Regulation of the dopamine transporter - Addiction Research ...

DAT Regulation Schmitt & Reith
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
320 Ann. N.Y. Acad. Sci. 1187 (2010) 316–340 c○ 2010 New York Academy of Sciences.