a lever-CS that predicts food reward becomes attractive, wanted, and elicits reward-seeking behavior to a greater extent in some rats (“sign-trackers”; STs), than others (“goal-trackers”; GTs). Variation in dopamine (DA) neurotransmission in the nucleus accumbens (NAc) core is thought to contribute to such individual variation. Given that the DA transporter (DAT) exerts powerful regulation over DA signaling, we characterized the expression and function of the DAT in the accumbens of STs and GTs. STs showed greater DAT surface expression in ventral striatal synaptosomes than GTs, and ex vivo fast-scan cyclic voltammetry recordings of electrically-evoked DA release confirmed enhanced DAT function in STs, as indicated by faster DA uptake, specifically in the NAc core. Consistent with this, systemic amphetamine (AMPH) produced greater inhibition of DA uptake in STs than in GTs. Furthermore, injection of AMPH directly into the NAc core enhanced lever-directed approach in STs, presumably by amplifying the incentive value of the CS, but had no effect on goal tracking behavior. On the other hand, there were no differences between STs and GTs in electrically-evoked DA release in slices, or in total ventral striatal DA content. We conclude that greater DAT surface expression may facilitate the attribution of incentive salience to discrete reward cues. Investigating this variability in animal sub-populations may help explain why some people abuse drugs, while others do not.

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Repeated intermittent amphetamine enhances efflux of dopamine through the dopamine transporter in rat basal ganglia and through the norepinephrine transporter in rat pheochromocytoma PC12 cells. Extracellular Ca 2ϩ is required for the detection of this enhancement in the rat. In this study, we examined the role of Ca 2ϩ and Ca 2ϩ channels in the enhanced amphetamine-induced dopamine efflux that develops in PC12 cells following repeated intermittent amphetamine. Repeated pretreatment of PC12 cells with 1 M amphetamine followed by a drug-free period increased amphetamine-induced efflux of dopamine compared with controls. The enhancement in amphetamine-induced dopamine efflux depended upon the presence of extracellular Ca 2ϩ and was inhibited by the blockade of N-type and L-type Ca 2ϩ channels. The enhanced dopamine efflux was not altered by tetanus toxin or reserpine, treatments that abrogate synaptic vesicle-mediated, exocytotic dopamine efflux. Measurement of intracellular Ca 2ϩ concentrations using fura-2/acetoxymethyl ester revealed that amphetamine increased intracellular Ca 2ϩ by a transporter-dependent mechanism. In amphetamine-pretreated cells, amphetamine elicited a greater increase in intracellular Ca 2ϩ ; this increase depended upon the presence of extracellular Ca 2ϩ and N-and L-type Ca 2ϩ channel activity. The enhanced amphetamine-induced dopamine efflux requires Ca 2ϩ /calmodulin kinase activity. In vehicle-treated cells, 1 M amphetamine inhibited the calmodulin kinase activity although it did not in amphetamine-pretreated cells. This study suggests that repeated intermittent amphetamine couples norepinephrine transporter activity and Ca 2ϩ signaling.