Cocaine Increases Dopamine Uptake and Cell Surface
Expression of Dopamine Transporters
Lynette C. Daws,* Paul D. Callaghan,* Jose´ A. Moro´n,*
,
† Kris M. Kahlig,*
Toni S. Shippenberg,† Jonathan A. Javitch,‡ and Aurelio Galli*
,1
*Department of Pharmacology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive,
MC 7764, San Antonio, Texas 78229-3900; †Integrative Neuroscience Section, NIDA IRP, 5500 Nathan Shock Drive,
Baltimore, Maryland 21224; and ‡Center for Molecular Recognition, College of Physicians and Surgeons,
Columbia University, 630 West 168th Street, New York, New York 10032
Received December 23, 2001
In HEK 293 cells expressing the human dopamine
transporter (DAT), a 10-min incubation with 10
M
cocaine followed by extensive washing resulted in a
30% increase in [
3
H]dopamine (DA) uptake as well as
an increase in cell surface DAT in biotinylation exper-
iments. Consistent with this novel regulation, [
3
H]DA
uptake into synaptosomes prepared from the nucleus
accumbens of rats sacrificed 30 min after a single co-
caine injection (30 mg/kg) was significantly increased
compared to controls (56% increase in V
max
, no change
in K
m
). In addition, DA clearance in the striatum of
anesthetized ratswas increased after local application
of a low (3 pmol) but not high (65 pmol) dose of cocaine,
presumably as a result of mobilization of DAT to the
cell surface. Cocaine-induced increases in cell surface
expression of DAT and associated changes in DA clear-
ance represent a novel mechanism that may play a
role in its addictive properties.
© 2002 Elsevier Science (USA)
Key Words: cocaine; dopamine transporter; traffick-
ing; uptake; biotinylation; chronoamperometry; stria-
tum; nucleus accumbens.
The dopamine transporter (DAT) is a critical site of
cocaine’s action and is thought to mediate the acute
behavioral and reinforcing effects that contribute to its
sensitization and subsequent abuse liability. In partic-
ular, altered dopamine (DA) neurotransmission in the
nucleus accumbens (NAcc) and striatum has been rec-
ognized as an important element of the abuse potential
of cocaine (e.g., 1). There are conflicting reports, how-
ever, concerning changes in DAT function as a mech-
anism contributing to addiction. Some studies have
found changes consistent with reduced activity of DAT,
including reduced DA uptake (2), down-regulation of
DAT (3) and elevated extracellular fluid (ECF) concen-
trations of DA (4). In contrast, other groups have re-
ported changes consistent with increased activity of
DAT, including increased DA uptake (5–7) and an at-
tenuation of cocaine-induced increases in ECF levels of
DA (8). The variable effect of cocaine on uptake likely
reflects the use of different dosing regimes, routes of
administration, brain regions, ECF concentration of
DA at the time of cocaine administration, as well as the
techniques to quantify DAT function (5, 9, 10).
A potential mechanism by which cocaine could in-
crease DA uptake is by mobilizing DAT to the plasma
membrane, thereby increasing the uptake capacity of
the system. We made use of HEK 293 cells stably
expressing the human DAT (hDAT) to examine
cocaine-induced trafficking of hDAT. In addition, we
used both in vitro and in vivo systems to measure DA
clearance. The results indicate that cocaine-induced
increases in DA clearance can be mediated through an
increase in the number of DATs on the cell surface.
MATERIALS AND METHODS
Plasmid construction and cell culture. A fluorescently tagged
hDAT was constructed by fusing the C-terminus of the coding region
of enhanced yellow fluorescent protein (YFP) from pEYFP-N1 (Clon-
tech) to the N-terminus of a human synthetic DAT cDNA (11),
thereby creating the fusion construct YFP-hDAT. This construct was
subcloned into the bicistronic vector, pCIHyg (11). EM4 cells, a HEK
293 cell line stably transfected with macrophage scavenger to in-
crease adherence to tissue culture plastic (12), were kindly provided
by R. Horlick (Pharmacopeia, Cranberry, NJ). EM4 cells were stably
transfected with YFP-hDAT (see 11).
Uptake of [
3
H]DA into EM4 cells expressing YFP-hDAT. YFP-
hDAT cells were seeded into 24-well plates ϳ24 h prior to experi-
ments and grown to confluence (approximately 100,000 cells per
well). After5hofserum starvation, the cells were incubated at 37°C
for 5 to 60 min. with 10
M cocaine in uptake buffer (120 mM NaCl,
4.7 mM KCl, 10 mM Hepes, 100
M ascorbic acid, 5 mM Tris base,
2.2 mM CaCl
2
, 10 mM glucose, pH 7.4) and uptake of 50 nM [
3
H]DA
(50 Ci/mmol NEN), plus 15
M cold DA, was then measured imme-
1
To whom correspondence and reprint requests should be ad-
dressed. Fax: (210) 567 4300. E-mail: galli@uthscsa.edu.
Biochemical and Biophysical Research Communications 290, 1545–1550 (2002)
doi:10.1006/bbrc.2002.6384, available online at http://www.idealibrary.com on
1545 0006-291X/02 $35.00
© 2002 Elsevier Science (USA)
All rights reserved.