Amphetamine-induced loss of human dopamine transporter activity: An internalization-dependent and cocaine-sensitive mechanism 2018/4/16, 3+57 PM

Proc Natl Acad Sci U S A. 2000 Jun 6; 97(12): 6850–6855. PMCID: PMC18764
Published online 2000 May 23. doi: 10.1073/pnas.110035297 PMID: 10823899
Neurobiology

Amphetamine-induced loss of human dopamine transporter activity: An

internalization-dependent and cocaine-sensitive mechanism
Christine Saunders,* Jasmine V. Ferrer,† Lei Shi,† Jiayun Chen,† Gerald Merrill,‡ Maria E. Lamb,*
L. M. Fredrik Leeb-Lundberg,* Lucia Carvelli,§ Jonathan A. Javitch,†¶ and Aurelio Galli§ǁ
Departments of §Pharmacology and *Biochemistry, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX
78248; ‡Brooke Army Medical Center, San Antonio, TX 78234-6200; and †Center for Molecular Recognition and Departments of
Pharmacology and ¶Psychiatry, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, New York, NY 10032
ǁ
To whom reprint requests should be addressed. E-mail: galli@uthscsa.edu.
Edited by H. Ronald Kaback, University of California, Los Angeles, CA, and approved April 4, 2000

Received 2000 Jan 27

Copyright © The National Academy of Sciences

This article has been cited by other articles in PMC.

ABSTRACT Go to:

The dopamine transporter (DAT) is a target of amphetamine (AMPH) and cocaine. These
psychostimulants attenuate DAT clearance efficiency, thereby increasing synaptic dopamine (DA)
levels. Re-uptake rate is determined by the number of functional transporters at the cell surface as well
as by their turnover rate. Here, we present evidence that DAT substrates, including AMPH and DA,
cause internalization of human DAT, thereby reducing transport capacity. Acute treatment with AMPH
reduced the maximal rate of [3H]DA uptake, decreased AMPH-induced currents, and significantly
redistributed the immunofluorescence of an epitope-tagged DAT from the plasma membrane to the
cytosol in human embryonic kidney 293 cells. Conversely, DAT inhibitors, such as cocaine, mazindol,
and nomifensine, when administered with AMPH, blocked the reduction in [3H]DA uptake and the
redistribution of DAT immunofluorescence to the cytosol. The reductions of [3H]DA uptake and
AMPH-induced DAT internalization also were inhibited by coexpression of a dominant negative
mutant of dynamin I (K44A), indicating that endocytosis modulates transport capacity, likely through a
clathrin-mediated pathway. With this mechanism of regulation, acute application of AMPH would
reduce DA uptake not only by direct competition for uptake, but also by reducing the available cell-
surface DAT. Moreover, AMPH-induced internalization might diminish the amount of DAT available
for DA efflux, thereby modulating the cytotoxic effects of elevated extracellular DA.

Dopamine (DA) signaling in the central nervous system mediates a wide variety of physiologic
functions such as movement, motivational control of voluntary behavior, and lactation (1, 2). The
magnitude and duration of DA signaling is defined by the amount of vesicular release, the sensitivity of
the DA receptors, and the efficiency of DA clearance. The DA transporter (DAT) is largely responsible
for regulating DA clearance (3).

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Psychostimulants, such as cocaine and amphetamine (AMPH), induce DA overflow into the synaptic
cleft by acting on the DAT, thereby enhancing dopaminergic transmission (4). Cocaine acts by
inhibiting the re-uptake of released DA (5, 6). AMPH-like drugs, however, are thought to promote the
release of the transmitter (carrier-mediated efflux) as well as to inhibit its uptake (7, 8). Repeated
administration of AMPH has been shown to sensitize monoaminergic synapses to subsequent
psychostimulant challenge (9). Furthermore, administration of a single, high dose of AMPH acutely (1
h) decreased DAT function in vivo as assessed in striatal synaptosomes prepared from drug-treated rats
(10). In contrast, administration of a high dose of cocaine had no effect on subsequent transporter
activity (10).

To explore the mechanism for the differential effects of AMPH and cocaine on the homeostatic uptake
capacity of the human DAT (hDAT), we stably expressed a FLAG-tagged hDAT in EM4 cells (see
Materials and Methods). The use of the FLAG fusion protein has provided the opportunity for confocal
microscopy analysis of trafficking of the transporter in cells. Here, we report that AMPH caused the
hDAT to redistribute intracellularly in a dynamin-dependent manner, consequently reducing subsequent
DA transport capacity. These results provide a mechanism for the AMPH-induced elevation of synaptic
DA mediated through a reduction of the number of transporters on the cell surface.

MATERIALS AND METHODS Go to:

Cell Culture. We created a synthetic hDAT gene, which was tagged at the amino terminus with a
FLAG epitope. The gene encodes a protein with an amino acid sequence identical to that of wild-type
hDAT with the Met at position 1 replaced by MDYKDDDDKA, but the nucleotide sequence was
altered to increase the number of unique restriction sites and to optimize codon utilization. The
nucleotide sequence of this construct and its creation will be described elsewhere. The FLAG-tagged
syntheticDAT was subcloned into a bicistronic expression vector that expresses the syntheticDAT from
a cytomegalovirus promoter and the hygromycin resistance gene from an internal ribosomal entry site.
This vector, pCIHyg, was constructed by replacing the NsiI to XhoI fragment from pCIN4 (11) (kindly
provided by S. Rees, Glaxo Wellcome) with the NsiI to XhoI fragment from pIREShygro
(CLONTECH). The final construct is referred to as FLAG-hDAT, whereas the non-FLAG-tagged
hDAT construct is referred to as hDAT. (Some biotinylation experiments also were done in a FLAG-
hemagglutinin hDAT construct, in which the first 22 residues of the hDAT sequence in FLAG-hDAT
were replaced by a hemagglutinin tag.)

EM4 cells, human embryonic kidney (HEK) 293 cells stably transfected with macrophage scavenger to
increase their adherence to tissue culture plastic (12), were kindly provided by R. Horlick
(Pharmacopeia, Cranbury, NJ). EM4 cells were stably transfected with the FLAG-hDAT with
Lipofectamine (GIBCO/BRL) as described (13), and a stably transfected pool was selected in
hygromycin (250 µg/ml). Cells were grown in DMEM supplemented with 10% FBS at 37°C and 5%
CO2. In addition, in some experiments, HEK 293 cells stably transfected with the wild-type hDAT
cDNA in pCIN4 as described (13) were used.

Uptake of [3H]DA. EM4 cells expressing FLAG-hDAT were seeded in 96- or 24-well plates ≈48 h
before the experiments and grown to confluence (approximately 25,000 cells per well in 96-well plates,
100,000 cells per well for 24-well plates). The FLAG-hDAT cells were preincubated with or without 2
µM D-AMPH (Sigma) in serum-free DMEM (GIBCO) for 1 h at 37°C, unless stated otherwise. One
millimolar tropolone (Sigma) was added during the last 10 min of the incubation. The plate of cells was
placed on ice and washed three times with 200 µl ice-cold uptake buffer (130 mM NaCl/1.3 mM
KCl/10 mM Hepes/1.2 mM MgSO4/1.2 mM KH2PO4/2.2 mM CaCl2/10 mM glucose, pH 7.4). After

3
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the washes, the cells were placed on a 37°C plate warmer, and [3H]DA (NEN) uptake was performed
immediately. In triplicate wells, 7 nM [3H]DA together with 3 µM DA was added in a final volume of
50 µl unless otherwise stated. All reagents were diluted in uptake buffer prewarmed to 37°C. The
mixture was incubated at 37°C for 1 min and then aspirated to terminate uptake. After three washes
with ice-cold buffer, cells were permeabilized with 50 µl 0.1% Triton X-100. Radioactivity was
measured in a Trilux scintillation counter with OptiPhase Supermix mixture (Wallac, Gaithersburg,
MD). Specific uptake was defined as total uptake less nonspecific in the presence of 5 µM mazindol
(MZ).

The experimental determination of Km and Vmax after treatment with AMPH or vehicle was done as
described above with several modifications. Cells were treated for 4 h with 40 µg/ml cycloheximide at
37°C in serum-free DMEM to block the potential contribution of newly synthesized transporter coming
to the cell surface during the uptake assay. To minimize internalization during the assessment of
uptake, the uptake experiments were done at 18°C for 2 min. [3H]DA (100 nM) was used with nine
concentrations of unlabeled DA between 0.03 µM and 60 µM, and the data were fit by homologous
competition by nonlinear regression (PRISM, GraphPad, San Diego).
Transient Expression of Dominant Negative Dynamin. The stable FLAG-hDAT expressing EM4
cells were transiently transfected with the human wild-type and K44A mutant dynamin I in pCB1 by
using the calcium phosphate transfection method with overnight incubation in the presence of 1 µg of
DNA per 35-mm dish (immunofluorescence assays) and 0.25 µg per well of a 24-well plate (uptake
assays). Forty-eight hours after transfection, the cells were treated with either AMPH or vehicle for 1 h
at 37°C in uptake buffer, and uptake was determined as described above except at 10°C for 2 min in a
final volume of 250 µl of uptake buffer containing 50 nM [3H]DA, 100 µM ascorbic acid, and 100 µM
pargyline. The transfection efficiency was 70–80% as assessed by fluorescence microscopy of green
fluorescent protein (GFP) in parallel transient transfection with pGREEN LANTERNTM-1
(GIBCO/BRL) (data not shown).
Immunofluorescence Analysis. To examine the localization and trafficking of the hDAT in the
FLAG-hDAT cell line, cells were treated with AMPH (2 µM) at 37°C for 1 h unless otherwise stated.
Preincubations of 20 min were done for experiments involving Con A (250 µg/ml), MZ (3 µM),
nomifensine (2 µM), and cocaine (3 µM), followed by the addition of AMPH. The immunostaining
steps were conducted at room temperature while rocking the cells on a rotating platform. The
coverslips containing the cells (50–70% confluent) were washed twice with PBS (154 mM NaCl/11
mM Na2HPO4/2.7 mM KCl/1 mM MgCl2/0.1 mM CaCl2, pH 7.4). They then were fixed for 25 min
with 4% paraformaldehyde made up in PBS. The cells were rinsed twice with PBS and blocked with
5% normal goat serum diluted in 0.05% Triton X-100/PBS (PBST) for 1 h. The blocking solution then
was aspirated and the cells were rinsed once with PBST. The coverslips were incubated with the
primary antibody (anti-FLAG M-2 mAb, Sigma #F3165) at a dilution of 1:3,000 in PBST for 1 h. The
primary antibody was aspirated, and the cells were washed three times with PBST for 5 min per
incubation. After washing, the cells were incubated with secondary antibody [goat anti-mouse IgG
(H&L) TriTC, Kirkegarrd & Perry Laboratories #03–18-06] diluted 1:200 in PBST for 1 h. The
secondary antibody was removed and the cells were washed three times with PBST (5-min incubations
each), and once with PBS. Coverslips then were mounted onto slides with Crystal Mount (Biomedia,
Foster City, CA) and allowed to dry. Confocal microscopy was performed by using a Bio-Rad
MRC1024 confocal imaging system operated via a Nikon Diaphot inverted microscope. Sample
illumination was via a krypton-argon laser with 568-nm excitation and a 598 ± 40-nm emission filter.
LASERSHARP ACQUISITION software (Bio-Rad) allowed for z-resolution imaging at 1-µm increments.

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Biochemical Analysis of Transporter Endocytosis Using Cleavable Biotin. Cell surface

biotinylation with cleavable sulfo-N-hydroxysuccinimide-S-S-biotin (0.25 mg/ml) (Pierce) and
glutathione strip to cleave all surface-localized biotinylated transporter were performed on stably
transfected EM4 cells expressing FLAG-DAT (or FLAG-hemagglutinin-DAT) as described (14). After
biotinylation but before the strip, cells were incubated for 1 h at 37°C in DMEM supplemented or not
with 2 µM D-AMPH. Cells were scraped into PBS-PI buffer (PBS supplemented with 1 µg/ml
leupeptin, 1 µg/ml pepstatin, 2 µg/ml aprotinin, 2 µg/ml pefablock, and 10 mM N-ethylmaleimide).
Cells were pelleted at 4°C and incubated in PBS-PI supplemented with 0.2% digitonin at 4°C for 20
min. Cells were pelleted and then incubated in lysis buffer (PBS-PI containing 1% Triton X-100) at
4°C for 45 min. The extract was centrifuged at 4°C (14,000 × g) for 30 min. An aliquot of the extract
was removed for determination of total DAT. The remaining extract was incubated with 50 µl
neutrAvidin Plus beads (Pierce) for 1 h at room temperature. The beads were washed twice with lysis
buffer and then eluted with SDS sample buffer containing 100 mM DTT. The crude extracts and the
eluted proteins were resolved by SDS/PAGE, transferred to poly(vinylidene difluoride) membranes
(Millipore), and blocked for 1 h in 5% dry milk, 1% BSA, 0.1% Tween-20 in Tris-buffered saline.
FLAG-DAT (or FLAG-hemagglutinin-DAT) was detected by anti-FLAG M-2 primary antibody and
anti-mouse-horseradish peroxidase secondary antibody (Santa Cruz Biotechnology), with ECL-Plus
(Amersham Pharmacia) and fluorescence detection and quantitation on a Storm 840 imager (Molecular
Dynamics).
Electrophysiology. Parental or stably transfected cells were plated at a density of 105 per 35-mm
culture dish. Before electrical recording (performed at 25°C) attached cells were washed three times
with bath solution at room temperature. The bath contained: 130 mM NaCl, 1.3 mM KCl, 1.3 mM
KH2PO4, 0.5 mM MgSO2, 1.5 mM CaCl2, 10 mM Hepes, and 34 mM dextrose. The solution was
adjusted to pH 7.35 and 300 mOsm with 1 M NaOH and dextrose. Pipette solutions for the whole-cell
recording contained: 130 mM KCl, 0.1 mM CaCl2, 2 mM MgCl2, 1.1 mM EGTA, 10 mM Hepes, and
30 mM dextrose adjusted to pH 7.35 and 270 mOsm. Free Ca2+ in the pipette was calculated as 0.1
µM. Electrodes were pulled with a programmable puller (Sutter Instruments, Novato, CA, p-2000).
Series conductance was 0.1 µS or greater, and cell capacitance was 25 to 80 pF. Voltage steps ranged
from −140 to +60 mV and lasted 500 msec. Between test pulses membrane potential was held at −40
mV for 4 sec. Values for steady-state currents were taken between 400 and 500 msec after the step. An
Axopatch 200B amplifier band-limited at 5,000 Hz was used to measure current. Data were stored
digitally on VCR and analyzed on a Nicolet Integra oscilloscope and a Pentium computer, using
instrumentation and programs written by W.N. Goolsby (Emory University, Atlanta, available on
request).

RESULTS AND DISCUSSION Go to:

DA uptake was not observed in EM4 cells not transfected with the hDAT. Moreover, neither 2 µM
AMPH, 100 µM DA, nor 2 µM AMPH together with 3 µM MZ produced whole-cell inward currents in
untransfected cells. Thus, the EM4 cells provided a suitable null background in which to study the
function and trafficking of hDAT stimulated by AMPH.
Characterization of the FLAG-hDAT Cell Line. The regulation of DAT by intracellular signaling,
and in particular by protein kinase C, has been described (15–18). Using biochemical and
electrophysiological approaches, those authors demonstrated that trafficking of DAT can play a role in
the function of the transporter. The ability to use a FLAG-hDAT fusion protein for
immunocytochemistry in parallel with uptake and electrophysiological strategies offers a means to
understand whether hDAT cell surface distribution is regulated by substrates and by inhibitors of the

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carrier.

Addition of the N-terminal FLAG tag did not significantly alter [3H]DA uptake by the transporter,
which had a Km of 1.2 ± 0.3 µM and a Vmax of 239 ± 42 fmol/min per well (n = 3). The function of the
transporter was further assessed electrophysiologically. As seen in Fig. 1A, the FLAG tag did not
perturb the ability of the transporter to produce substrate-induced currents. In the whole-cell
configuration, the membrane potential of the cell was held at −40 mV and then the voltage was stepped
to a new potential between −140 mV and 0 mV in 20-mV increments. The application of DA or AMPH
induced a substrate-mediated current (19, 20) in both the hDAT and the FLAG-hDAT cells. The
substrate-induced current was defined as the current recorded at a particular voltage in the presence of
substrate (in these experiments, 100 µM DA) at steady state, minus the current recorded at the same
potential in the presence of the substrate and 10 µM MZ. The substrate-induced current had a similar
steady-state voltage dependence in the hDAT and the FLAG-hDAT cell line.

Open in a separate window

Figure 1

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Transporter function and expression of the FLAG-hDAT cells. (A) Current-voltage relationships of the DA-
induced whole-cell currents from either hDAT (○) or FLAG-hDAT (■) cells. The DA-induced current is
defined as the whole-cell steady-state current recorded upon DA bath application minus the current obtained
in the presence of DA and MZ at a defined membrane voltage. The membrane potential was held at −40 mV,
stepping the voltage between −140 and 0 mV for 500 msec, with an interval of 4 sec. Data were normalized at
−140 mV. Confocal microscopy images of hDAT cells reveal no significant fluorescence (B), whereas
confocal images of the FLAG-hDAT cells show a strong cell surface staining pattern (C). Z sections of the
FLAG-hDAT (D), 1 µm in thickness, go from the top to the bottom of the cell; no intracellular
immunofluorescence was seen in any of the six focal planes.

Confocal microscopy images of the immunofluorescence of the non-FLAG-tagged hDAT cell showed
no staining with the antibody directed against the FLAG epitope, as seen in Fig. 1B. In Fig. 1C,
however, a strong peripheral cell surface staining pattern was observed with the FLAG-hDAT cells.
This is consistent with the steady-state localization of a GFP-hDAT fusion construct in MDCK cells
(18). The immunofluorescence in Fig. 1C is almost exclusively on the cell surface, as evidenced by
sectioning through the cells at 1-µm steps (Fig. 1D) in the x-z plane. A very small amount of
intracellular fluorescence was observed, however, which might be caused by anterograde and
retrograde trafficking of the transporter under steady-state conditions.

These data show that the FLAG-hDAT retained the functional characteristics of the hDAT, including
affinity for DA, maximal rate of transport, voltage dependence of the substrate-induced current, and
predominant steady-state cell surface localization.

Acute AMPH Treatment Causes a Decrease in [3H]DA Uptake, AMPH-Induced Current, and
Cell Surface FLAG-hDAT.
In cells pretreated with 2 µM AMPH for 1 h, the uptake of [3H]DA was significantly decreased (78 ±
5% relative to control) (n = 5; P < 0.01 by paired Student's t test). This decrease in uptake was caused
by a decrease in Vmax, without a change in Km [Km was 1.2 ± 0.3 µM for both vehicle and AMPH
treatment; Vmax was 239 ± 42 and 189 ± 35 fmol/min per well for vehicle and AMPH treatment,
respectively (n = 3; P < 0.025 by paired Student's t test)]. A similar decrease in uptake was observed
after preincubation with 10 µM DA (73 ± 4% relative to control) (n = 5; P < 0.01 by paired Student's t
test).

To further evaluate the reduction of DAT functional activity upon AMPH treatment, we evaluated the
time dependence of AMPH-induced currents. Fig. 2A shows that a bath application of 2 µM AMPH
generated an inward current in FLAG-hDAT cells. In the whole-cell configuration, the voltage was
stepped from a holding potential of −40 mV to −120 mV for 500 msec while recording a control
current (control). After perfusing the cell with 2 µM AMPH, an increase of the steady-state inward
current (AMPH) was detected. Both currents were blocked by addition of 3 µM MZ with AMPH still
present (AMPH & MZ). MZ by reducing the control current is unmasking the existence of an hDAT
leak current that has been described both for the hDAT and other neurotransmitter transporters (19–22).
The AMPH-induced current was defined as the current recorded in the presence of AMPH, minus the
current recorded after addition of MZ to the bath with AMPH still present, and was analyzed as a
function of time (between 0 and 40 min). The AMPH-induced current reached stability between 1 and
2 min after the addition of AMPH, and then decreased in a sigmoidal fashion over time. The time until
the onset of this decreasing phase varied from cell to cell [between 5 and 14 min, averaging 8.75 ± 1.93
min (SEM, n = 4) after AMPH application]. To compare the rates of decline between different cells,
time 0 was defined as the experimental time point 2 min before the AMPH-induced current began to

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decline. The current was normalized to the AMPH-induced current recorded at time 0 and plotted over
time (Fig. 2B). Because the equilibrium potential of the AMPH-induced current was stable at −7.9 ±
1.7 mV during the duration of the experiments, it is unlikely that the loss of functional hDAT activity
estimated by measuring the AMPH-induced current was caused by either a loss of ion gradients or by
an intracellular accumulation of AMPH. It is noteworthy that AMPH reduced the substrate-induced
currents to a greater extent than it reduced [3H]DA uptake. Thus, AMPH may have additional effects
on the ionic movements generated by substrate-induced DAT activity that cannot be detected in uptake
assays (see below). Alternatively, the difference in the magnitude of the effect may result from
differences between single cells assayed under voltage clamp as compared with determinations of
[3H]DA uptake in unclamped populations of cells.

Figure 2

AMPH-induced loss of hDAT function and hDAT cell surface expression. (A) Whole-cell currents recorded
from FLAG-hDAT cells. In this representative experiment, the cell was held at −40 mV and then the voltage
was stepped to −120 mV for 500 msec. The labels refer to: control current before addition of substrate
(control), current after addition of 2 µM AMPH to the bath (AMPH), and inhibited current after addition of 5
µM MZ to the bath (AMPH & MZ) with AMPH still present. (B) AMPH induced a loss of hDAT whole-cell
current over time. Cells were held at −40 mV and then the voltage was stepped to −120 mV for 500 msec
every 1–2 min. Upon AMPH bath application, the hDAT inward current increased, reaching the maximum
value over a time of 1–2 min. After several minutes of stability, the AMPH-induced current began to
decrease. From this point, the AMPH-induced current recorded at −120 mV was plotted against time (each
symbol type represents a single cell; n = 4). The current recorded during the different experimental time
points was normalized to the AMPH-induced current recorded at a virtual time 0 (2 min before the onset of
the decreasing phase). (C) Confocal microscopy images in the absence (control) and presence of 2 µM AMPH
added for 1 h. The galleries are of 1-µm sections of patches of cells or single cells treated with AMPH
showing extensive intracellular immunofluorescence compared with control conditions.

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AMPH-induced loss of transporter activity was further investigated by using confocal microscopy.
AMPH (2 µM) caused a significant intracellular accumulation of FLAG-hDAT, as seen in Fig. 2C: the
cell surface fluorescence intensity became weaker with a significant amount of transporter found in the
cytosol. Confocal microscopy images of 1 µm z-sectioning (Fig. 2C) illustrate that the fluorescence
shifted substantially from the cell surface to the intracellular compartment, and intracellular vesicles
are now clearly visible in the gallery of the 1-µm steps from the top to the bottom of the cells. The loss
of cell surface transporter caused by 2 µM AMPH was seen as early as 20 min (data not shown) and
was maximal at 1 h, the incubation time chosen for further studies. The AMPH phenomenon was also
concentration dependent, as a greater loss of cell surface transporter was seen at 50 µM compared with
2 µM AMPH (data not shown). However, 2 µM AMPH, a value approximating the Km for AMPH (20),
was chosen to facilitate the removal of AMPH in subsequent uptake assays. Cell surface redistribution
of hDAT was also evident with DA at 100 µM (data not shown). Taken together, these data imply that
there is a substrate-induced down-regulation of the hDAT activity, which is likely a consequence of cell
surface redistribution of the transporter.
Inhibitors of DAT, Such as Cocaine and MZ, Block the AMPH-Induced Loss of Cell Surface
FLAG-hDAT.
Pretreatment of the FLAG-hDAT cells with the DAT uptake inhibitors cocaine and MZ (Fig. 3C–F)
and nomifensine (data not shown) blocked the AMPH-mediated redistribution of FLAG-hDAT. The
effect of AMPH alone is shown in Fig. 3B. The lack of significant intracellular fluorescence in the
presence of either AMPH plus cocaine or AMPH plus MZ is evident in the 1-µm z-sections (Fig. 3 E
and F) from the top to the bottom of the cells. Neither MZ nor cocaine caused internalization when
applied in the absence of AMPH (data not shown).

Figure 3

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AMPH-induced loss of cell surface FLAG-hDAT was inhibited by blockers of hDAT activity. Confocal
microscopy images of FLAG-hDAT in the presence of vehicle (A), 2 µM AMPH (B), 2 µM AMPH and 3 µM
cocaine (C and E), and 2 µM AMPH and 3 µM MZ (D and F). The galleries (E and F) are 1-µm z-sections of
patches of FLAG-hDAT from the top to the bottom of the cells. (G) Uptake studies of [3H]DA were done in
the absence and presence of 2 µM AMPH and in the presence of 2 µM AMPH and 3 µM cocaine. AMPH
significantly inhibited [3H]DA uptake, and this effect was significantly attenuated by coincubation with
cocaine (* = P < 0.05; ** = P < 0.01 by paired Student's t test). [3H]DA uptake was expressed as a percentage
of inhibition of uptake under control conditions. The data are the mean ± SEM of five experiments performed
in triplicate.

Cocaine, which impaired the AMPH-induced trafficking of hDAT, also blocked the AMPH-induced
inhibition of [3H]DA uptake (Fig. 3G). Fig. 3G shows that the AMPH-mediated inhibition of [3H]DA
uptake (22 ± 5%) (n = 5; P < 0.01 by paired Student's t test) was significantly attenuated when the
FLAG-hDAT cells where pretreated (20 min) and coincubated with 3 µM cocaine (5 ± 4%) (n = 5; P <
0.05 by paired Student's t test). Thus, different classes of psychostimulants, such as AMPH and
cocaine, appear to have opposite effects on the cell surface distribution and functional activity of
hDAT.

AMPH-Induced Loss of Cell Surface hDAT Is an Internalization Event That Is Dynamin

Dependent and Results in Reduced Transporter Capacity.
The increase in intracellular FLAG-hDAT caused by AMPH may be caused by sequestration and
internalization of the transporter or result from nascent transporter being held up in the cytosol (i.e.,
hDAT never reached the cell surface). To test this, FLAG-hDAT expressing cells were pretreated with
250 µg/ml of Con A to prevent internalization by stabilizing cell surface integrity caused by tetravalent
lectin contacts (23). As seen in Fig. 4, pretreatment with Con A (Fig. 4B) largely prevented the AMPH-
mediated loss of cell surface FLAG-hDAT (Fig. 4A). Preincubation with Con A in the absence of
AMPH resulted in an immunofluorescence profile similar to that of control (data not shown).

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Open in a separate window

Figure 4

AMPH-induced internalization of FLAG-hDAT is dynamin dependent. Confocal microscopy images of

FLAG-hDAT cells incubated in the presence of either 2 µM AMPH for 1 h (A) or 2 µM AMPH plus 250
µg/ml Con A (B). Confocal microscopy images from FLAG-hDAT cells transiently transfected with either the
dominant negative dynamin I mutant, K44A (C), or the wild-type dynamin (D) in the presence of 2 µM
AMPH. (E) Uptake of [3H]DA (at 10°C), expressed as a percent of control, after 1 h preincubation with 2 µM
AMPH in FLAG-hDAT cells, FLAG-hDAT cells transiently expressing the mutant dynamin, K44A, or the
wild-type dynamin. The data are the mean ± SEM of three different experiments performed in triplicate (* =
P < 0.05 by paired Student's t test).

Cell-surface biotinylation experiments also demonstrated the presence of AMPH-induced

internalization. Incubation with 2 µM AMPH for 1 h resulted in 27.1 ± 7.9% internalization (n = 4).
This demonstrates that AMPH produced its effect through hDAT internalization and not by interfering
with the delivery of the protein to the cell surface. The extent of internalization was similar to the

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extent of inhibition of uptake.

To examine the cellular mechanism by which this endocytotic phenomenon occurred, we explored the
possible role of clathrin-coated pits by coexpressing the FLAG-hDAT with a dominant negative mutant
of dynamin I (K44A) or wild-type dynamin I. Dynamin is a GTPase that catalyzes the pinching off of
clathrin-coated pits from the cell surface (24, 25). Coexpression with K44A (Fig. 4C) inhibited the
AMPH-mediated internalization of FLAG-hDAT whereas wild-type dynamin (Fig. 4D) did not.
Similarly, transient overexpression of K44A has been shown to block internalization of G protein-
coupled receptors (26, 27). These data suggest that AMPH-mediated internalization of hDAT may
require clathrin-coated pits. Although dynamin also has been suggested to play a role in the budding of
caveolae (28, 29) as well as in signal transduction (30, 31), a clathrin-mediated internalization of the
hDAT by AMPH seems more likely. A similar block by K44A dynamin of phorbol ester-mediated
internalization of GFP-hDAT in MDCK cells has been reported (18), and this was interpreted as a
clathrin-mediated mechanism as well.

We examined whether the AMPH-induced decrease in DA uptake (Fig. 3G) was a direct effect of the
FLAG-hDAT internalization process itself, rather than a reduction in the maximal rate of transport
mediated by enzymatic modification of the transporter, such as phosphorylation. The FLAG-hDAT cell
line was transiently transected with either wild-type or K44A dynamin, or neither. After 1 h
preincubation with 2 µM AMPH, DA uptake was significantly decreased, both in FLAG-hDAT cells
(56 ± 17% relative to control) (n = 3; P < 0.05 by paired Student's t test) and FLAG-hDAT cells
transiently transfected with wild-type dynamin 1 (52 ± 20% relative to control) (n = 3; P < 0.05 by
paired Student's t test). In contrast, uptake in cells transiently transfected with K44A was unaffected by
pretreatment with AMPH (103 ± 3% relative to control) (n = 3). These data further support AMPH-
induced internalization of hDAT as being responsible for the decrease of transporter activity observed
with acute AMPH treatment.

Although it is well known that AMPH increases the concentration of DA at the synaptic cleft, the
underlying mechanism has been under considerable debate. Changes in hDAT activity and the
extracellular DA concentration caused by AMPH have been ascribed to a counter transport mechanism
by the carrier (7, 32). Regulation of transporter surface distribution may represent an additional
mechanism by which neurons modulate transport capacity in response to psychostimulants such as
AMPH as well as elevated levels of extracellular DA. In fact, our data show that acute exposure to
AMPH in FLAG-hDAT cells reduced surface transport activity, as assessed by [3H]DA uptake, AMPH-
induced currents (Figs. 2B and 3G), and cell surface localization of FLAG-hDAT as determined with
both confocal microscopy (Fig. 2C) and cell surface biotinylation.

Single high doses of AMPH and methamphetamine were found to reduce [3H]DA uptake in
synaptosomes prepared from the striatum of rat brains 1 h after drug administration without altering the
amount of overall hDAT protein (10, 33). This decrease resulted from a decrease in transporter Vmax
that recovered within 24 h. Our in vitro finding of AMPH-induced hDAT internalization may provide a
potential mechanism for these observations. Curiously, methamphetamine administration did not
reduce uptake in the nucleus accumbens (33), suggesting that this effect may not occur in all brain
regions and may be subject to complex regulation.

Because hDAT controls DA clearance from the extracellular fluid, a decrease in the number of
transporters on the cell surface because of AMPH exposure would result in an increase in extracellular
DA. Therefore, loss of membrane transporter number might contribute to neurotoxicity by decreasing
DA clearance, and consequently increasing extracellular DA (34, 35). Conversely, a reduction of cell
surface hDAT may be a neuroprotective mechanism in that it could diminish the amount of carriers

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Amphetamine-induced loss of human dopamine transporter activity: An internalization-dependent and cocaine-sensitive mechanism 2018/4/16, 3+57 PM

available for DA efflux upon AMPH stimulation.

Cocaine inhibits the ability of hDAT to traffic in response to AMPH stimulation, indicating that there
are different mechanisms of action for these two psychostimulants. Transport blockers, such as MZ and
nomifensine, appear to behave like cocaine. Therefore, hDAT ligands capable of inhibiting DAT
activity (e.g., cocaine, MZ, and nomifensine) may not only act by preventing the reuptake of substrate,
but also by preventing the internalization of transporter itself.

A number of studies have implicated internalization of neurotransmitter transporters as a possible

means of modulating transporter activity (15, 36–39). In human embryonic kidney 293 cells, serotonin
(5HT) was found to inhibit the phorbol ester-mediated phosphorylation, and the associated
internalization, of the serotonin transporter (SERT) (39). A homeostatic mechanism was proposed for
this effect, in that elevated synaptic levels of 5HT could prevent the internalization of SERT and
thereby maintain effective levels of the transporter at the cell surface where it would help to clear the
elevated 5HT. Similarly, a 1-h exposure to γ-aminobutyric acid was shown to slow internalization of
GAT1, another member of this gene family (40). Interestingly, the effect we observed in DAT, in the
same parental line of cells as the SERT studies, is opposite to that reported with SERT and GAT1,
suggesting a markedly different mechanism of regulation of these closely related transporters.

A recent study with a GFP-hDAT fusion protein stably expressed in MDCK cells reported that 10 µM
DA did not significantly affect the cellular distribution of GFP fluorescence (18). Whether this
discrepancy with our data relates to the concentration of DA, to an impairment in substrate-mediated
internalization of GFP-DAT related to the presence of the N-terminal GFP “tag” compared with the
much smaller FLAG tag, or from differences in the cell lines used is not yet clear.

It should be noted that AMPH decreased FLAG-hDAT-mediated currents more rapidly and to a greater
extent than it reduced DA uptake or produced internalization. Thus, it is possible that AMPH caused a
modification of the transporter, such as phosphorylation, which rapidly decreased substrate-induced
currents. Indeed, the regulation of DAT by phorbol esters, presumably mediated by protein kinase C,
has been described (15–18). Clearly, however, at 1 h AMPH-induced internalization of hDAT and
decreased uptake was blocked by dominant negative dynamin I, suggesting that these effects also
depend on endocytosis and cannot be fully explained simply by a modification of the transporter.
Moreover, we see evidence of internalization by cell surface biotinylation of a magnitude relatively
consistent with the observed changes in uptake, but less than the AMPH-induced reduction in current.
Thus, the inactivation of hDAT upon AMPH application is largely but perhaps not exclusively
controlled by the trafficking of this carrier.

Taken together our data suggest that cell surface redistribution of hDAT is a mechanism that
contributes to the enhancement of extracellular DA levels in response to psychostimulants such as
AMPH. Greater understanding of this pathway and its mechanistic details may lead to novel cellular
targets for substance abuse therapies.

ACKNOWLEDGMENTS Go to:

We are very grateful to Eileen Grass for her technical support and superb maintenance of the cell lines
and to Drs. Alan Frazer, Richard Lamb, William Clarke, Lynette Daws, and Myles Akabas for their
helpful comments and review of this manuscript. We also thank Dr. Marc Caron for the kind gift of
wild-type dynamin and dynamin (K44A) DNA. This work was supported by a grant from the National
Alliance for Research on Schizophrenia and Depression (to A.G.), National Institutes of Health Grant
GM41659 (to L.M.F.L.-.L.), and by a Grant-in-Aid from the American Heart Association and National

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Amphetamine-induced loss of human dopamine transporter activity: An internalization-dependent and cocaine-sensitive mechanism 2018/4/16, 3+57 PM

Institutes of Health Grants MH57324, DA11495, and DA12408 (to J.A.J.).

ABBREVIATIONS Go to:

DA dopamine

DAT dopamine transporter

hDAT human DAT

AMPH amphetamine

MZ mazindol

GFP green fluorescent protein

PBST 0.05% Triton X-100/PBS

FOOTNOTES Go to:
This paper was submitted directly (Track II) to the PNAS office.
Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073/pnas.110035297.
Article and publication date are at www.pnas.org/cgi/doi/10.1073/pnas.110035297

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