Amphetamine-induced Dopamine Efflux
A VOLTAGE-SENSITIVE AND INTRACELLULAR Na+-DEPENDENT
MECHANISM*
Habibeh
Khoshbouei
§,
Hongwei
Wang
§,
James D.
Lechleiter¶,
Jonathan A.
Javitch
, and
Aurelio
Galli
**
From the
Department of Molecular Physiology and
Biophysics and the Center for Molecular Neuroscience, Vanderbilt
University, Nashville, Tennessee 37232, the ¶ Department of
Cellular and Structural Biology, University of Texas Health Science
Center, San Antonio, Texas 78229, and the
Center for Molecular
Recognition and the Departments of Pharmacology and Psychiatry, College
of Physicians and Surgeons, Columbia University,
New York, New York 10032
Received for publication, December 17, 2002, and in revised form, January 27, 2003
 |
ABSTRACT |
Amphetamine (AMPH) elicits its behavioral effects
by acting on the dopamine (DA) transporter (DAT) to induce DA overflow
into the synaptic cleft. Facilitated exchange diffusion is the
classical model used to describe AMPH-induced DA efflux. This model
hypothesizes that AMPH-induced DA efflux is mediated by DAT and results
from the transport of AMPH into the cell followed by a counter movement of DA out to the extracellular compartment. To further characterize the
action of AMPH, we used the patch clamp technique in the whole-cell configuration combined with amperometry on human embryonic kidney HEK-293 cells stably transfected with the human DAT (DAT cells). In DAT
cells, AMPH-induced DAT-mediated currents were blocked by cocaine. We
demonstrate that DA efflux mediated by DAT is
voltage-dependent, electrogenic, and dependent on
intracellular Na+ concentration in the recording
electrode. Intracellular Na+ fluorescence, as measured by
confocal microscopy using a Na+-sensitive dye, was enhanced
by AMPH application. Furthermore, the ability of AMPH to induce DA
efflux was regulated by intracellular Na+ concentration and
correlated with the size of the DAT-mediated, AMPH-induced ion flux
across the plasma membrane. In the absence of intracellular
Na+ but the presence of high intracellular
Cl
, AMPH-induced inward currents elicited DA efflux
proportionally to their dimension and duration. Thus, we propose that
AMPH-induced DA efflux depends on two correlated transporter processes.
First, AMPH binds to the DAT and is transported, thereby causing an
inward current. Second, because of this AMPH-induced inward current, Na+ becomes more available intracellularly to the DAT,
thereby enhancing DAT-mediated reverse transport of DA.
 |
INTRODUCTION |
The dopamine transporter
(DAT)1 is thought to control
the temporal and spatial action of released dopamine (DA) by rapid
reuptake of the neurotransmitter into presynaptic terminals. The
interaction of amphetamine (AMPH) with DAT, which induces DA overflow
into the synaptic cleft, is thought to mediate the acute behavioral and
reinforcing effects of this psychostimulant (1). Although some of the
initial steps of AMPH action have been clarified, the mechanism
driving the AMPH-induced reverse transport of DA at the plasma membrane
remains controversial. The reverse transport of DA occurs by a
carrier-mediated release, which is not dependent on action potential
depolarization and is only slightly calcium-dependent (2).
One of the models used to explain this AMPH effect is the facilitated
exchange diffusion model (3, 4). This model proposes that AMPH-induced
DA release is mediated by DAT and results from translocation of AMPH
into the cell followed by a counter movement of DA out to the
extracellular compartment. By acting as a substrate, AMPH increases the
number of inward-facing transporter binding sites and thus increases
the rate of reverse transport. In contrast, the weak base or vesicle
depletion model (5, 6) proposes that the elevated cytoplasmic DA
concentration and its altered gradient across the plasma membrane
caused by the AMPH-mediated depletion of synaptic vesicles induce a
reversed transport of DA. This process could be independent of AMPH
interacting with DAT (7). Based mainly on the observation that DA
displacement from vesicles by Ro4-1248 and reserpine-like compounds
does not cause DA efflux, Jones et al. inferred that,
although vesicular depletion is rate-limiting, the facilitated exchange
diffusion mechanism is critical for AMPH-induced DA release (8).
Therefore, simply increasing the intracellular DA concentration was not
enough to cause DA efflux, and an interaction of AMPH with DAT was
essential as well.
However, other intriguing experiments have challenged a simple model of
facilitated exchange diffusion (9-13). For example, it has been shown
that PKC activation leads to an immediate increase in outward transport
of DA from the homologous norepinephrine transporter (13) and that
protein kinase C (PKC) inhibitors block AMPH-induced DA release via DAT
in the striatum in a synaptic vesicle-independent process (14). In
addition to signaling pathways, intracellular ions such as
Ca2+ (13), Na+, and Cl
(9, 10,
15, 16) have been implicated in AMPH-induced DA efflux. In particular,
a relationship between intracellular Na+ and the mechanism
of substrate-induced monoamine efflux has been suggested (9, 10,
16-18), and an increased intracellular Na+ concentration
has been proposed to be an important step for reversing the action of
monoamine transporters (9, 18, 19). The ability of AMPH to stimulate
reverse transport of DA was further proposed to result from its ability
to stimulate inward ion fluxes through DAT (9, 10).
In such a model, the inward current generated by DAT substrates such as
AMPH, which is most likely produced by the flow of Na+ into
the cell, is sufficient to stimulate the efflux of intracellular substrate because of an elevation of intracellular Na+.
Indeed, results from Sitte et al. support the hypothesis
that the releasing properties of DAT substrates are not proportionally related to their ability to be taken up by DAT but instead to their
ability to elicit DAT-mediated inward currents (see above) (9). To
date, it has not been possible to directly test the role of
intracellular Na+ on AMPH-induced DA efflux. Here we
demonstrate that the following events occur in DAT-expressing cells. 1)
AMPH increases intracellular Na+. 2) Intracellular
Na+ regulates AMPH-induced DA efflux. 3) AMPH-induced DA
efflux is voltage-dependent and electrogenic. 4)
AMPH-induced inward currents elicit DA efflux proportionally to their
dimension and duration.
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MATERIALS AND METHODS |
Plasmid Construction, Transfection, and Cell Culture--
The
synthetic human DAT gene, modified to express a FLAG epitope fused to
the N terminus of DAT, was subcloned into a bicistronic expression
vector (20) modified to express the synthetic DAT from a
cytomegalovirus promoter and the hygromycin resistance gene from
an internal ribosomal entry site as described previously (pciHyg) (21). EM4 cells, which are from a human embryonic
kidney HEK-293 cell line stably transfected with macrophage scavenger (R. Horlick, Pharmacopeia, Cranberry, NJ), were transfected with the
FLAG-DAT using LipofectAMINE (Invitrogen), and a stably
transfected pool was selected in 250 µg/ml hygromycin as described
(22). Cells were grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum at 37 °C and 5%
CO2. Previous studies have shown that addition of the
N-terminal FLAG tag does not alter the ability of the transporter to
produce substrate-induced currents (21). A fluorescently tagged DAT was
constructed by fusing the C terminus of the coding region of enhanced
yellow fluorescent protein (YFP) from pEYFP-N1
(Clontech) to the N terminus of the human synthetic
DAT cDNA, thereby creating the fusion construct YFP-DAT. This
construct was subcloned into pciHyg, and stable pools of EM4 cells
expressing YFP-DAT (YFP-DAT cells) were obtained as described above.
Electrophysiology--
Before recording from parental or stably
transfected cells, cells were plated at 105 per 35-mm
culture dish. Attached cells were washed three times with bath solution
at room temperature. The bath solution contained 130 mM
NaCl, 10 mM HEPES, 34 mM dextrose, 1.5 mM CaCl2, 0.5 mM MgSO4,
and 1.3 mM KH2PO4 adjusted to pH
7.35. The pipette solution for the whole-cell recording contained 120 mM KCl, 0.1 mM CaCl2, 2 mM MgCl2, 1.1 mM EGTA, 10 mM Hepes, and 30 mM dextrose plus DA (2 mM or 100 µM, as specified in the text)
adjusted to pH 7.35. Free Ca2+ was 0.1 mM. In
ion replacement experiments, the intracellular Na+
concentration was adjusted by iso-osmotically changing the
concentration of KCl in the pipette solution. Patch electrodes were
pulled from quartz pipettes on a P-2000 puller (Sutter Instruments,
Novato, CA) and filled with the pipette solution. Whole-cell currents were recorded using an Axopatch 200B with a low-pass Bessel filter set
at 1,000 Hz. Current-voltage relations were generated using a voltage
step (1 s) protocol ranging from
160 to 100 mV separated by 20 mV
from a given holding potential. Current and oxidative (amperometric)
signals were measured simultaneously. Data were recorded and analyzed
off-line using the software pCLAMP 8 from Axon Instruments.
Amperometry--
A carbon fiber electrode connected to a second
amplifier (Axopatch 200B) was attached to the plasma membrane of the
cell held at 700 mV for all experiments unless noted otherwise. The
carbon fiber electrodes (ProCFE; fiber diameter is 5 µm) were
obtained from Axon Instruments. Oxidative (amperometric)
current-voltage relationship was generated as above. At voltages more
negative than
60 mV the amperometric signal approaches zero, making
80 mV a convenient membrane potential to hold the YFP-DAT cells. Unlike the usual amperometric calibration, which requires conversion to
concentration, we report the current directly without considering the
effective volume. Thus, our requirements are a defined baseline, and
our data represent a lower limit to the DA efflux because some
transmitter is lost to the bulk solution. The amperometric currents
were low pass filtered at 100 Hz. Data were recorded and analyzed
off-line using the software pCLAMP 8 from Axon Instruments. Current-voltage relations were generated by plotting against the test
voltage the values of the amperometric currents between 800 and 1000 msec after the step.
Confocal Microscopy--
EM4 cells expressing FLAG-DAT or
YFP-DAT were cultured on round glass coverslips in 35-mm dishes
~48-72 h prior to the experiments and grown to 50-70% confluence.
The cells were incubated with 5 µM Sodium
GreenTM-AM (Molecular Probes) in Me2SO
(Sigma) at room temperature for 30 min under light-protected
conditions. The coverslips containing the cells were washed twice in
bath solution and placed into the confocal chamber (Bioptechs). Real
time confocal imaging was performed on an Olympus IX-70 inverted
microscope using an Olympus Fluoview 500 confocal microscope equipped
with an argon laser. Images were acquired every 15 s using a 488-nm
excitation wavelength with a 505 long pass filter. After acquiring two
basal images, seven images were recorded during continuous perfusion of
10 µM AMPH or vehicle. To determine the concentration of
free intracellular Na+, the following equation was
used,
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(Eq. 1)
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where F is the value of the average fluorescent intensity within
the cytoplasm of a cell, Kd is the dissociation constant of the fluorescent probe for sodium, and Fmax is
the maximum fluorescent intensity, calculated as described above, after
permeabilizing the cell with digitonin (15 µM) in a bath solution containing 300 mM NaCl, 10 mM HEPES,
34 mM dextrose, 1.5 mM CaCl2, 0.5 mM MgSO4, and 1.3 mM
KH2PO4 (n = 12 and 10 for FLAG
and YFP-DAT cells, respectively). Fmin is the minimum
fluorescent intensity calculated from FLAG and YFP-DAT cells after
incubating the cells for 6 h, in a Na+-free medium
solution. Confocal imaging was conducted at 25 °C. Image analysis
was performed using the public domain ImageJ imaging program
(rsb.info.nih.gov/ij/).
 |
RESULTS |
DA uptake was not observed in EM4 cells not transfected with the
YFP-DAT (data not shown). Furthermore, in untransfected cells neither
10 µM AMPH nor 10 µM AMPH together with 10 µM cocaine produced either whole-cell currents or DA
efflux, as measured by amperometry with DA in the whole-cell pipette
(see below). Thus, the EM4 cells provided a suitable null background in
which to study DA-efflux mediated by DAT and stimulated by AMPH.
To facilitate the selection of cells expressing adequate DAT for
electrophysiological and amperometric studies, we created a pool of
cells stably expressing a YFP-DAT fusion construct and selected for
analysis cells with easily visualized plasma membrane fluorescence
(Fig. 1A, inset).
Addition of the N-terminal YFP tag to DAT did not significantly alter
[3H]DA uptake (data not shown) and did not perturb the
ability of the transporter to produce substrate-induced currents (Fig.
1A). YFP-DAT-mediated currents were recorded in the
whole-cell configuration, and the membrane voltage was stepped from a
holding potential of
20 to
120 mV for 500 msec while acquiring a
control current (Fig. 1A, CO). Perfusion of the
cell with 10 µM AMPH caused an increase of the
steady-state inward current (Fig. 1A, AMPH),
which was blocked in the presence of 10 µM cocaine (COC)
with AMPH still present (Fig. 1A, AMPH + COC).
Cocaine also reduced the control current because of its block of a
DAT-mediated leak current, which has been described both for DAT and
other neurotransmitter transporters (9, 23-25). Therefore, the
AMPH-induced current was defined as the current recorded in the
presence of AMPH minus the current recorded after the addition of
cocaine to the bath with AMPH still present. Fig. 1B, shows
a current voltage relationship for the AMPH-induced current. The
membrane potential was held at
20 mV, and then the voltage was
stepped to a new potential between
160 and 100 mV in 20 mV
increments. Outward currents were recorded at membrane voltages more
positive that
20 mV.

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Fig. 1.
AMPH-induced whole-cell currents.
A, compared with control (CO), 10 µM AMPH induces an inward current at 120 mV
(AMPH) in YFP-DAT cells. The AMPH current was blocked by
concomitant application of 10 µM AMPH plus 10 µM cocaine (AMPH + COC), indicating that the
current observed is DAT-mediated. The inset shows a
z section obtained from a YFP-DAT cell by confocal
microscopy; the YFP fluorescence is localized mainly at the plasma
membrane. B, current-voltage relationship of the
AMPH-induced whole-cell current from YFP-DAT cells. The AMPH-induced
current is defined as the whole-cell steady-state current recorded upon
bath application of AMPH minus the current obtained in the presence of
AMPH and cocaine at a defined membrane voltage. Data were normalized at
140 mV. The recording electrode contained 2 mM DA in a
zero Na+ intracellular solution.
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The effects of AMPH were further investigated by following real time
changes in intracellular Na+ concentration by fluorometric
determination with a cell-permeant Na+ green tetraacetate.
We collected confocal microscopy images every 15 s, and for each
time point we subtracted the background (fluorescence measured from a
confocal plane in the control condition (rest)) from the fluorescence
recorded in the same z section upon the addition of AMPH. An
increase in intracellular fluorescence was detected within 15 s of
AMPH application (Fig. 2A). In
both YFP-DAT and FLAG-DAT cells, AMPH increased intracellular
Na+ with similar kinetics (Fig. 2B). No
significant changes in intracellular fluorescence were detected either
in EM4 cells treated with AMPH or in untreated YFP-DAT cells (Fig.
2C). A 60 s application of 10 µM AMPH
increased intracellular free Na+ to 25.9 ± 3.3 mM (n = 10) and 46 ± 12 mM (n = 12) in YFP-DAT and FLAG-DAT,
respectively. These data suggest that AMPH is able to modify ionic
gradients across the plasma membrane in cells expressing DAT. To test
whether such AMPH-induced changes in intracellular Na+
concentration regulate reverse transport of DA by DAT, we combined patch clamp recording with amperometry. YFP-DAT cells were voltage clamped with a whole-cell patch pipette while an amperometric electrode
was placed onto the plasma membrane (Fig.
3A). The whole-cell electrode
was filled with a solution containing 2 mM DA and different concentrations of NaCl substituted with KCl to maintain a constant osmolarity of 270 mOsm. The amperometric electrode was held at 700 mV,
a potential greater than the redox potential of DA. We recorded
DAT-mediated currents with the whole-cell pipette by stepping the
membrane voltage from a holding potential of
80 mV to potentials
between
100 and 100 mV. To isolate the DAT current, we subtracted the
current recorded in the presence of external COC from the current
recorded in the control condition. Fig. 3B shows an example
of transporter currents recorded between
100 and 100 mV with a
whole-cell pipette filled with an internal solution containing DA and
90 mM NaCl. DAT-mediated outward currents were voltage-dependent and increased at positive voltages in an
exponential fashion. For a fixed voltage, the DAT-mediated outward
current was increased by increasing the intracellular Na+
concentration. Thus, at an intracellular Na+ concentration
equal to 90 mM, the whole-cell current recorded at 100 mV
was 12.4 ± 4.2-fold greater than the current recorded with no
Na+ in the pipette solution. At the same time, we monitored
DA efflux with the amperometric electrode (Fig. 3C). We
isolated the DAT-mediated DA efflux by subtracting the background
currents (traces recorded in the presence of 10 µM
cocaine) from amperometric traces recorded under control conditions (DA
and high intracellular Na+) for each potential tested. An
upward deflection in the amperometric current corresponds to an outward
flux of DA. At the "on" of the voltage step, for voltages more
positive than
60 mV the amperometric electrode recorded an oxidation
current (positive), which is indicative of DA efflux. At the
termination "off" of the voltage step, the amperometric
current relaxed to baseline. The on and off of the voltage step
are defined by vertical arrows in Fig. 3C. Moving the carbon
fiber away from the patch caused the oxidative response to become
smaller and slower. Furthermore, in the absence of intracellular Na+ in the whole-cell pipette, a barely detectable signal
was recorded with the amperometric electrode. Finally, as expected for
DA oxidation, the oxidative response diminished upon reduction of the
carbon fiber voltage to 300 mV and disappeared completely with further reduction of the voltage. In Fig. 3D, we plot oxidation
currents obtained using different concentrations of Na+
(between 0 and 90 mM) in the whole-cell pipette against the
test voltage applied to the cell. For each single concentration of intracellular Na+ used, DA efflux increased at positive
voltages without reaching saturation in the range of voltages studied.
Similarly, at a defined membrane voltage, increasing intracellular
Na+ concentration increased DA efflux. The dependence of DA
efflux on internal Na+ was determined by fitting the values
of the steady-state amperometric currents recorded at different
intracellular Na+ concentrations by nonlinear regression to
a Hill equation. The Kms obtained were 54 ± 8 mM at 100 mV, 46 ± 6 mM at 80 mV, and
50 ± 12 mM at 60 mV, with a Hill coefficients of
2 ± 0.2, 3 ± 0.7, and 3.3 ± 0.7, respectively. At
voltages more negative than 60 mV, the reduced values of the
amperometric currents, obtained at low Na+ concentrations,
impaired our ability to estimate the affinity of DAT for intracellular
Na+. Finally, we were also able to record amperometric
currents with 100 µM DA (data not shown), and these, like
those measured with higher DA, were voltage dependent and sensitive to
intracellular Na+ concentration as well.

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Fig. 2.
AMPH induces an increase of intracellular
Na+ that is DAT-mediated. A, intracellular
Na+ fluorescence obtained from a single z
section of a patch of FLAG-DAT cells. Sodium GreenTM
fluorescence was measured from confocal images and used to monitor
temporal changes of intracellular Na+ levels upon AMPH
application. Background fluorescence (rest) was subtracted from each
single time point. Upon AMPH application (Amph), an increase
of intracellular fluorescence was detected. B, the relative
changes ( F/F) in Na+ sensitive
green fluorescence induced by AMPH were evaluated by ImageJ imaging
analysis by tracing a region of interest defining the cytoplasmic area
of a single z section of the cell. The images were collected
every 15 s for 2 min. The ratio F/F was measured for YFP-DAT
(n = 10) and FLAG-DAT cells (n = 12)
for each time point. C, changes in the ratio F/F over 2 min in EM4 cells after the application of AMPH (open
circles) and in YFP-DAT cells (open
squares) under control conditions in the absence of
AMPH.
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Fig. 3.
Intracellular Na+ stimulates DA
efflux. A, Schematic illustrating the
experimental configuration. The DAT cells were voltage clamped with a
whole-cell patch pipette while an amperometric electrode was placed
onto the cell membrane. The amperometric electrode measures the
concentration of catecholamine by oxidation/reduction reactions.
B, DAT-mediated currents recorded by stepping the membrane
voltage from a holding potential of 80 mV to potentials between 100
and 100 mV. The internal solution of the whole-cell pipette contained 2 mM DA and 90 mM Na+. C,
oxidation currents acquired concomitant to the whole-cell currents
represented in panel B by voltage clamping the cell between
100 and 100 mV with the whole-cell patch pipette. For voltage steps
higher than 80 mV, the amperometric electrode recorded an oxidation
current (positive) that increased during the entire duration of the
voltage step. D, amperometric currents obtained by using
different Na+ concentrations (symbols) plotted
against the test voltage applied to the cell. DA efflux increased at
positive voltages without reaching saturation in the range of the
voltages studied.
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To determine whether AMPH-induced DA efflux was also regulated by
intracellular Na+, we recorded DA efflux using the
experimental configuration described in the legend for Fig.
3A in the presence of 10 µM AMPH in the bath
solution. At a DAT saturating concentration of intracellular DA (2 mM), the steady-state amperometric currents recorded in the
presence of 10 µM AMPH were plotted against different
voltages (Fig. 4). DA efflux increased
exponentially at voltages more positive than
60 mV in a manner
dependent on the intracellular Na+ concentration. Although
DAT did not require the presence of AMPH to mediate DA efflux (Fig.
3D), AMPH substantially enhanced its ability to cause
carrier-mediated DA release at low concentrations of intracellular
Na+ (Fig. 4). For example, with 10 mM
intracellular Na+, the amperometric current recorded at 100 mV in the present of AMPH was twice the current magnitude recorded in
its absence (0.12 ± 0.015 pA versus 0.06 ± 0.02 pA, respectively, n = 4, p < 0.005 by
paired Student's t test) (compare Figs. 3 and 4,
open diamonds). Concomitantly, we recorded DAT-mediated
whole-cell currents. At 100 mV in the presence of AMPH, the DAT
whole-cell current increased 1.72 ± 0.23 times with respect to
the current recorded in its absence. In contrast, at an intracellular
Na+ concentration of 90 mM, no significant
increase of DA efflux or DAT-mediated outward currents was detected
upon AMPH application.

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Fig. 4.
Intracellular Na+ regulates
AMPH-induced DA efflux. Amperometric currents recorded at
different voltages using the experimental configuration described in
the legend of Fig. 3A. The average amperometric currents
obtained after bath application of 10 µM AMPH were
plotted against the different test voltages. Different
symbols correspond to different concentrations of
Na+ in the whole-cell pipette.
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Our data show that DA efflux mediated by DAT is
voltage-dependent, electrogenic, and dependent on the
intracellular Na+ concentration (Fig. 3). Moreover, DAT
does not require AMPH to generate DA efflux, but AMPH appears to play a
major role in stimulating the reverse transport mechanism of DAT at low
intracellular Na+ concentrations. In contrast, at high
intracellular Na+ concentrations the presence of AMPH did
not affect DAT-mediated DA efflux, which was already substantial.
Therefore, we considered the possibility that substrate-induced DA
efflux depends on two independent transporter processes. First,
substrate (e.g. AMPH) translocation by DAT produces an
inward current (Fig. 1). This inward current, in part carried by
Na+, results in an increase in intracellular
Na+ concentration (Fig. 2), thereby enhancing the ability
of DAT to produce DA efflux.
To test this hypothesis, we used a whole-cell patch pipette containing
an internal solution with 2 mM DA and no Na+
while 10 µM AMPH and a physiological concentration of
NaCl (130 mM) were present in the bath solution. The
concentration of Cl
was 124.2 mM
intracellularly and 133 mM extracellularly. The membrane
potential of the cell was held at
20 mV, a voltage close to the
equilibrium potential of the AMPH-stimulated, DAT-mediated outward
current recorded under these experimental conditions. In the absence of
Na+ and the presence of intracellular DA, we were able to
record only a small AMPH-induced outward current and a small
amperometric signal at 100 mV (Fig. 5,
left side of panels A and
B, respectively). In the same cell, with AMPH still present
in the bath solution, we stepped the membrane potential to
140 mV for
3 s (negative prepulse) and then again to 100 mV for 1 s. As
expected, AMPH stimulated an inward current at
140 mV (Fig.
5A, center). This is consistent with cations
entering the cell via DAT. In fact, we detected an increase of
intracellular Na+ upon AMPH application (Fig. 2). The
DAT-mediated current caused by the negative prepulse stimulation
substantially enhanced the ability of DAT to carry an outward current
at 100 mV (Fig. 5A, right side). Simultaneously,
we recorded DA efflux with the amperometric electrode. The negative
prepulse also facilitated the reverse transport of DA mediated by DAT
(Fig. 5B, right side). At 100 mV, upon negative
prepulse stimulation the AMPH-induced whole-cell and amperometric
current increased 1.63 ± 0.19-fold and 2.08 ± 0.14-fold,
respectively, with respect to the current obtained in the absence of a
negative prepulse (Fig. 5C). In contrast, the negative
prepulse stimulation failed to significantly increase either the
amperometric or whole-cell currents recorded at 100 mV at
concentrations of intracellular Na+ equal to or greater
than 30 mM (n = 10). In Fig. 5D,
we show the correlation between the amount of charge crossing the
plasma membrane during the negative prepulse stimulation (Q) and
the increase (fold) of the amperometric and whole-cell currents
recorded at 100 mV. The coefficient of correlation (R) between Q and
the amperometric and whole-cell currents was 0.73 and 0.8, respectively (n = 8), indicating that upon AMPH exposure we were
able to increase DAT-mediated currents and DA efflux by increasing the
amount of cotransported ions crossing the plasma membrane during the
negative prepulse.

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Fig. 5.
DAT-mediated inward currents stimulate
AMPH-induced DA efflux. The whole-cell patch pipette internal
solution contained 2 µM DA and no Na+. The
bath solution was an external solution plus 10 µM AMPH.
The membrane potential of the cell was held at 20 mV. In the absence
of Na+ and the presence of intracellular DA, a small
AMPH-induced DAT outward current was recorded at + 100 mV
(A, left side). In the same cell, with AMPH still
present in the bath solution, the membrane potential was stepped to
140 mV and then rapidly to 100 mV. An AMPH-stimulated inward current
was recorded at 140 mV (A, center). The
DAT-mediated current caused by the negative prepulse stimulation
enhanced the subsequent DAT-mediated outward current recorded at 100 mV
(A, right side). Similarly, the cocaine-sensitive
amperometric signal also increased at 100 mV upon negative prepulse
stimulation (B). The whole-cell current increased on average
1.63 ± 0.19-folds with respect to the outward current obtained in
the absence of negative prepulse, whereas the amperometric signal
increased on average 2.08 ± 0.14-folds (C).
Panel D illustrates the correlation between the amount of
charge carried by the AMPH-induced current during the negative prepulse
(Q) and either the increase of whole-cell (open
circle) or the amperometric (open square) current. Q
was normalized to the maximum movement of charge obtained during the
negative prepulse (n = 8), whereas the amperometric and
whole-cell currents were normalized to the respective currents recorded
at 100 mV in the absence of the negative prepulse (I).
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To further demonstrate that the ability of AMPH to induce DA efflux
also relies on its ability to cause ion fluxes across the plasma
membrane, we varied the duration of the negative prepulse and,
therefore, the dimension of the AMPH-induced current. Using an
experimental setting as in Fig. 5, we increased the duration of the
negative prepulse between 0.5 and 3 s and then recorded the
amperometric signal at 100 mV (Fig. 6).
The amperometric currents were normalized to currents recorded at 100 mV in the absence of the negative prepulse in the same cell
(y axis) and plotted against the duration of the negative
prepulse stimulation (x axis). The amperometric signal
increased without reaching saturation in the range of prepulse duration
studied, confirming that the amount of elementary charges crossing the
plasma membrane stimulated by AMPH regulates DAT-mediated DA
efflux.

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Fig. 6.
Increasing the duration of the negative
prepulse increases AMPH-induced DA efflux. YFP-DAT cells were
perfused with a bath solution plus 10 µM AMPH. The
whole-cell patch pipette contained an internal solution with no
Na+ plus 2 mM DA. The membrane potential of the
cell was stepped from a holding potential of 20 to 140 mV for
different time periods (between 0.5 and 3 s) and then rapidly to
100 mV to record amperometric currents. The amperometric currents
recorded at 100 mV after the negative prepulse were normalized to the
amperometric currents obtained in the absence of negative prepulse in
the same cell. The increase of the amperometric signals, generated by
negative prepulses, is represented on the y axis. The
x axis represents the duration of the negative prepulses.
Asterisk indicates significant changes in DA efflux compared
with a prepulse stimulation of 0.5 s (analysis of variance (ANOVA)
followed by Tukey's test; level of significance equal to
p < 0.01 for *).
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DISCUSSION |
This study shows for the first time that AMPH increases
intracellular Na+ availability and that the intracellular
Na+ concentration and transmembrane potential regulate
AMPH-induced, DAT-mediated DA efflux. Because changes in DA
neurotransmission are thought to play an important role in the
addictive properties of psychostimulants such as AMPH, the AMPH-induced
increase of intracellular Na+ and its regulation of DA
efflux may be an important mechanism in the development of its
psychostimulant action. Our initial experiments were designed to assess
the ability of AMPH to increase intracellular Na+
concentration. Our finding that incubation of YFP-DAT cells with AMPH
evoked a significant increase in intracellular Na+ is
consistent with the hypotheses of previous reports (9, 10). In the
present study we have shown that a 60 s of exposure to 10 µM AMPH increases intracellular Na+ to ~46
mM. It is likely that the increase in cytoplasmic
Na+ induced by AMPH is because of the substrate-like
properties of AMPH (3, 9, 16, 26). Indeed, extracellular substrates (e.g. AMPH) induce an inward current (Fig. 1), which is most
likely partially produced by a flow of Na+ into the cell
generated by DAT (9, 23, 27). In fact, although DAT-mediated currents
have been shown to be comprised primarily of anions (25), a
contribution of Na+ ions to the response mediated by DAT
substrates has not been excluded. In support of such a role for
Na+ ions, our real time confocal data showed that cocaine
prevented the AMPH-induced increase in intracellular Na+ in
DAT cells (data not shown) and that AMPH failed to increase intracellular Na+ in HEK-293 cells not expressing DAT (Fig.
2C). From our data it may also been inferred that the
increase of Na+ green fluorescence upon AMPH application is
greater near the plasma membrane, the location of DAT (Fig.
2A).
Dopamine uptake by DAT has been shown to be electrogenic in a
heterologous expression system (24), but a recent study described voltage-independent DA uptake in mesencephalic, dopaminergic neurons (28). The slow turnover rate of DAT relative to neuronal firing and its
voltage independence has been suggested as a possible mechanism for
increased DA signaling during burst firing. In contrast, reversal of DA
transport by depolarization has recently been proposed in experiments
conducted in neurons ex vivo (29). Mintz and co-workers
suggested that glutamate-gated channels and Na+ channels
could eventually depolarize the membrane potential of the neuron beyond
the reversal potential of DAT to induce reversal of DA transport (29).
Here, we showed that DA efflux mediated by DAT is
voltage-dependent. The DA transport process reversed at
voltages more positive than
60 mV, increasing exponentially without
reaching saturation in the range of membrane potentials studied (Fig.
3C). As expected, DAT-mediated whole-cell currents reversed
at positive voltages as well (Fig. 1B). Several lines of
evidence suggested the possibility that the influx of extracellular Na+ could be the trigger for the transporter-mediated
release of the neurotransmitter (9, 10, 29-31). Substrate-induced
release of norepinephrine from the dog saphenous vein was augmented by inhibition of the Na+,K+-ATPase (32).
Inhibition of the Na+,K+-ATPase produces
increased internal Na+ concentration as well as membrane
depolarization. These two events might contribute to the enhanced amine
efflux. Stimulation of DA efflux by ouabain, an inhibitor of
Na+,K+-ATPase, was also reported in LLC-PK
cells stably expressing the human norepinephrine transporter (18). The
authors inferred that the ouabain stimulation of efflux was most likely
because of an increase of intracellular Na+, although they
were unable to demonstrate a voltage dependence of substrate-mediated
DA efflux (18). In addition, a strong correlation has been found
between the ability of a substrate to induce DAT-mediated currents,
which are Na+, Cl
- and
substrate-dependent, and its releasing action (9). Our studies
demonstrate that increasing the cytoplasmic Na+
concentration increases the ability of DAT to mediate DA efflux, particularly at voltages more positive than
60 mV (Fig.
3D). At a saturating concentration of DA over the range of
voltages studied (between 60 and 100 mV), the Km for
intracellular Na+ was ~50 mM and was
voltage-independent (Fig. 3). These results suggest that the voltage
dependence of DA efflux does not result from altered DAT affinity for
intracellular Na+ and that intracellular Na+
regulates DA efflux.
We found that DA efflux was barely detectable, even in the
presence of AMPH, at a level of intracellular Na+
approaching zero (Figs. 3 and 4, open circles). In this
experimental configuration with zero Na+ solution in the
whole-cell pipette, AMPH may not increase the intracellular
Na+ sufficiently to drive DA efflux. In contrast, at an
intracellular Na+ concentration of ~10 mM,
the amperometric current recorded at 100 mV in the present of AMPH was
twice the current magnitude recorded in its absence (compare Figs. 3
and 4, open diamonds). With an intracellular
Na+ concentration equal to or higher than 30 mM, large amperometric and whole-cell currents were
recorded, and no significant increase of DA efflux or DAT-mediated
outward currents was detected upon AMPH application (compare Figs. 3
and 4). Thus, we conclude that normal intracellular Na+
concentration is required for AMPH to reverse the DAT cycle and that high intracellular Na+ can substitute for AMPH in
causing DA efflux.
Sitte et al. demonstrated a correlation between the
releasing properties of a substrate and its ability to induce
DAT-mediated inward current (9). To test the hypothesis that
AMPH-induced Na+ influx regulates AMPH-induced DA reverse
transport, we performed amperometric experiments without
Na+ in the whole-cell patch pipette (Fig. 5). The
relatively small DA efflux recorded in the presence of AMPH at 100 mV
doubled in size when preceded by a negative prepulse that stimulated an
AMPH-induced inward current. Because we used a saturating concentration
of Cl
both intracellularly and extracellularly, these
data suggest that the potentiation of the AMPH-induced DA efflux
results from an influx of Na+. Moreover, the negative
prepulse failed to potentiate DA efflux when the whole-cell patch
pipette contained Na+ at a concentration equal to or
greater than 30 mM. This was because the signal recorded at
100 mV was already reasonably sustained by the high intracellular
Na+ concentration. We also demonstrated that DA efflux
increased in proportion to the number of elementary charges crossing
the plasma membrane through DAT upon AMPH stimulation (Fig.
5D). Because transport of substrate through DAT is coupled
to both Na+ and Cl
ions, these data further
suggest the role of the activity of DAT and the influx of
Na+ in the DAT-mediated release of DA. Increasing the
duration of the negative prepulse and, therefore, the amount of
cotransported ions crossing the plasma membrane also increased DA
efflux (Fig. 6). The negative prepulse failed to increase the
amperometric signal in the absence of AMPH, suggesting that
substrate-induced DAT activity is essential for the stimulation of DA efflux.
Based on these data, we propose a model for AMPH-induced DA efflux in
which the ability of AMPH to increase intracellular Na+
concentration is essential for its stimulation of DA efflux. Future
experiments will be required to define the role of Cl
in
the regulation of this AMPH action.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants DA-13975 and DA-14684 (both to A. G.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
These authors contributed equally to this work.
**
To whom correspondence should be addressed. Tel.: 615-936-3891;
Fax: 615-936-3745; E-mail: Aurelio.Galli@Vanderbilt.edu.
Published, JBC Papers in Press, January 29, 2003, DOI 10.1074/jbc.M212815200
 |
ABBREVIATIONS |
The abbreviations used are:
DAT, dopamine
transporter;
DA, dopamine;
AMPH, amphetamine;
YPF, yellow fluorescent
protein.
 |
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