Regulation of the dopamine transporter - Addiction Research ...

Schmitt & Reith DAT Regulation
around 1 min, before quickly dropping back to baseline
levels within 3 min. In accordance with most
substrate-induced trafficking studies, DAT surface
expression began to drop below vehicle levels
after 20 min of amphetamine exposure. Curiously,
dopamine itself was not found to induce the same
immediate upregulation of surface DAT expression
as amphetamine, indicating that this biphasic
trafficking pattern might not extend to all DAT
substrates and may contribute to amphetamine’s
ability to stimulate dopamine efflux via the DAT. 31
However, a more recent study from this group—
using total internal reflection fluorescent microscopy
to visualize hDAT trafficking dynamics in
N2Aneuroblastomacellswithreal-timetemporal
resolution—shows evidence that both dopamine
(10 �M) and d-amphetamine (5 �M) induce
changes in surface DAT expression within seconds,
but the effect of amphetamine persisted longer after
substrate washout. 32
Effects of DAT inhibitors
Compared with the robust literature on DAT regulation
by phenethylamine substrates, there is a paucity
of studies investigating the acute regulatory effects
of DAT inhibitors. The few in vitro regulatory studies
using heterologous cell systems, as well as the bulk
of in vivo and clinical studies, have focused solely
on the classical DAT inhibitor cocaine, doubtless a
result of its notorious reputation as one of the most
addictive compounds known to humans. 33 Atypical
DAT inhibitors lacking cocaine-like abuse potential
34,35 remain almost entirely uninvestigated;
thus, it is not yet possible to make global statements
regarding the regulatory effects of inhibitors
other than cocaine. However, at least for cocaine,
it appears that nontranslocated DAT inhibitors not
only block the acute regulatory effects of substrates,
they may exert the opposite effect: insertion of DATs
from the endosomic recycling pool into the plasma
membrane. 16,36
Chronic administration of cocaine upregulates
striatal DAT expression in rhesus monkeys, an
effect that persists for more than 30 days after
cocaine withdrawal. 37 Increased DAT expression
has also been shown in postmortem analyses of
brain tissue from human cocaine addicts 38 and
synaptosomes prepared from this tissue exhibit
greater [ 3 H]dopamine uptake than synaptosomes
from age-matched cocaine-naïve individuals. 39 In
addition, cyclic voltammetry studies of rats that
exhibit a self-administration preference for high
doses of cocaine reveal a specific increase in the
V max of dopamine uptake with no effect on the
Km for dopamine, suggesting an upregulation of
DAT expression. 40 Interestingly, these results are
exactly inverted compared with the findings for
d-methamphetamine, indicating that the two psychostimulants
possess opposing effects on DAT trafficking,
despite their comparably high abuse potentials.
Different effects on plasmalemmal DAT
expression may play a role in the observed efficacy
of d-amphetamine for the treatment of cocaine
dependence in preclinical and early clinical
trials. 41–44
In studies of hDAT-expressing cultured cells,
preincubation with cocaine has been routinely
demonstrated to prevent the DAT-internalizing effects
of substrates (e.g., Refs. 23, 24, and 45), as well
as the putative amphetamine-induced brief, transient
upregulation of surface DAT levels. 31,32 The
acute effects of cocaine on DAT trafficking in the
absence of substrates are less consistent: whereas
some research groups have shown that cocaine elicits
a rapid increase in plasmalemmal DAT expression,
46,47 others have found no effect of cocaine pretreatment
on DAT activity or surface expression. 24,48
In the study by Little et al., treatment of N2A-hDAT
cells with 1 �M cocaine for 24 h increased cell
surface DAT expression by approximately 30%. 47
Remarkably, this same magnitude of increase was
detected using a wide array of techniques, including
biotinylation, intact-cell [ 3 H]CFT binding, and
visualization of plasma membrane anti-DAT immunofluorescence
with confocal microscopy. 47 The
long incubation time used in this study (24 h) may
underlie some of the discrepancy with other reports
showing no effect of cocaine, because most studies
have used incubation times on the order of 30 min to
1 h—curiously, however, Daws et al.demonstrateda
cocaine-mediated increase in surface expressed DAT
in HEK-hDAT cells after only 1 h of treatment. 46
Cellular signaling cascades,
phosphorylation, and DAT regulation
DAT phosphorylation and trafficking
Members of the neurotransmitter sodium symporter
superfamily are large proteins—the hDAT
possesses 620 amino acid residues—containing
Ann. N.Y. Acad. Sci. 1187 (2010) 316–340 c○ 2010 New York Academy of Sciences. 321