Regulation of the dopamine transporter - Addiction Research ...

ANNALS OF THE NEW YORK ACADEMY OF SCIENCES 

Issue: Addiction Reviews 2 

Regulation of the dopamine transporter 

Aspects relevant to psychostimulant drugs of abuse 

Kyle C. Schmitt 1 and Maarten E. A. Reith 1,2 

Ann. N.Y. Acad. Sci. ISSN 0077-8923 

1 2 Department of Pharmacology, New York University School of Medicine, New York, New York, USA. Department of 

Psychiatry, New York University School of Medicine, New York, New York, USA 

Address for correspondence: Maarten E. A. Reith, New York University School of Medicine, 550 First Ave., New York, NY 

10016. maarten.reith@med.nyu.edu 

Dopaminergic signaling in the brain is primarily modulated by dopamine transporters (DATs), which actively 

translocate extraneuronal dopamine back into dopaminergic neurons. Transporter proteins are highly dynamic, 

continuously trafficking between plasmalemmal and endosomal membranes. Changes in DAT membrane trafficking 

kinetics can rapidly regulate dopaminergic tone by altering the number of transporters present at the cell surface. 

Various psychostimulant DAT ligands—acting either as amphetamine-like substrates or cocaine-like nontranslocated 

inhibitors—affect transporter trafficking, triggering rapid insertion or removal of plasmalemmal DATs. In this 

review, we focus on the effects of psychostimulants of addiction (particularly d-methamphetamine and cocaine) 

on DAT regulation and membrane trafficking, with an emphasis on how these drugs may influence intracellular 

signaling cascades and transporter-associated scaffolding proteins to affect DAT regulation. In addition, we consider 

involvement of presynaptic receptors for dopamine and other ligands in DAT regulation. Finally, we discuss possible 

implications of transporter regulation to the putative toxicity of several substituted amphetamine derivatives 

commonly used as recreational drugs, as well as to the design of therapeutics for cocaine addiction. 

Keywords: dopamine transporter; trafficking; regulation; amphetaminergic substrates; internalization; cocaine; 

methamphetamine; MDMA 

Introduction 

The neuronal dopamine transporter (DAT)—a 

member of the neurotransmitter sodium symporter 

protein superfamily (the SLC6 gene family)— 

regulates dopaminergic neurotransmission in the 

brain by actively clearing extraneuronal dopamine, 

using the energy of cellular ionic electrochemical 

gradients. 1 Members of the neurotransmitter 

sodium symporter protein family are glycoproteins 

with 12 transmembrane-spanning domains, with 

both the amino and carboxyl termini located on 

the inner face of the membrane. 2 The DAT plays 

an integral role in cognition, affect, behavioral reinforcement, 

and motor function, and DAT pathologies 

are suspected to contribute to disorders, such as 

depression, attention deficit–hyperactivity disorder, 

anhedonia, Parkinson’s disease, and addiction. 2,3 

The DAT is a target of several clinically used drugs, 

such as the psychostimulants methylphenidate, d- 

amphetamine, and modafinil, as well as the antidepressant 

bupropion. In addition, the reinforcing and 

euphoric effects of the powerfully addictive psychostimulants 

cocaine and d-methamphetamine 

(“crystal meth,” a vastly more potent analogue of 

amphetamine, often administered in large doses by 

vaporization) are primarily mediated by interaction 

with the DAT. 4 Researchers in several disciplines 

have empirically demonstrated the integral nature of 

the DAT protein in both the acute and chronic effects 

of cocaine and in cocaine addiction (see, e.g., Mash 5 

for review). A major recent finding is that DATknockout 

mice show an attenuated response to cocaine 

and amphetamines and a reduced preference 

for cocaine under self-administration paradigms. 6 

These mice, however, still self-administer cocaine— 

although more sessions were needed to meet 

self-administration criteria 7 —indicating that developmentally 

compensatory nondopaminergic mechanisms 

can mediate cocaine-taking behavior in 

doi: 10.1111/j.1749-6632.2009.05148.x 

316 Ann. N.Y. Acad. Sci. 1187 (2010) 316–340 c○ 2010 New York Academy of Sciences.

Schmitt & Reith DAT Regulation 

DAT-lacking animals. In particular, cocaine’s interaction 

with the serotonin transporter (SERT) 

has been hypothesized to contribute to continued 

self-administration in DAT-knockout animals. 7,8 

Nevertheless, convincing preclinical evidence implicating 

specific activity at the DAT in cocaine dependence 

comes from the observation that the reinforcing 

effect of cocaine is lost in transgenic mice 

expressing a triple point–mutated DAT with preserved 

(albeit somewhat reduced) substrate translocation 

but little appreciable affinity for cocaine. 9 

It is conceivable that elevated basal dopaminergic 

tone in these mice (a result of their reducedfunction 

DAT isoform) causes adaptive changes, 

altering the response to cocaine; however, knockdown 

mutant mice with a ∼90% reduction in 

DAT expression—but with functionally unmodified 

DATs and elevated basal dopamine—still exhibit robust, 

wild-type–like preference for cocaine, indicating 

that interaction with the DAT is necessary for 

the reinforcing effects of cocaine in animals that 

carry the DAT. 10 Moreover, a recent investigation 

by Thomsen et al. showed that a different strain 

of DAT –/– (DAT knockout) mice generally fail to 

acquire intravenous cocaine self-administration behavior. 

11 Importantly, in the minority of mice that 

do initially show self-administration, this behavior 

is quickly extinguished as the response requirement 

(the “work” required to obtain a dose of cocaine) is 

increased. In contrast, both wild-type and SERT –/– 

mice maintain consistent self-administration, even 

when the response requirement for a single dose of 

intravenous cocaine is increased 50- to 100-fold. Unlike 

cocaine, other dopaminergic compounds (i.e., 

the dopamine D1-like receptor agonist SKF82958) 

and food rewards are comparably administered by 

DAT –/– and wild-type mice, indicating that elimination 

of the DAT does not globally hinder behavioral 

reinforcement. These data strongly suggest that cocaine 

is not a reliable reinforcer in the absence of the 

DAT, despite some intersubject differences in initial 

responses among DAT –/– mice. 11 

The DAT governs both the duration and magnitude 

of dopaminergic neurotransmission by actively 

translocating dopamine from the extracellular space 

into presynaptic neurons. Extracellular dopamine is 

also subject to enzymatic catabolism—thus terminating 

its signaling potential—however, studies of 

DAT-knockout mice 6,12 have demonstrated that the 

DAT is the chief arbiter of dopaminergic signal- 

ing flux (for review, see Gainetdinov and Caron 3 ). 

Dopaminergic transmission is volume transmission 

with an extrasynaptic predominance of dopamine 

receptors and transporters; the function of the DAT 

is not to remove dopamine from the synapse but 

rather to regulate dopamine’s action at extrasynaptic 

dopamine receptors (see Rice and Cragg 13 for 

review and highly recommended, updated cartoon 

of a dopamine synapse). The overall rate of de novo 

DAT protein synthesis is a relatively slow process— 

the half-life of striatal transporter proteins, for example, 

is approximately 2–3 days. 14,15 Hence, it is 

not surprising that cells use an assortment of posttranslational 

regulatory strategies to rapidly alter 

DAT function—without requiring de novo protein 

synthesis or transcriptional-level modifications—to 

dynamically shift the tone of dopaminergic activity 

in response to various endogenous and exogenous 

stimuli. There are two conceivable manners 

in which a cell could modulate DAT function “on 

the fly” (on a time scale of seconds to minutes) in 

response to changing environmental conditions— 

either via direct modification of single-transporter 

parameters, such as intrinsic substrate permeation 

efficacy, or by affecting the trafficking of the transporter, 

redistributing DATs between the plasma 

membrane and intracellular endosomal compartments. 

4 Experimental evidence has demonstrated 

that both processes may play some role in acute DAT 

regulation, but that redistribution of transporters 

to and from the cell surface is the predominant 

mechanism. 16,17 

This regulatory method—rapid insertion or removal 

of plasmalemmal transporter proteins—may 

seem like a cumbersome way of acutely controlling 

neurotransmitter signaling; however, it makes logistical 

sense when we consider the kinetics of the 

substrate translocation cycle. That is, transporters 

must undergo a conformational shift between the 

discrete phases of extracellular substrate binding 

(outward facing) and cytosolic substrate release (inward 

facing), limiting the potential maximum uptake 

velocity to around one substrate molecule per 

second per transporter. 4 Ion channels, by contrast, 

can achieve flux velocities on the order of millions of 

ions per second per channel. 18 Because they offer a 

far wider dynamic range in “substrate” flux rate, ion 

channels are more amenable to regulation strategies 

involving posttranslational modification at the individual 

protein level than transporters, such as the 

Ann. N.Y. Acad. Sci. 1187 (2010) 316–340 c○ 2010 New York Academy of Sciences. 317

DAT Regulation Schmitt & Reith 

Figure 1. The many parallel factors influencing DAT trafficking and membrane distribution. (A) The DAT undergoes 

continuous constitutive recycling between the plasma membrane and early endosomal compartments. (B) and (C) 

Substrates, such as amphetamine trigger internalization of plasmalemmal DATs, whereas cocaine-like inhibitors 

upregulate surface DAT expression. (D) and (E) Alterations in the phosphorylation state of DAT and associated 

scaffolding proteins by intracellular signaling cascades modulates transporter trafficking—although direct phosphorylation 

of DAT proteins is probably not caused by PKC, this kinase is an integral participant in DAT dynamics. (F) 

Physical coupling of the DAT to dopamine D2 receptors encourages recruitment of DAT to the plasma membrane. 

(G) PKC is also involved in ubiquitin-mediated DAT regulation, with monoubiquitination hypothesized to target 

internalized DAT to the lysosomal degradation pathway. (In color in Annals online.) 

DAT. Moreover, as Deken et al. have proposed, the 

ability to rapidly alter transporter trafficking may 

allow cells to regulate the exocytic release of neurotransmitters 

and the insertion of surface transporters 

in parallel. 19 This hypothesis was prompted 

by the finding that the GABA transporter (GAT1) 

is trafficked to and from the plasma membrane in 

recycling vesicles similar in size and morphology 

to synaptic vesicles (but lacking synaptophysin and 

the vesicular GABA transporter) and that the rate 

of recycling depends on intracellular Ca 2+ concentration, 

indicating that transporter trafficking may 

indeed be coupled to transmitter release. 19 

RegulationofDATactivitycanbetriggeredby 

a myriad of exogenous factors and cellular events, 

such as (1) small-molecule ligands targeting either 

the DAT itself (i.e., substrates and inhibitors) or 

presynaptic G protein-coupled receptors (GPCRs) 

that affect DAT, (2) enzymatic modification (e.g., 

phosphorylation) involving intracellular secondmessenger 

cascades, and (3) protein–protein 

interactions between the DAT and other transmembrane 

or cytoskeletal scaffolding proteins. A graphical 

overview of the putative factors influencing 

DAT membrane trafficking is given in Figure 1. Our 

primary focus in this review will be on short-term 

DAT regulation in response to acute exposure to psychostimulants, 

such as cocaine- and amphetaminelike 

drugs. Although the complete signaling mechanisms 

underlying substrate- and inhibitor-mediated 



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 



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 



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 



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 



transport, in addition to non–raft-associated DATs 

responding to PKC by a classical clathrin-dependent 

mechanism. 

On the basis of the preceding studies, it would 

be tempting to posit that trafficking of the DAT is 

mediated (at least in part) by PKC, with increased 

phosphorylation signaling that a given DAT protein 

is ready to be internalized. Recent work by 

Cervinski et al. 45 shows that d-methamphetamine 

increases DAT phosphorylation in addition to triggering 

internalization—both of which are prevented 

by PKC inhibitors. Taken together, these findings 

prompt a tantalizing hypothesis positing a link 

between substrate interaction, transporter phosphorylation 

by PKC, and subsequent membrane 

redistribution. Unfortunately, several lines of evidence 

suggest that the relationship between PKC, 

transporter phosphorylation, and membrane trafficking 

is far more complex. For example, removal 

of three classical PKC consensus sites from the DAT 

protein by mutagenesis of the target residues (Ser- 

262, Ser-586, and Thr-613) to glycine prevents the 

phosphorylation normally observed after PKC activation 

with �-PMA but fails to prevent �-PMA– 

induced internalization and endosomal trafficking 

of the DAT. 62 Similarly, truncation of the transporter 

distal N terminus—which bears several serine 

residues implicated in phosphorylation after PKC 

activation—also eliminates �-PMA–induced DAT 

phosphorylation without hindering the typical endocytic 

response in HEK cells. 63 Furthermore, although 

removal of the serine-rich distal N terminus 

prevents methamphetamine-induced DAT phosphorylation, 

it does not affect internalization of the 

DAT in response to the substrate. 45 That is, although 

PKC clearly plays a role in the mechanism underlying 

the trafficking effects of amphetaminergic substrates, 

direct phosphorylation of the DAT is not part 

of that role. It is possible that activation of PKC results 

in DAT phosphorylation via a more circuitous 

route, involving thus far unidentified downstream 

kinases; however, the observation that substrateinduced 

trafficking can occur without appreciable 

phosphorylation of the DAT protein begs the 

question of whether DAT phosphorylation actually 

serves as a “proendocytosis” signal. Instead, it may 

be that PKC regulates the action of a DAT-associated 

scaffolding or cytoskeletal protein that is ultimately 

responsible for determining the trafficking fate of 

a given DAT. 49,51 Data suggest that scaffolding 

proteins can control the trafficking dynamics of the 

noradrenaline transporter (NET): the cytosolic scaffolding 

protein syntaxin 1A—a well-known mediator 

of plasmalemmal vesicle docking and fusion— 

directly interacts with the NET and is involved with 

regulation of surface NET expression levels. 64 Moreover, 

exposure to amphetamine results in cytosolic 

redistribution of plasmalemmal NETs with a concomitant 

increase in the association of the NET protein 

and syntaxin 1A at the plasma membrane. 65 It 

is not clear whether syntaxin 1A promotes the internalization 

of the NET or stabilizes its presence 

at the plasma membrane (classically, syntaxin 1A is 

considered an exocytosis-promoting vesicle fusion 

protein), but the mere interaction between the two 

indicates that scaffolding proteins involved in regulated 

vesicular neurotransmitter release may also 

play a role in regulating transmitter transporters. 

Although explicit syntaxin 1A-dependence in 

amphetamine-mediated trafficking has not yet been 

shown with the DAT, syntaxin 1A does interact with 

the N terminus of the DAT, 66 and this interaction 

has been recently shown to promote the efflux of 

intracellular dopamine by amphetamine. 67 Interestingly, 

PKC (the classical isoform, PKC-�) is known 

to promote amphetamine-stimulated dopamine 

efflux via interaction with a DAT-associated 68 

protein complex; PKC-� is also necessary for the 

rapid short-term increase in surface DAT levels 

upon substrate exposure. 32 In the syntaxin study by 

Lee et al., 66 the authors also noted an interaction 

between the N terminus of the DAT and a protein 

known as the receptor for activated C kinases 

(RACK1). RACK1 can bind to activated PKC 

molecules and other intracellular signaling kinases 

and hence may help transduce increased intracellular 

PKC activity into a DAT proendocytosis signal 

without the need for a direct phosphotransferase 

interaction between the DAT and PKC. This 

protein–protein interaction might also contribute 

to amphetamine-induced dopamine efflux by 

attracting activated PKC to the transporter, 

promoting an inward-facing, “efflux-favoring” 

conformational state. 68,69 Furthermore, the 

conformational state of the DAT protein itself may 

influence its internalization rate, because disruption 

of the outward-facing conformational state by mutation 

of membrane-proximal N-terminal residues 

Arg-60 or Trp-63 to alanine results in increased 

DAT endocytosis. 70 Substrates could encourage an 



inward-facing transporter conformation via the 

process of translocation, suggesting a potential 

link between high concentrations of substrate and 

eventual DAT endocytosis. 

Thus, even if the identity of the terminal protein 

mediator of substrate-mediated DAT internalization 

is obfuscated, PKC still appears to be a requisite 

middleman in the endocytic signaling cascade. 

However, another significant question remains: how 

might amphetaminergic substrates activate PKC in 

the first place? PKC activity is regulated by Ca 2+ 

and diacylglycerol, a phospholipid metabolite generated 

in concert with inositol triphosphate by the 

enzyme phospholipase C (PLC), which is also Ca 2+ 

dependent. Stimulation of PKC activity by diacylglycerol 

can be amplified by arachidonic acid (for 

references, see Zhang and Reith 71 ), which has been 

shown to affect DAT function (e.g., Refs. 71–73); 

however, the effect of amphetamine on endogenous 

arachidonic acid—potentially through regulation 

of phospholipase A2, releasing arachidonic 

acid—is not known. Usually, PLC is activated by 

agonist binding at GPCRs coupled to the Gq/11type 

�-subunit; however, compounds, such as amphetamine 

and methamphetamine, lack significant 

affinity for GPCRs other than the trace amine– 

associated receptor 1 (TAAR1), a Gs-coupled receptor. 

74 It is conceivable that in certain experimental 

conditions and cell types, endogenous dopamine 

released by amphetamines could activate dopamine 

receptors, which can affect DAT function (see section 

on GPCR-mediated DAT regulation). However, 

Cervinski et al. 45 observed no differences in 

methamphetamine-induced DAT phosphorylation 

or internalization when cells were simultaneously 

pretreated with D1- andD2-like receptor antagonists. 

How, then, could amphetamine induce PLC 

activity? Although there have been few investigations 

into the non–transporter-mediated actions 

of amphetamines, research by Giambalvo indicates 

that treatment with 10 �M amphetamine enhances 

PLC activity in rat striatal synaptosomes and that 

pharmacological inhibition of PLC attenuates the 

usual activation of PKC by amphetamine. 75 Interestingly, 

inhibition of the Na + /Ca 2+ antiporter with 

amiloride also blocked amphetamine-induced PKC 

activation, suggesting that the ionic effects of DAT 

substrate translocation can directly influence activation 

of intracellular signaling cascades. When substrates 

are transported across the plasma membrane 

via the DAT, sodium ions are cotransported, and 

this increase in intracellular sodium concentration 

may cause Na + /Ca 2+ antiporters at mitochondrial 

and endoplasmic reticulum membranes to operate 

in reverse, favoring a net flux of Ca 2+ into the cytosolic 

compartment. Because PLC is also a Ca 2+ - 

dependent enzyme, the increase in intracellular calcium 

may catalyze PKC activation via stimulation 

of PLC in addition to its direct effect on PKC. 75 

PI3K, MAPK, and other protein kinases 

Protein kinases other than PKC are also involved in 

DAT phosphorylation, but again, evidence of definitive 

phosphotransferase reactions has proven elusive. 

76 As with PKC, both the PI3K and MAPK signaling 

pathways influence phosphorylation of DAT 

serine and threonine residues, 49 but their regulatory 

effects on DAT function appear to be opposite that 

of PKC, with kinase inhibition resulting in downregulation 

of and kinase activation resulting in upregulation 

of DAT activity, respectively. PI3K can be activated 

by GPCRs and by receptor tyrosine kinases, 

such as the insulin receptor. Although the classical 

isoform of PI3K itself is not considered a serine/threonine 

protein kinase, it is directly upstream 

of the serine/threonine kinase Akt (also known as 

protein kinase B). Inhibition of PI3K decreases the 

V max of dopamine uptake and causes loss of surface 

expressed DAT, which—like PKC- and substrateinduced 

downregulation—is dependent on endocytosis 

by clathrin-coated vesicles. 77,78 Conversely, 

treatment with insulin or expression of constitutively 

active forms of PI3K increases [ 3 H]dopamine 

uptake, a sign of putative surface DAT upregulation. 

77 Unlike inhibition of PKC, inhibition of PI3K 

has been recently demonstrated to attenuate the 

dopamine-releasing effects of amphetamine; however, 

this is most likely due to reduction in cell 

surface DAT expression rather than a direct effect 

of PI3K on transporter function. 79 The MAPK 

pathway is a complex signaling network consisting 

of a three-tiered series of various serine/threonine 

protein kinases: primary members of the MAPK 

protein family (such as extracellular signal– 

regulated kinases-1 and -2 [ERK1/2]) are activated 

by secondary upstream kinases known as 

MAP/ERK kinases (MEKs) which are in turn activated 

by a tertiary group of further upstream kinases 

known as MEK kinases. 80 Reducing ERK1 and ERK2 



phosphorylation by using small-molecule inhibitors 

of MEK affects DAT function and trafficking similarly 

to PI3K inhibition, decreasing the V max of 

dopamine uptake without altering the Km and 

inducing cytosolic redistribution of plasmalemmal 

DAT into endosomal compartments. 78,81 The 

MAPK and PI3K signaling cascades are probably 

involved in the regulation of DAT activity by various 

classes of transmembrane receptors, such as 

GPCRs (discussed in the following section) and receptor 

tyrosine kinases. For example, in addition 

to insulin—which has been shown to increase plasmalemmal 

DAT function—activation of the neurotrophin 

receptor tyrosine kinase TrkB with brainderived 

neurotrophic factor also upregulates DAT 

activity, an effect that hinges on both the MAPK and 

PI3K pathways. 52 MAPK family members ERK1 and 

ERK2 have also been implicated in cocaine addiction 

and relapse after cocaine withdrawal, as central 

mediators of long-term sensitization to cocaine associated 

cues (for review, see Lu et al. 82 ). 

Ubiquitination and other direct 

posttranslational modifications 

Covalent attachment of the small soluble protein 

ubiquitin to the ε-amino moiety of lysine residues 

in target cellular proteins is known as ubiquitination. 

The ubiquitination reaction is catalyzed by a 

multistep enzymatic system, with the final covalent 

attachment mediated by members of a family of proteins 

known as E3 ubiquitin ligases (the multifarious 

functions of ubiquitin are reviewed in Welchman 

et al. 83 ). The most widely recognized function of 

ubiquitination is polyubiquitination, which occurs 

when several ubiquitin subunits are attached to one 

lysine, forming a chain. Polyubiquitin chains serve 

as a signaling motif, indicating that a target protein 

is to be trafficked to the 26S proteasome for proteolytic 

destruction. However, attachment of one 

ubiquitin subunit to a target protein (monoubiquitination) 

is also a signaling motif and is thought 

to encourage retention of target proteins in sorting 

endosomes for eventual trafficking to the lysosomal 

degradation pathway. 84 Ubiquitination can 

fulfill roles other than those directly serving protein 

breakdown. For example, ubiquitination of the 

yeast �-factor receptor Ste2 promotes endocytosis 

of the receptor–ligand complex prior to degradation 

in the vacuole. 85 The yeast multidrug transporter 

Pdr5, 86 and the Ste6 �-factor pheromone 

transporter, 87 both members of the ATP-binding 

cassette multidrug transporter family, appear to be 

prepared by ubiquitination for endocytic delivery to 

the vacuole for proteolytic turnover. In these cases, 

ubiquitination may serve as a signal for protein trafficking 

rather than a signal for protein degradation 

itself. Work by Hicke’s group 88 has provided 

more information on ubiquitin as a signal for endocytosis 

of the plasma membrane protein Ste2p, 

the mating pheromone �-factor receptor. Unlike 

the ubiquitin proteasome recognition signal, the 

internalization signal does not require polyubiquitin 

formation through Lys-18 but rather relies on 

monoubiquitination of one lysine residue of the 

Ste2p. 

There is evidence that, like phosphorylation, 

ubiquitination of the DAT affects cell surface expression 

and membrane trafficking of the transporter. 

Interestingly, many of the same intracellular signaling 

kinases involved in DAT phosphorylation may 

also be responsible for signaling via ubiquitination. 

For example, Miranda et al. demonstrated that activation 

of PKC by treatment with �-PMA increases 

ubiquitination of the DAT and that the major proportion 

of ubiquitin-conjugated DAT was present 

as the monoubiquitinated species. 89 Using Förster 

resonance energy transfer, the authors also showed 

that fluorescent-tagged hDAT and ubiquitin are associated 

in late endosomes and multivesicular bodies 

destined for lysosomal degradation. Much like 

PKC-associated transporter phosphorylation, ubiquitination 

of the DAT depends upon residues residing 

in the N terminus of the transporter, because 

simultaneous mutation of three amino-terminal lysine 

residues (Lys-19, Lys-27, and Lys-35) inhibits 

PKC-mediated ubiquitination. 90 Ubiquitin conjugation 

to the DAT is catalyzed by the ubiquitin ligase 

Nedd4-2. 91 Although it is clear that ubiquitination 

is involved in the regulated endocytosis of the DAT, a 

relationship between substrate-induced transporter 

trafficking and ubiquitination has not yet been 

established. 

Presynaptic G protein-coupled receptors 

affecting DAT function 

Because DAT protein function and membrane distribution 

are acutely regulated via several different 

intracellular signaling cascades, it is not surprising 

that activation of various types of GPCRs expressed 



by dopaminergic neurons can affect the DAT. Of the 

possible GPCR subtypes that can affect DAT function, 

dopamine D2-like autoreceptors are the most 

logical candidates, with their presynaptic localization 

and clearly defined role in regulatory inhibition 

of both tyrosine hydroxylase 92 and vesicular 

dopamine release. 93 However, although D2-like autoreceptors 

are the most thoroughly investigated, 

other GPCRs also appear to modulate dopaminergic 

neurotransmission by altering DAT function. 

In particular, activation of both �-opioid receptors 

and TAAR1 receptors (a member of a recently discovered 

family of receptors for endogenous trace 

amines) has been shown to elicit changes in DAT 

function. 

Dopamine D2 and D3 autoreceptors 

The D2-like family of dopamine receptors comprises 

D2,D3,andD4 receptor subtypes. In addition 

to their role as classical postsynaptic receptors, the 

D2 and D3 subtypes are expressed by dopaminergic 

neurons themselves, serving as presynaptic autoreceptors. 

Activation of D2-like autoreceptors attenuates 

dopaminergic neurotransmission via several 

parallel mechanisms: in PC12 cells, for example, 

treatment with the D2/D3 agonist quinpirole causes 

feedback inhibition of tyrosine hydroxylase—the 

rate-determining enzyme in dopamine synthesis— 

and decreases K + -evoked dopamine release. 93 In 

addition to reducing stimulated dopamine release, 

recent evidence suggests that D2 and D3 receptor 

activation also reduces extracellular dopamine concentration 

via acute upregulation of DAT function. 

This effect was first demonstrated in the early 1990s 

by using rotating disk electrode voltammetry to 

measure the rate of extracellular dopamine clearance. 

That is, Meiergerd et al. demonstrated 94 that 

the D2/D3 agonist quinpirole increases the V max of 

dopamine transport into rat striatal synaptosomes, 

an effect that is reversed by concomitant administration 

of the antagonist sulpiride. The authors also 

showed in vivo evidence of the inverse situation: a 

reductionintheV max of dopamine clearance in rats 

chronically treated with the potent D2 antagonist 

haloperidol. In support of this finding, localized intrastriatal 

application of the selective D2 antagonist 

raclopride has also been shown to decrease clearance 

of exogenous dopamine applied locally via a 

second micropipette. 95 Studies using selective D3 

receptor ligands paint a similar picture: the selective 

D3 agonist PD128907 increases electrochemically 

measured dopamine clearance in rat nucleus accumbens 

slices, producing an increase in transport V max 

with no effect on the K m of dopamine after a 10min 

preincubation. 96 In contrast, the D3 receptor 

antagonist GR103691 decreased dopamine significantly 

below vehicle levels. In Xenopus oocytes coexpressing 

hDAT and D2R transcripts, activation of 

D2 receptors with apomorphine for as little as 5 min 

results in a 40% increase of whole-cell [ 3 H]CFT 

binding (Bmax) with no alteration in the Ki value 

for CFT, suggesting that activation of D2 receptors 

triggers rapid trafficking of intracellular DATs to the 

plasma membrane. 97 Upregulation of surface DAT 

expression by D2 receptor agonists requires Gi/Go 

protein coupling, because pretreatment with pertussis 

toxin (PTX) blocked the increase in [ 3 H]CFT 

binding. In our collaborative study with the group 

of Garris, in vivo voltammetry was used to monitor 

dopamine uptake and release in rat striatum 

and nucleus accumbens. 98 We found that D2R antagonism 

reduced uptake at all frequencies used to 

stimulate the medial forebrain bundle, whereas release 

was enhanced only at lower frequencies. Be 

that as it may, both processes—D2R regulation of 

dopamine uptake as well as release—serve as parallel 

feedback mechanisms aimed at reducing extracellular 

dopamine levels when high dopamine levels 

activate D2 receptors. 

Most investigations into the effects of D2-like receptor 

ligands on DAT function in vitro have used 

electrochemical methods to assess dopamine uptake 

in animal tissues, in lieu of more commonly 

used measurement of [ 3 H]dopamine uptake by heterologous 

cells. The reason for this methodological 

preference is purely technical: dopamine is a 

high-affinity agonist at D2 and D3 receptors, creating 

an obvious confound because the radioligand 

will activate the receptors as well. Although electrochemical 

detection has greater temporal resolution 

than is possible with cell models, 16 it does not allow 

direct observation of effects on DAT trafficking 

in concert with kinetic measures of DAT function. 

Recent studies have overcome this issue by using a 

novel synthetic DAT substrate that is easily detected 

yet exhibits negligible affinity for dopamine receptors. 

The substrate 4-(4-(dimethylamino)styryl)- 

N-methylpyridinium (ASP + )—a styryl analogue 

of the neurotoxic substrate MPP + —has the added 

benefit of possessing a fluorescent �-conjugated 



diarylethene moiety (with excitation and emission 

spectra of 488 nm and 609 nm, respectively 99 ). 

Hence, unlike other potential surrogate DAT substrates 

(e.g., [ 3 H]MPP + and [ 3 H]amphetamine), it 

is possible to measure the cellular uptake and accumulation 

of ASP + in real time by using fluorescent 

microscopy. In EM4 cells coexpressing hDAT and 

D2R transcripts, receptor activation with quinpirole 

increases the rate of ASP + uptake compared 

with vehicle treatment—this effect was abolished 

in cells treated with PTX, corroborating the necessity 

of Gi/Go protein–coupled signaling for functional 

DAT upregulation. 100 Moreover, inhibition of 

MEK (which, in turn, prevents ERK1/2 activation) 

blocked the effects of quinpirole, whereas PI3K inhibition 

had no effect. Treatment with quinpirole 

also increases surface expressed DAT (with a concomitant 

decrease in intracellular-localized DAT), 

as measured by biotinylation. Similarly, quinpirole 

also increases plasmalemmal DAT expression and 

ASP + uptake in EM4 cells coexpressing hDAT and 

D3 receptors. 101 Like the DAT upregulation mediated 

by D2 receptor activation, this effect is PTX 

sensitive; however, both the MAPK and PI3K pathways 

are required for the quinpirole-induced increase 

in DAT function. Interestingly, much like the 

observations of Johnson et al. 31 for amphetaminergic 

substrates, D3 activation results in a rapid increase 

in surface DAT expression that eventually 

gives way to significantly reduced plasmalemmal 

transporter expression upon prolonged D3 agonist 

exposure. 101 As a tritiated alternative to fluorescent 

ASP + , it also appears possible to use tracer amounts 

of [ 3 H]tyramine for monitoring DAT function in 

experiments disentangling transporter and receptor 

phenomena, because tyramine has been shown to 

display rather low potency (micromolar) in activating 

D2 dopamine receptors. 100 It will be important 

to show that the tritiated ligand does not appreciably 

bind to dopamine receptors; caution is required 

because the thorough study of Zapata et al. 101 

shows considerable binding of [ 3 H]dopamine to D3 

dopamine receptors that can be displaced by 0.1 �M 

spiroperidol in cells that do not express hDAT compared 

with cells subjected to the same conditions 

with hDAT on board. 

One unexpected finding in this series of receptormediated 

ASP + -uptake experiments was the 

observation of positive resonance energy transfer 

between fluorescent-tagged hDAT and a biolumi- 

nescent D2R–luciferase construct, suggesting that 

the DAT and D2R proteins are in proximity (


that they are bona fide neuromodulators. �- 

Phenethylamine (�-PEA), p-tyramine, and DMT 

act as full agonists at the trace amine receptor 

TAAR1, triggering the production cAMP. 106 

Interestingly, like �-PEA, the DAT substrates damphetamine 

and d-methamphetamine are also 

full agonists at TAAR1. 74 In HEK cells expressing 

hDAT and TAAR1, exposure to �-PEA causes a 

rapid reduction in [ 3 H]dopamine uptake in both 

HEK cells and mouse synaptosomes—importantly, 

this effect does not appear to be due to generalized 

DAT substrate activity of �-PEA, because the effect 

is absent in synaptosomes prepared from TAAR1knockout 

mice. 107 Later analysis of transfected HEK 

cells demonstrated that TAAR1 and dopamine D2 

receptors may have opposing effects on DAT regulation: 

cells expressing DAT and D2 receptors show the 

expected increase in [ 3 H]dopamine uptake, whereas 

cells expressing DAT and TAAR1 show a decrease 

in [ 3 H]dopamine uptake under identical conditions. 

108 However, the relevance of this finding is 

difficult to interpret, because dopamine was used as 

both the D2R and TAAR1 agonist, but as discussed in 

the foregoing, radiolabeled [ 3 H]dopamine used to 

measure uptake would also probably activate receptors. 

Because the neuromodulatory roles of these 

once-mysterious trace amines are just now beginning 

to be uncovered, further investigations into 

the effects of TAAR1 receptor activation on transporter 

trafficking are clearly needed. Subtle modulation 

of monoaminergic neurotransmission by 

TAAR1 agonists has, for example, been speculated 

to underlie the anxiolytic effects of small doses of 

d-amphetamine and DMT in humans. 109 

Downregulation of DAT activity by κ-opioid 

receptors 

Interest in the effects of �-opioid receptor activation 

on DAT function stems from initial observations 

that �-opioid agonists attenuate the dramatic 

rise in extracellular dopamine levels after cocaine 

administration. In the rat nucleus accumbens, for 

example, a single high-dose injection of cocaine 

(20 mg/kg) causes a 10-fold increase in extracellular 

dopamine concentration—pretreatment with 

the �-agonist U50488 decreases this effect by more 

than 50%. 110 Using voltammetry and in vivo microdialysis, 

Thompson et al. demonstrated that the 

DATmodulatoryeffectsof�-opioid agonists differ 

between acute and chronic agonist administra- 

tion. 111 That is, whereas acute administration of the 

�-agonist U69593 increases dopamine uptake, repeated 

administration results in a decrease in uptake 

with a concomitant decrease in surface DAT 

labeling by the tropane radioligand [ 125 I]CIT (2�carbomethoxy-3�-(4-iodophenyl)tropane) 

but no 

loss in total DAT protein. Importantly, the effect 

of repeated cocaine treatment was opposite that of 

U69593 (a significant acceleration of dopamine uptake, 

indicating an upregulation of surface DATs), 

but the effect of both drugs in combination was not 

significantly different from control. 111 The mechanism 

underlying �-opioid–induced DAT regulation 

is unknown, but it is hypothesized that activation 

of PI3K by �-receptor agonists plays a role. 16 The 

DAT-modulating effects of �-opioids prompt consideration 

of their receptors as potential targets for 

novel cocaine addiction therapeutics. For example, 

�-opioid receptor agonists attenuate the acute reinforcing 

effects of cocaine, and repeated �-opioid 

agonist treatment may oppose some of the alterations 

in dopaminergic transmission observed after 

withdrawal from chronic cocaine. 111 

Possible implications of DAT regulation to 

the putative toxicity of several substituted 

amphetamines 

Historically, neurotransmitter transporters, such as 

the DAT, were regarded as relatively static entities: 

“molecular vacuums” expressed at a constant level 

at presynaptic terminals. 16 This preconceived notion 

has changed dramatically over the last decade, 

because many studies have indicated that—akin to 

classically defined transmembrane receptors—the 

activity and membrane trafficking of transporter 

proteins is rapidly and differentially regulated by 

interaction with chemically distinct ligands. In fact, 

current research suggests that DAT trafficking is 

the primary mechanism by which cells modulate 

extracellular dopaminergic tone. That is, dynamic 

change in the rate of dopamine uptake is achieved 

by shifting the distribution of DAT proteins between 

the plasmalemmal and endosomal compartments, 

rapidly altering the number of DATs at the cell surface. 

As discussed earlier, the most robust example 

of ligand-mediated transporter redistribution is the 

internalization response triggered by amphetaminergic 

substrates. Long before it was known that 

transporter proteins undergo dynamic trafficking 



and that this trafficking can be rapidly regulated, 

findings of in vitro amphetamine-induced reduction 

in transporter radioligand binding were interpreted 

as evidence of the in vivo neurotoxicity of 

these compounds. With the new knowledge on DAT 

trafficking phenomena, such findings will need to be 

reinterpreted. In the following, we will discuss the 

possible implications of transporter protein regulation 

to the putative toxicity of several substituted 

amphetamine derivatives that are often encountered 

as drugs of addiction (the prototypical example being 

methamphetamine) and/or recreational drugs. 

We will also discuss the potential utility of agents 

that reduce plasmalemmal DAT expression in the 

rectification of dopaminergic neuroadaptation that 

occurs in chronic cocaine addiction. 37,40 

The amphetamines are an immensely diverse class 

of compounds and the structure-activity relationship 

of the amphetaminergic structural framework 

has been thoroughly investigated. 112 Amphetamine 

derivatives can elicit a wide range of phenomenological 

and neurochemical effects; however, for this 

discussion, we will focus solely on stimulant-like 

amphetamine derivatives that act as monoamine 

transporter substrates and will not consider any of 

the psychedelic amphetamine derivatives, which act 

primarily at serotonin 5-HT2 receptors. 113 Whereas 

amphetamine itself exhibits roughly equipotent 

substrate activity at the DAT and NET with little 

meaningful activity at the SERT—amphetamine inhibits 

[ 3 H]DA and [ 3 H]NE uptake with 112- and 98fold 

greater potency than [ 3 H]5-HT, respectively— 

certain amphetamine analogues bearing substitutions 

on the phenyl ring and amine nitrogen have 

unique activity profiles at the three monoamine 

transporters. 114 For example, aromatic substitution 

with a bulky, electron-rich moiety (such as a 

methoxy group or a halogen larger than fluorine) at 

the 3- or 4-position usually decreases substrate activity 

at the DAT but concomitantly increases affinity 

toward the SERT. 115,116 Hence, amphetamine 

derivatives, such as 4-chloroamphetamine (PCA), 

4-methoxyamphetamine (4-MA; more traditionally 

known as PMA, but we shall refer to it as 4-MA 

to prevent confusion with the phorbol ester �- 

PMA) and 3-trifluoromethyl-N-ethylamphetamine 

(fenfluramine) are often labeled as “serotonergic 

amphetamines” (although PCA still possesses 

significant affinity for both the DAT and 

NET 116 ). Alkylation of the amphetamine nitro- 

gen to the secondary amine generally increases 

SERT affinity without drastically altering potency 

at either the DAT or NET; however, alkyl 

moieties longer than N-ethyl impede substratelike 

activity, with d-N-butylamphetamine being 

virtually inactive in behavioral assays. 117 

Therefore, d-methamphetamine exhibits roughly 

2.5-fold greater potency than d-amphetamine 

as a SERT substrate. 115 The empathogen 3,4methylenedioxyamphetamine 

(MDA) and its Nmethyl 

analogue (MDMA)—both widely used 

recreational drugs, often sold (with any number of 

admixes) under the name “ecstasy”—have affinity 

for all three of the monoamine transporters, with 

slightly greater substrate activity at the SERT and 

NET than at the DAT. 118–120 SERT-affecting substituted 

amphetamine analogues decrease plasmalemmal 

SERT expression, paralleling the endocytic effect 

that amphetamine itself has on the DAT. Acute 

administration of high doses of d-fenfluramine or 

PCA to rats decreases the Bmax for SERT radioligand 

binding by 30–60% (depending upon the interval 

after drug administration), without significant 

changes in whole-cell SERT protein levels, 121 as 

measured by Western blot analysis (but also see Xie 

et al. 122 ). MDMA has also been frequently shown 

to cause loss of both radiolabeled SERT and DAT 

binding sites in experimental animals. 123–125 

Because monoaminergic neurotransmission is so 

intimately involved in cognition, affect, behavioral 

reinforcement, and motor function, 2,126 it is not surprising 

that the implications of substrate-induced 

monoamine transporter downregulation on human 

physiological and psychical function are highly debated 

topics. The amphetaminergic substrate that 

has received the most attention in this debate is 

MDMA. Because of its widespread popularity as a 

recreational drug and nearly ubiquitous presence in 

the party scene, ascertaining the potential of recreational 

doses of MDMA to elicit neurotoxicity in 

humans is an important public health question. 

Furthermore, elucidation of the mechanism underlying 

MDMAergic neurotoxicity will also provide 

insight into strategies to minimize potential damage 

dealt by recreational MDMA use. 

Initial neurochemical studies in rats revealed a 

dramatic and protracted reduction in the density of 

SERT expression and in total brain 5-HT content 

after high doses of either MDA or MDMA. 123 Similar 

losses of SERT expression were observed after 



treatment with the well-established serotonergic 

neurotoxin 5,7-dihydroxytryptamine (5,7-DHT). 

Hence, authors of these early studies concluded 

that MDA-like drugs produce neurotoxic insult to 

serotonergic neurons and suggested that the magnitude 

and duration of the decline in SERT expression 

constitutes a method of quantifying the 

extent of this damage. The reduction in SERT expression 

that occurs after MDMA administration 

is quantitatively similar to reductions caused by 

4-substituted amphetamines, such as 4-MA and 

PCA. 127,128 In light of the fact that membrane trafficking 

of monoamine transporter proteins is altered 

upon exposure to amphetaminergic substrates— 

and that such changes persist beyond the duration 

of acute drug exposure—the question is 

thus whether or not reductions in SERT expression 

represent mere changes in transporter trafficking 

and membrane distribution or true damage to 

serotonergic neuronal function. Evidence in support 

of both conclusions exists. Experiments by 

Wang et al. 125,129 showed that MDMA administration 

reduces radioligand binding to plasmalemmal 

SERTs in rats; but unlike the specific serotonergic 

toxin 5,7-DHT, it neither alters total SERT 

protein expression nor induces microglial activation. 

In contrast, the results of Xie et al. 122 —who 

used a different, more specific antibody for immunochemical 

detection of the SERT protein— 

do indicate substantial loss of SERT protein after 

treatment with either 5,7-DHT or the serotonergic 

amphetamines MDMA, PCA, and fenfluramine, 

suggesting functional damage to serotonergic nerve 

terminals (Ref. 122, Figs. 8–10). However, some 

of the differences seen by these two groups may 

be related to respective MDMA dosing regimens: 

22.5 mg/kg total MDMA (3 × 7.5 mg/kg, each 2 h 

apart) was given in the studies by Wang et al., 125,129 

whereas a total dose of 45 mg/kg (3 × 15 mg/kg, 

each 1.5 h apart) was used in the Xie et al. 

study. 122 Callaghan et al. compared the effects of repeated 

administration of either MDMA or 4-MA on 

in vitro measures of neurodegeneration (such as 

plasmalemmal SERT binding) with in vivo assessment 

of 5-HT clearance by using real-time microamperometry. 

128 Here, the authors found that 

[ 3 H]cyanoimipramine binding to the SERT was 

significantly reduced 2 weeks after repeated administration 

of either high-dose 4-MA or MDMA 

(15 mg/kg, twice daily for 4 days); however, clear- 

ance of locally applied 5-HT was reduced only in 

rats treated with a high dose of 4-MA. The observation 

that 5-HT clearance in vivo was unaltered by 

even a high-dose MDMA treatment regimen suggests 

that in vitro measures, such as plasmalemmal 

SERT radioligand binding, do not necessarily predict 

the functional state of serotonergic neurons in 

vivo. Moreover, these findings highlight that the toxicity 

of substituted amphetamines can vary greatly 

depending upon their individual chemical structure. 

Here 4-MA appears to have a greater potential 

than MDMA to elicit toxicity in vivo. This finding 

is certainly consistent with epidemiological data, 130 

which show a high incidence of mortality after consumption 

of clandestine pressed pills containing 4- 

MA sold as “ecstasy.” By contrast, fatalities caused 

by consumption of moderate doses of unadulterated 

MDMA (as opposed to ecstasy pills) in the absence 

of other drugs are exceedingly rare. 

Paradoxically, localized direct administration of 

MDMA in rats by in vivo reverse microdialysis— 

at concentrations equivalent to those attained after 

high-dose systemic administration—does not result 

in the typical indicators of serotonergic neurotoxicity, 

despite eliciting dramatically greater increases 

in extracellular monoamine levels. 131 Surprisingly, 

MDMA was demonstrated to exert neuroprotective 

effects in cultured rat fetal neurons, indicating that 

direct exposure to the chemical itself is not necessarily 

cytotoxic. 132 In addition, whereas systemically 

administered MDA and MDMA acutely increase 

the concentration of radical reactive oxygen 

species (ROS) and produce oxidative stress in the 

brain, direct intracerebroventricular administration 

is devoid of such effects. 131,133 

The difference in toxicological profile between 

direct microdialysis and systemic routes of administration 

suggests that much of the toxicity of 

MDA and its N-alkylated cousins is the result of 

metabolic transformation of parent compounds 

into more potent neurotoxins. 134 In humans, 

MDA and MDMA are largely metabolized via 

demethylenation by the hepatic CYP2D6 enzyme 

to the respective 3,4-dihydroxyamphetamine 

species (�-methyldopamine and �,Ndimethyldopamine). 

135 In recent years, several 

investigators have revealed that—much like 

dopamine—these catechol species readily undergo 

oxidation to semiquinone and quinone species 

that can form neurotoxic thioether compounds 



Figure 2. Metabolic reaction pathway of MDA-like compounds leading to the formation of neurotoxic prooxidant 

thioether species and the relative toxicity of metabolites. The parent compounds MDA and MDMA are not directly 

cytotoxic. 131,132 However, demethylenation of the 3,4-methylenedioxy moiety by the hepatic CYP2D6 enzyme 135 

results in the formation of 3,4-dihydroxyamphetamines, which are readily oxidized to quinones. 134 These quinones 

can form conjugates with GSH, greatly potentiating neurotoxicity. 138 Further metabolism in the brain via the 

mercapturic acid pathway results in toxic NAC-conjugated species. Although the quinones, GSH-conjugates, and 

NAC-conjugates all promote the formation of reactive oxygen species, NAC-substituted compounds appear to possess 

the greatest relativistic potential for toxicity owing to their protracted rate of elimination from the brain. 137 (In color 

in Annals online.) 

in vivo. 136,137 Quinones are strong electrophiles: 

they can serve to propagate ROS via a one-electron 

redox reaction to a stable semiquinone radical 

or form conjugates with reducing agents via 

two-electron nucleophilic attack. 138 In particular, 

the dopaminoquinone-like metabolite of 

MDA/MDMA can undergo conjugation with 

the cysteinyl thiol moiety of the endogenous 

reductant glutathione (GSH) to form the thioether 

5-(glutathionyl)-�-methyldopamine (5-(GSH)- 

�MeDA) or its N-methyl analogue. 136 In the central 

nervous system, the 5-(glutathionyl)-thioethers 

are ultimately metabolized via the mercapturic 

acid pathway to form 5-(N-acetyl-cysteinyl)- 

�-methyldopamine (5-(NAC)-�MeDA) and its 

N-methyl analogue (5-(NAC)-�,N-diMeDA). 

The structural formulae of these metabolites 

and their relative potencies as neurotoxins are 

displayed in Figure 2. The thioether metabolites are 

potent serotonergic neurotoxins that can induce 

a dose-dependent increase in ROS formation and 

cause caspase-3–mediated apoptosis in cultured 

cortical neurons. 137 Moreover, direct intrastriatal 

administration of pure 5-(NAC)-�,N-diMeDA in 

rats fully recapitulates the serotonergic toxicity 

observed with systemic high-dose MDMA. 136 

These thioethers are detectable in the rat brain 

after systemic administration of a high-dose 

neurotoxic regimen of MDMA—repeated dosing 

leads to significant accumulation due to a 

nonlinear increase in elimination half-life. 139 

Interestingly, cotreatment with the antioxidant 

GSH precursor N-acetylcysteine (NAC) successfully 

protects cultured cortical neurons from 

thioether-induced apoptosis and attenuates the 

formation of ROS, even though NAC could 



obviously contribute to the formation of the 

5-(NAC)-thioether metabolites. 137 Pretreatment 

with the antioxidant compound acetyl-L-carnitine 

was also shown to prevent oxidative damage to 

neuronal mitochondria in vivo after high-dose 

MDMA administration in rats. 140 These data 

strongly indicate that the toxicity of MDMA and its 

analogues is due chiefly to oxidative stress induced 

by the potent and selective serotonergic neurotoxins 

formed during metabolism of the parent 

compound. Moreover, these findings suggest that 

avoidance of repeated dosing and concomitant use 

of central nervous system–penetrating antioxidant 

compounds are prudent harm reduction strategies 

for preventing or limiting the damage inherent to 

recreational MDMA use. 

If the neurotoxic effects of the MDA-type ring 

substituted amphetamines is due primarily to the 

formation of prooxidant quinone metabolites, what 

can be said of amphetamine derivatives that are 

unlikely to form such unstable metabolites, such 

as d-methamphetamine? Despite being similar to 

amphetamine itself in structure, pharmacokinetics, 

and metabolic fate, methamphetamine exhibits increased 

toxicity and significantly greater abuse potential 

than the parent compound. 20,141 Toxic insult 

to both serotonergic and dopaminergic neuronal 

systems after high doses of d-methamphetamine has 

been routinely demonstrated in experimental animals 

(see Cadet et al. 142 for a review). In humans, 

chronic high-dose methamphetamine addicts show 

moderate deficits in cognitive function, and in vivo 

neuroimaging techniques reveal decreased surface 

DAT concentration 143 and increased microglial activation 

compared with control subjects. 144 Eventual 

recovery from these effects appears possible, 

but normalization of dopaminergic function is an 

extremely slow process, taking months to years. 145 

Similarly to neurotoxicity associated with MDA-like 

substituted amphetamines, the toxic response to 

methamphetamine—particularly in dopaminergic 

neurons—also involves oxidative stress by quinonemediated 

formation of ROS and subsequent activation 

of proapoptotic signaling cascades. 142 

Findings that antioxidant compounds can reduce 

neurotoxic insult by methamphetamine support of 

this theory: for example, high doses of the aforementioned 

antioxidant NAC ameliorate the longterm 

loss of striatal DAT in moneys after repeated 

injections of methamphetamine. 146 However, un- 

like with MDA/MDMA, here the likely culprit is 

not a metabolic product of the parent stimulant but 

rather dopamine itself. 

Free-floating dopamine (that is, existing outside 

synaptic vesicles) can be spontaneously oxidized 

to dopaminoquinone and successively converted to 

5-cysteinyl-catechol thioethers in a manner identical 

to the reaction of the MDA metabolite 3,4dihydroxyamphetamine 

shown in Figure 2. 147 If sufficiently 

large concentrations of soluble dopamine 

are present, mechanisms to rapidly clear or safely 

inactivate dopamine—namely, the DAT and the 

catabolic enzyme COMT—will presumably be 

saturated. Excess dopamine will thus be nonspecifically 

oxidized, resulting in dose-dependent formation 

of dopaminoquinone. How might this oxidative 

mechanism explain the increased toxicity of 

methamphetamine compared with amphetamine 

itself? A series of experiments by Goodwin et al. 

systematically highlighted, for the first time, some 

vital differences between the two compounds in 

their respective effects on the DAT and dopaminergic 

function. 20 Notably, the authors demonstrated 

that d-methamphetamine is fivefold more effective 

at stimulating the DAT to release dopamine in 

hDAT-expressing HEK cells than d-amphetamine at 

identical concentrations. Also, in vivo chronoamperometry 

in the rat nucleus accumbens after 

administration of an equivalent dose of either 

methamphetamine or amphetamine (5 mg/kg) revealed 

that methamphetamine significantly prolonged 

dopamine clearance time compared with 

amphetamine, resulting in greater sustained external 

dopamine levels. However, no differences between 

the two were detected at a 1-mg/kg dose (Ref. 

20, Fig. 5). 

Thesedatasuggestthatathigh(yetequivalent) 

doses, methamphetamine has a far greater propensity 

than amphetamine to produce accumulation 

of excess external dopamine. The higher resultant 

concentrations of external dopamine conceivably 

contribute to the greater addictive potential 

of methamphetamine than with amphetamine and 

may also render methamphetamine more likely to 

cause oxidative neurodegeneration. Although the 

specific DAT-mediated pharmacology of methamphetamine 

contributes to its particularly high toxicity 

and abuse potential, another basic contributing 

factor is the dose and administration pattern typical 

of human methamphetamine addicts. Recreational 



methamphetamine users typically take upward of 

100 mg (a “point”) per dose session—for those 

that administer it via inhalation (as smoked “crystal 

meth” or “ice”), this can be even higher—often 

taking multiple subsequent doses in a “binge” pattern 

(every few hours); chronic methamphetamine 

addicts can easily consume doses on the order of 

grams per day. 145 Intranasal administration of a 50mg 

dose of d-methamphetamine results in an average 

plasma concentration of 758 nM in human 

subjects. 148 Sequential 100-mg doses could thus result 

in prolonged plasma drug concentrations ranging 

into the micromolar levels shown in animal 

and in vitro studies to elicit profound dopamine 

release, cytotoxicity, and long-lasting downregulation 

of DAT function. Methamphetamine addicts 

that consume gram quantities per day can 

achieve steady-state plasma concentrations exceeding 

13 �M. 74 Hence, it is likely that the transient reduction 

in plasmalemmal DAT expression observed 

after a single high-level dose of methamphetamine 

and the prolonged reduction seen in chronic 

high-dose methamphetamine users are two entirely 

different phenomena: the former representing a 

neuroadaptive feedback mechanism and the latter 

representing accumulated apoptotic oxidative injury 

to dopaminergic neurons. 

Conclusion 

Neuronal DATs are the primary regulators of 

extracellular dopamine in the brain and are thus responsible 

for both the duration and the magnitude 

of interneuronal dopaminergic signals. Biogenic 

amine transporter proteins, such as the DAT, are 

highly dynamic entities that rapidly cycle between 

the plasma membrane and early endosomal compartments, 

probably in specialized vesicles roughly 

similar to synaptic vesicles in size (e.g., Ref. 19). By 

altering the pattern of DAT trafficking, cells can adjust 

the number of active (surface localized) DATs 

in real time, allowing for condition-specific adaptive 

changes in dopaminergic signaling. DAT trafficking 

is regulated by several intracellular signaling cascades 

involving protein kinases and phosphatases. 

Activation or inhibition of PKC, PI3K, and members 

of the MAPK family can trigger transporter 

membrane redistribution; however, the identity of 

the kinase(s) ultimately responsible for direct phosphorylation 

of the DAT is currently unknown. With 

the recent evidence that syntaxin 1A and other scaffolding 

proteins interact with the DAT, it is certainly 

conceivable that central signaling kinases, such as 

PKC, affect DAT activity via DAT-associated scaffolding 

or cytoskeletal proteins that more directly 

control the transporter’s trafficking fate. In addition, 

ligands for certain presynaptic GPCRs found 

on dopaminergic neurons (such as dopamine D2like 

autoreceptors and �-opioid receptors) can also 

acutely regulate DAT surface presence, probably by 

modulating the activity of these cell signal transduction 

pathways. 

Finally, DAT trafficking is acutely regulated by ligands 

that directly interact with the DAT itself (substrates 

and inhibitors). Acute exposure to high doses 

of substrates (including several amphetaminergic 

compounds and dopamine itself) elicits a biphasic 

effect on DAT trafficking: initially, a rapid yet 

transient (beginning within seconds and lasting for 

several minutes) increase in surface DAT occurs, 

followed by significant transporter endocytosis that 

begins after approximately 15 min of substrate exposure. 

In direct contrast with the internalizing effects 

of substrates, cocaine-like DAT inhibitors appear 

to induce upregulation of surface DAT levels. Upregulation 

of surface DAT concentration might be 

responsible for much of the anhedonic, depressionlike 

symptoms that chronic cocaine addicts experience 

during withdrawal—doubtless triggering 

cravings for more cocaine. The DAT-internalizing 

effect of substrates, such as d-amphetamine, might 

thus be useful in ameliorating symptoms of cocaine 

withdrawal. In limited preclinical trials, 

d-amphetamine has shown efficacy in reducing cocaine 

consumption and craving. 43 Amphetaminergic 

prodrugs would be desirable for this purpose, 

because their requisite metabolic activation after 

oral administration and slow onset of action would 

prevent the possibility of parenteral abuse, a clear 

concern in psychostimulant-addicted patients. The 

atypical psychostimulant compound modafinil also 

shows preclinical promise as a potential therapeutic 

for stimulant addiction. Modafinil decreases cocaine 

craving during withdrawal 149 and attenuates 

the effect of concomitantly administered cocaine 150 

or methamphetamine, 151 warranting investigation 

into its effects on DAT trafficking. There is also evidence 

that withdrawal from psychostimulant use 

is associated with deficits in not only dopaminergic 

but also serotonergic function. Moreover, agents 



that increase serotonergic activity can attenuate reinforcing 

effects mediated by excessive dopamine 

release. 152 Therefore, novel amphetaminergic substrates 

functioning as dual dopamine/serotonin 

releasers—but lacking the prooxidant metabolites 

of MDA-like compounds or the 5-HT2B receptor agonist 

activity of fenfluramine and its metabolites— 

may be beneficial as treatment agents. 153 

Conflicts of interest 

The authors declare no conflicts of interest. 

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