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Volume 17, Number 3,
Issue of February 1, 1997
pp. 960-974
Copyright ©1997 Society for Neuroscience
Multiple Ionic Conductances of the Human Dopamine Transporter:
The Actions of Dopamine and Psychostimulants
Mark S. Sonders1, 2,
Si-Jia Zhu3,
Nancy R. Zahniser3,
Michael P. Kavanaugh2, and
Susan G. Amara1, 2
1 The Howard Hughes Medical Institute and
2 Vollum Institute, Oregon Health Sciences University,
Portland, Oregon 97201, and 3 Department of Pharmacology,
University of Colorado Health Sciences Center, Denver, Colorado 80262
ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Electrophysiological and pharmacological studies of a cloned human
dopamine transporter (hDAT) were undertaken to investigate the
mechanisms of transporter function and the actions of drugs at this
target. Using two-electrode voltage-clamp techniques with hDAT-expressing Xenopus laevis oocytes, we show that
hDAT can be considered electrogenic by two criteria. (1) Uptake of hDAT substrates gives rise to a pharmacologically appropriate
"transport-associated" current. (2) The velocity of DA uptake
measured in oocytes clamped at various membrane potentials was
voltage-dependent, increasing with hyperpolarization. Concurrent
measurement of transport-associated current and substrate flux in
individual oocytes revealed that charge movement during substrate
translocation was greater than would be expected for a transport
mechanism with fixed stoichiometry of 2 Na+ and 1 Cl
per DA+ molecule. In addition to the
transport-associated current, hDAT also mediates a constitutive leak
current, the voltage and ionic dependencies of which differ markedly
from those of the transport-associated current. Ion substitution
experiments suggest that alkali cations and protons are carried by the
hDAT leak conductance. In contrast to the transport-associated
functions, the leak does not require Na+ or
Cl, and DAT ligands readily interact with the transporter
even in the absence of these ions. The currents that hDAT mediates
provide a functional assay that readily distinguishes the modes of
action of amphetamine-like "DA-releasing" drugs from cocaine-like
translocation blockers. In addition, the voltage dependence of DA
uptake suggests a mechanism through which presynaptic DA autoreceptor
activation may accelerate the termination of dopaminergic
neurotransmission in vivo.
Key words:
Na+/Cl-dependent;
carrier;
cocaine;
amphetamine;
methamphetamine;
methylphenidate;
MPP+;
uptake;
release;
Xenopus oocyte;
psychomotor stimulant
INTRODUCTION
The dopamine transporter (DAT) is thought to be a
principal site of action for several psychomotor stimulants, including
cocaine, amphetamine, methamphetamine, and methylphenidate
drugs used
widely for both therapeutic and nontherapeutic purposes. After release of the neurotransmitter dopamine (DA), its extracellular concentrations are regulated primarily by reaccumulation into dopaminergic neurons through the action of DAT and by diffusion. Accordingly,
pharmacological inhibition of DA uptake may both prolong the duration
of DA action at its receptors and expand the spatial domain of its
actions in a manner comparable to that reported for GABA uptake
inhibition (Isaacson et al., 1993
). Numerous in vivo
microdialysis and electrochemical studies have shown that psychomotor
stimulants raise extracellular DA concentrations (for review, see Wise,
1996). Similarly, in vitro and in vivo
electrophysiological experiments demonstrate that stimulant actions are
consistent with elevation of DA concentrations (for review, see Lacey,
1993; White, 1996). Although cocaine, amphetamine, and many of their
congeners display comparable actions at serotonin and norepinephrine
transporters (SERT and NET), DAT is the pharmacological target best
correlated with their reinforcing properties and abuse potential (Ritz
et al., 1987; Nestler et al., 1993; Pulvirenti and Koob, 1994; Wise,
1996). Gene disruption experiments demonstrate that DAT may be the
principal mediator of the locomotor stimulatory effects of cocaine and
amphetamine, and that DAT plays a critical role setting dopaminergic
"tone" in the murine CNS (Giros et al., 1996). Thus, there is
considerable interest in understanding the molecular mechanisms by
which DAT functions.
DAT belongs to a recently cloned family of neurotransmitter and amino
acid transporters that are functionally related by their requirement
for extracellular Na+ and Cl (Amara and
Kuhar, 1993). These transporters operate by coupling the transmembrane
translocation of organic substrates to the movement of driving ions
down preestablished electrochemical gradients (Kanner and Schuldiner,
1987; Rudnick and Clark, 1993). Biochemical studies of the dependence
of DA uptake on Na+ and Cl concentrations
suggest that two Na+ ions and one Cl ion are
cotransported with each DA molecule (Krueger, 1990; McElvain and
Schenk, 1992; Kilty, 1993; Gu et al., 1994). These results predict that
the transport of each DA+ molecule will be accompanied by
the movement of two net positive charges (because DA is positively
charged at physiological pH), and will thereby generate an inward
current.
A number of neurotransmitter/ion cotransporters generate detectable
electrical currents during the process of substrate translocation both
in situ and in reconstituted systems. Certain transporters, furthermore, exhibit ion channel-like electrical activities that would
not be predicted from classical alternating-access models of carrier
function (for review, see Lester et al., 1994; DeFelice and Blakely,
1996; Sonders and Amara, 1996). To investigate the electrogenic
properties of DAT, we have studied a cloned human DAT with
two-electrode voltage-clamp methods. By expressing hDAT in
Xenopus laevis oocytes and applying a combination of
electrophysiological, pharmacological, and biochemical techniques, we
can assess its translocation activity in real time and examine in
greater detail the voltage dependence, ionic coupling, and channel-like
properties of this carrier. These studies also provide insights into
the actions of an important class of neuropharmacological agents.
MATERIALS AND METHODS
hDAT cloning and expression. Total RNA was extracted
according to the method of Chomczynski and Sacchi (1987) from a single human midbrain sample and reverse-transcribed into cDNA using SuperScript II (Life Technologies, Grand Island, NY) and the
oligonucleotide GTCTTCGTCTCTGCTCCC complementary to the sense strand of
hDAT subsequent to the termination codon (Giros et al., 1992;
Vandenbergh et al., 1992). hDAT cDNA was amplified by PCR using Vent
DNA polymerase (New England Biolabs, Beverly, MA) and primers
overlapping 23 and 21 nucleotides of the 5
and 3 ends of the 1860 nucleotide coding region. The PCR transcript was digested and
directionally ligated into the oocyte transcription vector pOTV (Arriza
et al., 1994). The insert was sequenced in its entirety and found to be identical at the nucleotide level to the hDAT allele
reported by Vandenbergh et al. (1992) (GenBank accession no. M95167[GenBank]). Capped cRNA was transcribed from linearized DAT plasmid using T7
polymerase (mMessage mMachine, Ambion), diluted with water, and
injected into defolliculated stage V or VI oocytes (~10 ng/oocyte). Oocytes were prepared as described by Quick and Lester (1994) and
maintained at 17 or 21°C for up to 3 weeks. The presence of hDAT in
cRNA-injected oocytes was confirmed by Western blot analyses, which
displayed immunoreactive bands not detected in water-injected oocytes.
Uptake assays. Transport of DA into individual oocytes was
quantitated either by liquid scintillation spectroscopy of
[3H]DA or HPLC-coupled electrochemical detection
(HPLC-EC) of nonradiolabeled DA. All uptake experiments were performed
at ambient temperature using frog Ringer's solution, containing (in
mM): 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES-NaOH, pH 7.4-7.5, or ion-substituted versions, as specified. Uptake kinetic experiments were initiated with
the addition of tritiated or unlabeled DA to groups of oocytes (3-6)
in a final volume of 500 µl and terminated by transferring the
oocytes through three sequential 5 ml baths of ice-cold Ringer's (total transfer time, <20 sec). Radiolabeled DA was quantitated by
liquid scintillation spectroscopy after dissolving individual oocytes
in 250 µl of 0.2% SDS. Intracellular DA concentration determination
by HPLC-EC (Gerhardt et al., 1989) was performed by sonicating
individual oocytes in 0.8 ml of 2 mM perchloric acid and
chromatographing aliquots equivalent to 1/50-1/160 of the
centrifugation supernatant (16,000 × g, 4°C, 15 min). Retention time of standards was used to identify peaks, and peak
heights were used to calculate absolute amounts of DA. Nominal
detection limits for DA and dihydroxyphenylacetic acid were 0.5 and
0.25 pg per injection, respectively.
The velocity of DA uptake was essentially constant over incubation
periods between 0.5 and 60 min using 75-200 nM
[3H]DA. Accordingly, incubation periods between 100 sec
and 30 min were used in uptake studies. Saturation experiments used at
least eight DA concentrations between 10 nM and 1 mM. Nonspecific uptake was determined by performing
parallel incubations of water-injected oocytes or of cRNA-injected
oocytes in the presence of saturating concentrations of uptake
inhibitors mazindol, cocaine, RTI-55, or GBR 12909. Nonspecific uptake
of radioligand was always <3% of total uptake, and for incubations
5 min, it was typically <1% of total uptake. Oocytes were
preincubated in drug/buffer solutions for at least 5 min before
addition of [3H]DA.
In ion substitution experiments, Na+ or Cl
ions in frog Ringer's solution were either partially or fully
replaced. LiCl and KCl were used to replace NaCl, and in most cases,
LiOH and KOH were used to adjust the pH of the buffers.
Cl substitution experiments were performed with
morpholinoethylsulfonate (MES)-NaOH and MES-LiOH in place of NaCl
at a concentration of 96 mM, either with 6.6 or 0 mM Cl remaining. In the latter case,
KNO3, Ca(NO3)2, and
MgSO4 were used in place of the corresponding chloride
salts.
For uptake and binding data, nonlinear regression analyses were
performed with KaleidaGraph 3.0 or GraphPAD software to generate estimates of kinetic constants KT (the apparent
affinity of transport), Vmax,
Ki, IC50,
KD, and Bmax.
Ki values were calculated from
IC50 using the Cheng-Prusoff equation. Geometric means
were determined for the apparent affinity values
KT, Ki, KD, and IC50. Error
values are given as SEMs or, where appropriate, 95 or 99% confidence
intervals (CI95 or CI99).
Drugs were obtained from National Institute on Drug Abuse Research
Technology Branch or were purchased from Research Biochemicals International (Natick, MA) or Sigma (St. Louis, MO). Radiochemicals were purchased from DuPont NEN (Boston, MA) and Amersham (Arlington Heights, IL).
Determination of hDAT density and turnover rate. The
turnover rate of hDAT was estimated from the ratio
Vmax:Bmax. In each of
three batches of oocytes, parallel measurements were made of the
velocity of DA uptake (Vmax) and the density of
hDAT sites (Bmax). Vmax
values were calculated from saturation analyses of assays using either
liquid scintillation spectrometry or HPLC-EC to quantitate accumulation
of tritiated or unlabeled DA, respectively (8 concentrations,
triplicate determinations). Saturation binding assays were performed on
intact ooctyes using 5 nM [3H]mazindol and
six concentrations of unlabeled mazindol (1 nM-1 µM, triplicate determinations) in frog Ringer's
solution. Oocytes were incubated in 1 ml of [3H]mazindol
solutions for 45-60 min on ice, washed for 5-10 sec in 4 ml of
ice-cold buffer, and then solubilized individually in 200 µl of 1%
SDS. Radioactivity was determined with liquid scintillation counting.
Nonspecific binding was determined with 1 µM GBR
12909.
Two-electrode voltage-clamp electrophysiology.
Two-microelectrode voltage-clamp recordings from oocytes were
performed at room temperature using glass microelectrodes filled with 3 M KCl solutions (resistance, <1 M
) and an
Ag/AgCl-pellet bath ground or an active bath probe. Dagan TV-200, Axon
GeneClamp 500, or Warner OC-725B amplifiers were used with AxoLab-1 or
DigiData1200 interfaces. The pClamp suite of programs (Axon
Instruments, Foster City, CA) was used to control stimulation
parameters, for data acquisition, and for analysis. MacLab data
acquisition software and a MacLab/2e interface (ADInstruments, Milford,
MA) were used simultaneously to monitor and record electrophysiological
experiments. Currents were low-pass filtered between 10 Hz and 2 kHz,
and digitized at rates between 1 and 5 kHz. Frog Ringer's buffer and
ion-substituted versions (described above) were superfused over
voltage-clamped oocytes at a rate of ~4 ml/min (bath volume, 0.5 ml).
During Cl substitution experiments, 3 M
KCl/agar bridges were used to avoid voltage offsets associated with
buffer changes.
The voltage dependence of hDAT-mediated currents was studied using two
voltage excursion protocols. In one protocol, membrane potentials were
ramped between 
130 mV and +80 mV over a 750 msec interval. The second
protocol used a sequence of jumps in membrane potential in 10 mV
increments to measure steady-state currents at potentials between 120
mV and +40 mV. Oocytes were held at 60 mV (600-750 msec) before
jumps to each test potential (duration 250-400 msec). Before each
application of drug, two voltage excursions were executed to measure
the control currents and establish that they were stable. For voltage
jump protocols, current values were measured and averaged during the
final 80 msec of the test interval when they had reached steady
state.
Currents attributable to the actions of drugs were determined by
performing off-line subtraction of currents recorded during buffer
perfusion (control) and currents recorded during drug perfusion. Substrate-elicited currents were measured by subtracting the control currents from those recorded during substrate application
(IDrug IControl).
However, changes in membrane current brought about by nonsubstrate
transport antagonists were determined by a subtraction using a reversed
order (IControl IDrug), because these drugs appear only to block
membrane conductances. This convention is obeyed in all figures except
Figure 3, in which the aim is to compare the actions of DA and cocaine,
and all data are plotted as IDrug IControl. The sole exception to the pattern of
DA-elicited currents being plotted as IDA IControl is found in Figure 7: under
Na+-free conditions, all DAT ligands only block a membrane
conductance; hence, the current-voltage (I-V) of
the conductances blocked by DA and cocaine are both plotted as
IControl IDrug.
Fig. 3.
Antagonism of DA transport-associated current by
cocaine. I-V plots of currents elicited during
consecutive applications to a single oocyte of 3 µM
cocaine (Coc; - - -), 2 µM DA (
),
and 3 µM cocaine plus 2 µM DA ( ).
Currents were measured during voltage ramps (130 to +80 mV in 750 msec), and subtractions all used the IDrug IControl convention. Comparison of the DA
I-V with the DA + Cocaine I-V reveals
the transport-associated component that is susceptible to blockade by
cocaine.
[View Larger Version of this Image (14K GIF file)]
Fig. 7.
Effects of Na+ substitution on
leak conductance I-V relations. A,
Sequential application to a single oocyte of 3 µM cocaine in Na+ Ringer's buffer (
), and then after a
change to 96 mM K+ (2 mM
Na+) Ringer's buffer, of 10 µM DA (
) and
3 µM cocaine (
). Subtractive currents
(IControl IDrug) determined during voltage jump
protocols represent the I-V of the leak conductance,
which is blocked by all three drug treatments. Cocaine
I-V plots display identical reversal potentials in the
two buffers (16 mV in this cell). In low Na+ buffer, the
DA I-V displays the same reversal potential. Moreover, DA elicits no inward transport-associated current at negative potentials. B, In 96 mM Li+ (2 mM Na+) Ringer's buffer, voltage jumps during
sequential applications of 10 µM DA (
) and 10 µM cocaine (
) reveal that both drugs block a
conductance with the same reversal potential (4 mV). Comparable results were observed when Cl was replaced with MES in
Li+ Ringer's (not shown). These data are representative of
four repetitions.
[View Larger Version of this Image (15K GIF file)]
Concentration-response data were collected by measuring
subtracted currents (IDA IControl) during voltage jump protocols. To six
oocytes, concentrations of DA were superfused at least twice (in
randomized order), and the two responses for each concentration were
averaged. The concentrations studied were 0.1, 0.3, 1.0, 3.0, 10.0, and
30.0 µM, although the 30 µM concentration
was examined in only five oocytes. To control for variation in
expression levels between oocytes, the concentration dependence and
voltage dependence of DA-evoked currents were analyzed by normalizing
current responses in an individual oocyte to that evoked by 10 µM DA at 120 mV. The DA concentrations evoking a
half-maximal current (K0.5) at single membrane
potentials were determined by nonlinear regression using the
Michaelis-Menten equation. At potentials greater than 20 mV,
responses evoked by 30 µM DA were omitted from the
regressions because at high concentrations, several phenethylamines
appeared to block endogenous channels. The voltage dependence of mean
normalized currents evoked at each DA concentration was studied by
nonlinear regression analysis. Comparable experiments were performed
using S(+)amphetamine over the concentration range 0.03-30
µM.
DA uptake under voltage-clamp conditions. The dependence of
DA uptake velocity on membrane potential was studied by measuring substrate accumulation in oocytes held in voltage-clamp at various potentials (+10, 0, 30, 60, 90, or 120 mV). DA uptake was measured in six batches of oocytes either by HPLC-EC after 3 or 5 min
perfusions of 20 µM DA, or by liquid scintillation
spectroscopy after 100 sec perfusions with 10.1 µM
[3H]DA (0.37 Ci/mmol). After incubations with DA or
[3H]DA, oocytes were briefly superfused with frog
Ringer's, voltage clamps were shut off, electrodes were withdrawn, and
the oocytes were transferred to either perchlorate or SDS solutions and
treated as described above (
30 sec). DA uptake velocities were
calculated on a per-second basis. The voltage dependence of uptake
velocity was quantified in each batch of oocytes by normalizing mean
velocities at each potential to that measured at 30 mV. Net charge
movement attributable to substrate translocation during
DA/[3H]DA perfusion was calculated off-line by
graphically integrating the DA-elicited current in each oocyte with
MacLab (5 of 6 oocyte batches). For each oocyte, an estimate of the net
charge:DA flux ratio was calculated as follows: Net Charges
Translocated/(Moles of Accumulated DA × Faraday Constant).
Ion substitution experiments. Reversal potentials were
determined by visual inspection of I-V plots generated with
Clampfit. After each cocaine perfusion, oocytes were perfused for at
least 8 min before subsequent drug applications to allow adequate drug washout. Ion permeability ratios were calculated from shifts in the
reversal potential of cocaine-elicited currents in different ion-substituted buffers using the Goldman-Hodgkin-Katz voltage equation (Hille, 1992).
RESULTS
DA uptake studies
To establish the validity of the Xenopus oocyte
expression system for investigating the electrophysiological properties
of hDAT, the kinetic and pharmacological properties of
[3H]DA uptake were studied. Levels of hDAT expression
were fairly consistent among oocytes of a single injection batch;
however, they varied widely between batches of oocytes. The time of
maximal hDAT expression also varied between batches, peaking between 4 and 14 d after injection. Oocytes expressing hDAT accumulated [3H]DA in a time- and ion-dependent manner. For instance,
when oocytes from four separate batches were incubated in
[3H]DA (30-150 nM) for 30 min, they
accumulated radioligand to levels >40-fold above external
concentrations, assuming an intracellular aqueous volume of 0.5 µl
(n = 12 assays). By contrast, control oocytes from
these and other batches did not concentrate [3H]DA
whatsoever, and excluded more than three-fourths the radioactivity found in a comparable volume of incubation bath (n > 60 assays). Control oocytes included uninjected and water-injected
oocytes, or cRNA-injected oocytes that were coincubated with 3
µM mazindol, 20 µM cocaine, 1
µM RTI-55, or 1 µM GBR 12909. [3H]DA uptake displayed strong Na+ and
Cl dependence: complete replacement of Na+
with Li+, K+, Cs+, or choline
reduced [3H]DA uptake by 97-98% (n 2 assays for each substitute). Complete replacement of external
Cl diminished [3H]DA uptake by 96.5%; in
comparison, parallel water-injected oocytes accumulated 98.9% less
than hDAT cRNA-injected oocytes (n = 2 assays).
Kinetic parameters for DA translocation by hDAT were similar in uptake
assays using either liquid scintillation spectroscopy to detect
accumulated [3H]DA or HPLC-EC to measure intracellular
DA. Saturation uptake experiments performed at 21°C with six
different oocyte batches yielded a mean KT value
of 1.7 µM (CI95 0.7-4.1 µM).
Greater batch-to-batch variability was observed in DA uptake velocity
than in apparent affinity, as would be expected to result from
differing levels of hDAT expression. (Vmax
values ranged between approximately 15 and 400 fmol/sec (21°C) over
all experiments.) Cocaine and S(+)amphetamine inhibited uptake of
[3H]DA in a concentration-dependent manner with mean
Ki values of 206 nM
(CI95 102-414 nM; n = 3) and
297 nM (CI95 137-647 nM;
n = 3), respectively. Cocaine exhibited a Schild slope
(Kenakin, 1987) of 0.94 (1 experiment), which is compatible with a
competitive interaction with DA at hDAT.
Estimation of hDAT turnover rate
To determine the turnover rate of hDAT, parallel measurements of
DA uptake velocity and hDAT density were made in three oocyte batches
to calculate the
Vmax:Bmax ratio (Table
1). Maximal DA uptake velocities were measured at room
temperature using radiolabeled flux or HPLC-EC. Catabolism of
accumulated DA to dihydroxyphenylacetic acid in hDAT oocytes was not
detected by HPLC-EC analysis, indicating that accumulated tritium
likely represents authentic [3H]DA. Endogenous DA was not
detected by HPLC-EC in uninjected or water-injected oocytes. The
Bmax of [3H]mazindol, which labels
DAT in rat striatum with nanomolar affinity (Javitch et al., 1984), was
determined on intact oocytes to estimate the number of transporters
expressed per oocyte. A single class of binding sites with an affinity
similar to that in rat brain was revealed. Assuming that each mazindol
binding site represents a functional DAT molecule on the oocyte
surface, the mean
Vmax:Bmax ratio estimates
the turnover rate to be 0.47 DA molecules/(sec × transporter
molecule), suggesting that each transport cycle requires ~2 sec at
21°C.
Table 1.
hDAT turnover rate determined from saturation analyses of
DA uptake velocity and [3H]mazindol binding to intact
oocytes
| DA
uptakea |
[3H]mazindol
binding |
Turnover
rate |
|
| Vmax |
KT |
Bmax |
KD |
Vmax/Bmax |
| (fmol/(oocyte × sec)) |
(µM) |
(fmol/oocyte) |
(nM) |
(sec1) |
| 66.9 ± 6.3 |
2.7b |
152.5 ± 7.0 |
5.2c |
0.47 ± 0.06 |
|
|
Mean values (±SEM) for Vmax and
Bmax were determined in three experiments using
different oocyte batches (not voltage-clamped). In each experiment,
parallel pools of oocytes were used for Vmax and
for Bmax measurements.
|
|
a
DA uptake was measured solely with
[3H]DA in one experiment and solely by HPLC-EC in a
second experiment. Both methods were used in a third experiment and
yielded Vmax values that differed by 7%.
|
|
b
CI95 0.2-36 µM.
|
|
c
CI95 2.9-9.2 nM.
|
|
Two-electrode voltage-clamp studies of hDAT currents
Preliminary calculations based on the Vmax
of DA uptake by hDAT-expressing oocytes indicated that if DA
translocation were accompanied by a net charge movement, then an inward
current attributable to hDAT transport activity should be detectable by
two-electrode voltage-clamp methods. Figure
1A shows the changes in membrane current elicited by drug application to a hDAT-expressing oocyte voltage-clamped at 60 mV. Superfusion with 20 µM DA
evoked a downward displacement in the current trace, consistent with a net inward current that occurs as a result of translocation of DA+ and Na+ ions. In contrast, 10 µM cocaine evoked outward currents (that only slowly
washed out) both on initial application and on reapplication after DA
superfusion of the same oocyte. The drug-elicited currents are
attributable to hDAT because they were not observed in uninjected or
water-injected oocytes (Fig. 1B).
Fig. 1.
Cocaine and DA applications to a voltage-clamped
hDAT oocyte evoke opposite changes in membrane current. Drug
applications (solid bars) to oocytes voltage-clamped at
60 mV. A, An hDAT-expressing oocyte was initially
superfused with 10 µM cocaine (Coc), which elicited a small outward current that slowly returned to baseline after
10 min of washout (flow rate 4 ml/min, chamber volume 0.5 ml).
Superfusion of 20 µM DA induced an inward current that
rapidly returned to baseline. Reapplication of cocaine caused an
outward current comparable in magnitude and kinetics to that evoked by its initial application. B, No responses were evoked by
drug application to a water-injected control oocyte.
[View Larger Version of this Image (15K GIF file)]
That cocaine could elicit any change in the holding current of an
hDAT-expressing oocyte in the absence of DA was somewhat unexpected,
because cocaine is generally viewed as a nontransported uptake
inhibitor that interacts with DA in an apparently competitive manner.
It was also intriguing that the cocaine-evoked current was opposite in
polarity to the DA-elicited current. Even though cocaine has
occasionally been reported to cause DA release (Baumann and Maitre,
1976; Pifl et al., 1995), the outward current elicited by cocaine does
not arise from DA extrusion because the outward current could be evoked
by cocaine before the initial presentation of extracellular DA (Fig.
1A), and oocytes were found by HPLC-EC to have little
or no endogenous DA (50 nM, data not shown). The opposite
DA- and cocaine-elicited currents suggest that hDAT mediates at least
two distinct steady-state ionic conductances, one of which is
associated with substrate transport, whereas the other can be observed
in the absence of translocation activity. Mager and colleagues (1994)
noted analogous currents manifested by rat SERT and termed them
transport-associated and leak currents. The following experiments
illustrate that the steady-state ionic conductances that give rise to
the hDAT transport-associated and leak currents are distinguishable
based on voltage dependence, ionic selectivity, and mode of drug
action.
I-V relations of transport-associated and
leak currents
To better understand the changes in ionic conductance that
underlie the currents mediated by hDAT, drug-evoked currents were studied across a voltage range, using both ramp and jump protocols to
control membrane potentials. The actions of drugs in changing membrane
conductance were identified by comparing membrane currents measured
during drug superfusion and during buffer superfusion (control
conditions). Subtraction of currents measured in the presence and
absence of hDAT ligands yielded I-V plots for the drugs.
Figure 2 shows results from a voltage
ramp experiment in which DA and cocaine were applied sequentially to a
single oocyte. At both negative and positive potentials, 3 µM cocaine superfusion diminished the magnitude of the
membrane current compared with that measured during the control voltage
ramp. This result is consistent with the cocaine-evoked outward current
observed in an oocyte clamped at 60 mV (Fig. 1A)
and suggests that the cocaine-elicited outward current likely reflects
the action of the drug in blocking a constitutive membrane conductance,
rather than an ability to activate either K+ ion efflux or
Cl ion influx. This point is made more obvious by
inspection of the difference between cocaine and control voltage
ramps.
Fig. 2.
Membrane conductance changes evoked by DA and
cocaine. Currents measured in a single voltage-clamped oocyte during
voltage ramps (130 to +80 mV in 750 msec). A, Membrane
currents measured during buffer (- - -) and 3 µM cocaine
() superfusion. B, I-V plot
describing current that is blocked by cocaine, determined by
subtraction of currents (IControl ICocaine) plotted in A. C, Membrane currents measured during buffer (- - -) and
20 µM DA () superfusion. D,
I-V of current elicited by DA, determined by
subtraction of currents (IDA IControl) plotted in
C.
[View Larger Version of this Image (21K GIF file)]
The I-V curve describing the tonic leak conductance that is
susceptible to blockade by cocaine is derived according to convention by taking the difference of IControl ICocaine (Fig. 2B). This current was voltage-dependent, outwardly rectifying, and reversed at
approximately 10 mV. The slope and reversal potential of this current
both argue against an action of cocaine to increase membrane permeability to K+ or Cl ions because: (1)
the amplitude of currents carried by either ion would be expected to
increase with depolarization between 60 mV and 10 mV, whereas the
observed current decreases over this range; and (2) the reversal
potential of approximately 10 mV is more positive than the Nernst
potentials for K+ or Cl in oocytes (95 mV
and 28 mV, respectively) (Dascal, 1987; Costa et al., 1989). That the
observed reversal potential is also more negative than the Nernst
potential for Na+ (61 mV) (Costa et al., 1989) suggests
that the tonic leak conductance may be nonselective for cations, an
assertion that is more directly supported by ion substitution
experiments presented below.
The effect of DA superfusion on hDAT oocyte membrane conductance is
more complex than that of cocaine because, at extremes of the potential
range, DA clearly evokes two opposite effects. At positive potentials,
DA acted to reduce the membrane conductance compared with control in a
manner analogous to cocaine; in contrast, at negative potentials, DA
superfusion increased the membrane conductance (Fig. 2C).
Because DA is believed to increase transport-associated ionic flux, the
DA-elicited current was determined by subtraction using the convention
of IDA IControl (Fig.
2D). The resulting DA I-V curve exhibited
an asymmetric inverted U-shape, with a positive slope conductance (at
hyperpolarized potentials) representing the transport-associated
current and a negative slope conductance (at depolarized potentials)
representing a cocaine-like blocking action on a tonic leak
conductance. As a result of the opposing conventions used to describe
DA-elicited and cocaine-inhibitable I-V relationships, the
similar inhibitory actions of the two drugs on a tonic leak conductance
are manifested as I-V plots with negative slope conductance
for DA but with positive slope conductance for cocaine over the range
of positive potentials (Fig. 2B,D). Despite this
artificial disparity in plotting, the suggestion that both drugs may
block an identical leak conductance is explored further in ion
substitution experiments presented below.
Over the range of negative potentials, Figures 1 and 2 suggest that
when applied separately, DA and cocaine may act on distinct hDAT ionic
conductances. However, because cocaine is a well established inhibitor
of DA translocation, it would be expected that cocaine should be able
to inhibit ionic flux (i.e., current) that accompanies substrate
transport. To test for the possibility of such an interaction, voltage
ramps were executed during superfusion with DA and cocaine in
combination. Figure 3 displays the I-V
values elicited by sequential application to a single representative
oocyte of 3 µM cocaine (~10 × Ki), 2 µM DA
(approximately KT), and the two drugs together.
(To facilitate comparison, only the IDrug(s) IControl convention was used for current
subtractions.) Coapplication of 2 µM DA and 3 µM cocaine gave rise to a voltage-sensitive current that
was not the sum of the individually elicited currents but that
virtually overlapped the cocaine I-V trace. Comparison of the DA I-V values in the absence and presence of cocaine
makes clear that cocaine fully antagonized the inward DA current
otherwise seen at negative potentials. That the DA-elicited current
observed at negative potentials was abolished by a near-saturating
concentration of cocaine supports the contention that this current is
closely associated with substrate translocation by hDAT.
From these data, it appears that the electrophysiological action of
cocaine on hDAT is limited to blocking ionic conductanceseither the
tonically active leak conductance when cocaine is applied alone, or the
transport-associated conductance when it is coapplied with DA. DA, on
the other hand, increases the transport-associated conductance, yet
also blocks the tonic leak conductance in a cocaine-like manner. Hence,
the observed substrate I-V plots likely represent the net
effect of substrates acting simultaneously on the two different hDAT
conductances: at negative potentials, activation of the
transport-associated conductance is the predominating effect, whereas
at positive potentials, blockade of the tonic leak conductance is most
apparent.
Concentration and voltage dependence of DA-elicited currents
To further explore the concentration, voltage,
and ionic dependence of the transport and leak conductances, as well as
their pharmacological sensitivity, steady-state currents were measured in oocytes during voltage jump protocols. Figure
4A displays the mean normalized
responses elicited by application of varying DA concentrations to six
oocytes. To control for variations in expression levels between cells,
the currents elicited in each oocyte were normalized to that observed
at 120 mV in response to 10 µM DA. For all DA
concentrations, the I-V relations took the shape of an
inverted U, the peak of which was approximately 20 mV and which
resembled the subtracted I-V plots generated in
non-steady-state ramp protocols (Fig. 2D). The
amplitudes of DA-elicited currents increased in a dose-related manner
in all oocytes. Comparable concentration-dependent responses were
observed in two oocytes treated with a range of S(+)amphetamine
concentrations (data not shown).
Fig. 4.
Concentration and voltage dependence of
DA-evoked steady-state currents. Concentration-dependent current
responses were measured in six oocytes using a voltage jump protocol
(see Materials and Methods). To control for differing levels of hDAT
expression between oocytes, IDA IControl subtractions for each DA
concentration and potential were normalized to the current elicited by
10 µM DA at 120 mV in the same cell (mean, 17.9 nA;
range, 7.9 to 43.5 nA). A, Nonlinear curve fitting
of mean current amplitudes for each DA concentration (x, 0.1 µM; , 0.3 µM; , 1.0 µM;
, 3.0 µM; , 10 µM; 
, 30 µM). Over the potential range 120 to 20 mV,
DA-elicited currents displayed an exponential dependence on voltage
(mean, e-fold per 67 mV, solid lines;
range, e-fold per 55-75 mV). At more positive
potentials, however, current amplitudes were better described as having
a linear relation to membrane potential (broken lines).
B, The DA concentration dependence of mean-normalized
currents (
) was well fit by a simple Michaelis-Menten equation for individual potentials over the range 120 to 20 mV.
Currents appeared to saturate with increasing [DA] and displayed K0.5 values of
1.9-2.9 µM. C, At potentials greater than
20 mV, mean normalized current amplitudes () were more poorly
described by Michaelis-Menten kinetics as a function of concentration.
In some oocyte batches, high DA concentrations affected an endogenous conductance (see Results), and therefore the 30 µM points
() were omitted from the curve fits. D, At each
membrane potential tested, geometric means of
K0.5 and CI95 values (error
bars) were determined from affinity values derived individually for
each of six oocytes (see Materials and Methods). The apparent
affinities (K0.5) of DA for eliciting
transport-associated current () and for blocking a leak current
() are displayed. The K0.5 for the transport-associated current (in the range of 120 to 20 mV) demonstrates little voltage sensitivity. For comparison, also plotted
is the DA apparent substrate affinity (KT,
; CI95 - - -) determined in uptake assays using six
different batches of oocytes. These oocytes were not voltage-clamped,
although other oocytes from the same batches displayed resting membrane
potentials in the range of 15 to 45 mV.
[View Larger Version of this Image (23K GIF file)]
Nonlinear curve fitting of the normalized I-V data
suggested that the two limbs of I-V plot divided at 20 mV
are best described by different functions. DA transport-associated
inward currents exhibited an exponential dependence on membrane
potential, increasing with hyperpolarization approximately
e-fold per 67 mV between potentials 20 mV and 120 mV. In
distinction, at voltages more depolarized than 20 mV, DA-evoked
currents displayed a dependence on membrane potential that was better
described by a linear than by an exponential function. At neither
extreme of the range studied did current magnitudes appear to saturate
as a function of potential.
The concentration-response relations of DA-evoked currents were
studied at individual potentials. Figure 4, B and
C, graphically represents the concentration dependence of
normalized DA-evoked currents at membrane potentials 120 to 20 mV
and 10 mV to 40 mV, respectively. For the transport-associated
currents, amplitudes appeared to saturate with increasing DA
concentration and were well described by Michaelis-Menten kinetics.
The DA concentrations that elicited half-maximal currents
(K0.5) were calculated in each oocyte for every
potential using the Michaelis function. The plot of mean
K0.5 versus membrane potential (Fig.
4D) illustrates that voltage has little influence on
the apparent affinity of DA between 120 mV and 20 mV, where the
mean values fluctuated only between 1.9 and 2.9 µM. These
K0.5 values are in good agreement with the
apparent affinity for DA transport (KT = 1.7 µM) determined in uptake assays using oocytes that were
not voltage-clamped (typical resting membrane potentials of hDAT
oocytes were 15 to 45 mV). The resemblance in apparent affinities
of DA for uptake and for eliciting transport-associated currents
supports the assertion that the currents are a manifestation of the
translocation process. The functional link between translocation and
the transport-associated current is reinforced by studies with
S(+)amphetamine. Normalized S(+)amphetamine concentration-response
curves generated in two oocytes yielded a mean
K0.5 of 0.5 µM over hyperpolarized
potentials (data not shown). This value closely corresponds to its
potency (Ki = 0.3 µM)
for inhibiting [3H]DA uptake, as would be expected for a
DA uptake inhibitor that is itself a transport substrate (Ross,
1976).
Although the Michaelis-Menten function provided a less satisfactory
fit to the DA concentration dependence of current at voltages more
positive than 20 mV (Fig. 4C), the estimates of mean DA K0.5 values (1.0-2.0 µM) at
depolarized potentials were in the same range as those found for
hyperpolarized potentials (Fig. 4D) and for the
KT. A number of factors may have contributed to the poorer fits of the DA currents to Michaelis-Menten kinetics at
potentials more positive than 20 mV, one of which is that in certain
batches of uninjected and water-injected oocytes, high concentrations
(30 µM) of DA and other phenethylamine congeners were
observed to block an endogenous ionic conductance (data not shown). In
particular, such a phenomenon might account for why the largest
divergences from the Michaelis-Menten model were observed at the
highest DA concentrations. Accordingly, only concentrations up to 10 µM were used to estimate the apparent affinity of DA for
blocking the leak conductance (Fig. 4C,D). Although there is
some uncertainty in determining the apparent affinity of DA in blocking
the leak conductance, the results presented in Figure 4D argue that DA has quite comparable potencies as a
transport substrate (KT), in evoking a
transport-associated current, and in blocking a leak current
(K0.5). In turn, the comparable affinities of DA
for influencing the three functions support the assertion that all
three are mediated by hDAT.
Dependence of DA transport velocity on membrane potential
The finding that DA uptake by hDAT is closely associated with an
inward current is consistent with the hypothesis that the translocation
process itself is electrogenic, because it couples the movement of some
net charge with DA flux, as has been proposed for rat DAT by Krueger
(1990), McElvain and Schenk (1992), Kilty (1993), and Gu et al. (1994).
If transport-associated currents arise from a fixed stoichiometric
coupling of driving ions to DA during translocation, two corollaries
should follow, i.e., that the net charge:DA flux ratio should not vary
with membrane potential, and that DA transport velocity should increase
with membrane hyperpolarization in an exponential manner akin to the steady-state, transport-associated current (Fig.
4A).
To examine the voltage dependence of DA uptake velocity and the
hypothesis of fixed stoichiometric coupling, the rates of DA
accumulation were quantified in individual oocytes held under voltage
clamp and superfused (300 sec) with 20 µM DA or 10.1 µM [3H]DA. In each of six batches of
oocytes, mean DA uptake velocity increased with membrane
hyperpolarization (3-8 oocytes/potential). Pooled results from six
batches (each normalized to its 30 mV value) are presented in Figure
5A, and these demonstrated a clear voltage
dependence of DA uptake velocity. The mean uptake velocity in oocytes
clamped at 120 mV was 85% faster than that observed in those clamped
at 30 mV. Although uptake velocities increased with hyperpolarization
in all experiments, marked batch-to-batch differences in the magnitudes
of increase were observed. In the different batches, uptake velocities
observed in oocytes held at 90 were between 12 and 108% higher than
the corresponding velocities measured at 30 mV. Moreover, these
variations did not correlate with variations in levels of hDAT
expression.
Fig. 5.
Voltage dependence of DA uptake velocity and of
charge:DA flux ratios determined in voltage-clamped uptake experiments.
Oocytes were voltage-clamped and superfused with DA or
[3H]DA for 100-300 sec periods, after which the DA
accumulated by each oocyte was quantitated using HPLC-EC or
scintillation counting (see Materials and Methods). A,
For each of six oocyte batches, uptake velocities at each potential
were normalized to that seen at 30 mV (dotted line).
The normalized values were pooled, and the mean values (±SEM) are
plotted. The number of oocyte batches and the total number of oocytes
studied at each potential are 1:3 at +10 mV, 5:25 at 0 mV, 6:28 at 30
mV, 6:31 at 60 mV, 6:28 at 90 mV, and 4:17 at 120 mV.
B, From current records available for five of the six
oocyte batches, net charge:DA flux ratios were calculated for each
oocyte from the time integral of currents elicited during periods of DA
perfusion and the corresponding measurements of accumulated DA. Mean
ratios (±SEM) for oocytes tested at each potential are graphed. Means
were compared with that determined at 30 mV (3.1 ± 0.26) using
one-way ANOVA with a post hoc Dunnett's multiple
comparisons test, and they differ with p < 0.01 (asterisk). The number of oocyte batches and the total
number of oocytes studied at each potential are 5:25 at 0 mV, 5:24 at
30 mV, 5:26 at 60 mV, 5:24 at 90 mV, and 3:10 at 120 mV. The
dashed line at 2.0 represents the net charge:DA flux
ratio predicted for a fixed transport stoichiometry of 1 DA+/2 Na+/1 Cl.
[View Larger Version of this Image (11K GIF file)]
Correlation of charge and substrate fluxes
Time integrals of the DA-elicited currents were calculated for
oocytes in five of the six batches used for clamped uptake studies.
These measures of charge movement were correlated with the quantity of
DA concurrently accumulated by the same oocytes, yielding a net
charge:DA flux ratio for each of the 84 oocytes studied. Because this
ratio eliminates the effect of differing expression levels between
oocytes, net charge:DA flux determinations could be pooled for all
oocytes held at each membrane potential. As Figure 5B
illustrates, the charge:DA flux ratio displayed a minimum at 30 mV of
3.1 ± 0.26 e0/DA molecule
(mean ± SEM; n = 24), and the ratio increased
with either hyperpolarization or depolarization from that potential
(60 mV, 3.8 ± 0.4, n = 26; 90 mV, 4.1 ± 0.3, n = 24; 120 mV, 6.6 ± 0.9, n = 10; 0 mV, 6.8 ± 0.9, n = 25).
The change with voltage in the ratio of net charge:DA flux indicated
that not all DA-elicited current was stoichiometrically coupled to DA
translocation. This point has already been suggested for voltages above
the leak conductance reversal potential (approximately 18 mV), where
the inhibitory action of DA on the outward cation leak inflates the
estimate of charges moving inward. In contrast, the rise in the net
charge:DA flux ratio with hyperpolarization from 30 mV cannot be
attributed to the same mechanism: at more negative potentials,
inhibition of a tonic cationic leak current by DA would tend to
diminish the net inward current. Hence, if DA elicits an inward
transport-associated current and simultaneously blocks a tonic inward
leak, the observed current will actually underestimate the magnitude of
the transport-associated charge movement and the net charge:DA flux
ratio. The elevation in the net charge:DA flux ratio attributable to
hyperpolarization supports the existence of a second ionic conductance
that is associated with DA transport (see Discussion).
Differentiation of pharmacological agents by
I-V plots
To examine whether the hDAT-mediated currents elicited by DA and
cocaine are also elicited by other DA uptake inhibitors (or releasers),
a spectrum of pharmacological agents was assayed using voltage-jump
protocols with hDAT-expressing oocytes. Examination of drug-elicited
steady-state currents revealed that the ligands could be classified as
either DA-like, cocaine-like, or electrically inactive based on their
I-V plots. The DA-like group all displayed inverted
U-shaped I-V curves with peaks at approximately 20 mV (Fig. 6A, compare Fig.
4A) and include the psychomotor stimulants S(+)amphetamine and S(+)methamphetamine, the neurotoxin
MPP+ (1-methyl-4-phenylpyridinium), as well as
()norepinephrine and the indirect sympathomimetic agents
p-tyramine, m-tyramine, 
-phenethylamine, and
()metaraminol. The catecholaminergic neurotoxin 6-hydroxydopamine elicited no transport-associated current at 10 µM, and at
a 30 µM concentration, the transport-associated current
(at 120 mV) was less than one-tenth the amplitude of that elicited by
10 µM S(+)amphetamine in the same oocytes.
Fig. 6.
I-V plots readily distinguish two
classes of pharmacological agents that act at hDAT. Nineteen hDAT
ligands that were studied in voltage jump protocols could be resolved
into two groups, displaying either DA-like or cocaine-like voltage
dependence of their subtractive currents. A, Substrates
for hDAT such as dopamine (), p-tyramine (), and
amphetamine () elicit a conductance increase at potentials below
20 mV, and their subtractive currents are plotted as
IDrug IControl. These and other compounds (listed
below I-V plot) thought to be substrates for hDAT
displayed a characteristic inverted U-shaped I-V curve
(see also Fig. 3A). B, In contrast,
nontransported uptake inhibitors such as cocaine (), GBR12909 (),
methylphenidate (), and other listed drugs all block an inward
current at potentials below 20 mV and are plotted as
IControl IDrug. The reversal potential of the
conductance blocked by these drugs was approximately 20 mV and was
independent of drug concentration (30 µM). The I-V plots represent data obtained from several
different batches of oocytes, and each drug was tested in at least two
hDAT oocytes. At the concentrations tested, none of the drugs affected
ionic conductances of control oocytes.
[View Larger Version of this Image (20K GIF file)]
In contrast, a number of drugs manifested cocaine-like I-V
curves and appeared to reduce membrane conductance over the entire range of membrane potentials. Their I-V plots
(IControl IDrug) reversed at approximately 20 mV and grew steeper at positive potentials (Fig. 6B, compare Fig.
2B). Drugs falling in this category include the
stimulant/anorectic drugs methylphenidate, pemoline, amfonelic acid,
phendimetrazine, aminorex, and mazindol, as well as GBR12909,
indatraline (Lu19-005), and the cocaine analog RTI-55. Drugs that
produced no detectable electrical responses in hDAT-expressing oocytes
included the tyramine congeners with low potency for inhibiting [3H]DA uptakeL-tyrosine (100 µM) and 3-methoxytyramine (30 µM)and the
anti-parkinsonian drug amantadine (100 µM). At the
concentrations tested, none of the drugs produced responses in
water-injected or uninjected oocytes.
Comparison of DA and cocaine actions on the steady-state
leak currents
It is clear from the I-V plots of the hDAT ligands
examined, that like cocaine and DA at depolarized potentials, they too block a tonic leak conductance (Fig. 6). Therefore, the question arises
whether substrates and nonsubstrate ligands inhibit an identical leak
conductance or whether they affect distinct conductances. One criterion
for determining whether two ligands affect the same conductance is
whether their I-V curves reverse at the same potential. Because I-V plots of substrates do not cross the
abscissaa result of the drugs' combined effects on the
transport-associated and leak conductanceswe reasoned that
elimination of the transport-associated component from the total
DA-evoked current might serve to isolate the membrane leak current that
is blocked by DA. To abolish the transport-associated currents, buffer
substitutions of K+ and Li+ were made for
Na+, because substrate translocation by hDAT shows strict
dependence on Na+ ions. By examining the DA and cocaine
I-V values in ion-substituted buffers, their reversal
potentials could be compared; furthermore, the relative permeability of
ions through the leak conductance could be characterized by measuring
the shifts in reversal potential brought about by ion
substitutions.
As expected, substitution of K+ for Na+
abolished the transport-associated component of the DA I-V,
altering its inverted U-shape to a more ohmic current, which does
indeed reverse and which now resembles the cocaine I-V.
Figure 7A illustrates that in 96 mM K+ (2 mM Na+)
Ringer's buffer, IControl IDrug subtractions for both DA and cocaine
generated I-V plots that had comparable slope conductances over the entire voltage range (contrast Fig. 3). The DA- and
cocaine-inhibited conductances both reversed at 16 mV in
96K+ bufferthe same potential at which the cocaine
I-V reversed when assayed in Na+-Ringer's
buffer during a previous trial on the same oocyte. This reversal
potential is not different from the mean reversal potential (18.4 ± 2.4 mV, mean ± SEM) for cocaine I-V
values in Na+ Ringer's buffer measured in 26 oocytes. When
cocaine reversal potentials were measured successively in individual
oocytes using both Na+ Ringer's and 48 mM
K+/50 mM Na+ Ringer's buffers,
these too showed that, despite a 24-fold increase in the extracellular
K+ concentration, the reversal potentials were not
appreciably different: respectively, 14.3 ± 5.7 mV and
15.4 ± 3.2 mV (n = 4 oocytes).
Substitution of Li+ for Na+ also
eliminated substrate-elicited transport-associated currents and
revealed that the leak currents blocked by substrates and nonsubstrate
hDAT inhibitors had indistinguishable reversal potentials. Successive
applications of DA and cocaine, both in 98 mM
Li+ (0 Na+) buffer, blocked conductances with
reversal potentials of 0.0 ± 3.1 mV and 0.8 ± 3.2 mV,
respectively (Fig. 7B, representative of 4 oocytes).
Overall, the mean reversal potential for cocaine in Li+
buffer was 4.2 ± 3.1 mV (n = 10 oocytes). Comparable
results were observed using Li+-substituted buffers with
the substrates S(+)amphetamine, p-tyramine, and
-phenethylamine, and the nonsubstrate blocker GBR12909 (data not
shown). Furthermore, similar experiments have been performed with DA
and cocaine in buffer lacking both Na+ and Cl
(Li+ and MES substitutions, respectively), and the results
are qualitatively the same as those in Figure 7B (data from
3 experiments not shown). That Cl substitutions did not
shift the cocaine reversal potentials strongly suggests that
Cl ions are not carried by the hDAT leak conductance.
The Goldman-Hodgkin-Katz voltage equation was used to characterize
the ionic selectivity of the leak conductance from the shifts in the
cocaine reversal potential observed in ion-substituted buffers.
Assuming that the intracellular concentrations of Na+,
K+, and Li+ do not change appreciably, the
relative permeabilities of these ions are 1, 0.96, and 2.4 for the hDAT
leak conductance. That removal of all external Cl did not
shift the cocaine reversal potential suggests that the tonic hDAT leak
conductance is Cl-impermeant, and yet it is fairly
nonselective for alkali cations. This ionic selectivity of the leak
conductance differs markedly from the comparatively strict dependence
on Na+ and Cl of the hDAT transport
functions, i.e., substrate flux and transport-associated currents.
The ionic selectivity of the leak conductance was explored further with
experiments in which reversal potentials were successively determined
in individual oocytes from cocaine I-V plots executed in
Ringer's buffer and in choline-substituted buffer. Complete replacement of Na+ with choline caused leftward shifts in
the I-V curves. The mean reversal potentials shifted from
15.4 ± 3.8 mV in Ringer's to 48.4 ± 4.0 mV in choline
(n = 7 oocytes), a value substantially more positive
than the predicted Nernst potential for K+ in oocytes (95
mV) (Dascal, 1987; Costa et al., 1989). These results raise the
possibility that choline or some other ion may permeate the leak
conductance aside from Na+ and K+. Although
removal of Ca2+ or Mg2+ from the Ringer's had
no effect, manipulating the buffer between pH 6.5 and 8.5 strongly
affected the cocaine reversal potentials, shifting them approximately
38.5 ± 3.5 mV per unit increase in pH (n = 10 oocytes). These data suggest that protons may be carried by the leak
conductance or, alternatively, that pH can alter the ionic selectivity
of the leak. Because a 10-fold change in
[H+]o had a greater effect than an
approximately 50-fold change in [Na+ + K+]o and that [Na+ + K+]o/[H+]o is
104, the Goldman-Hodgkin-Katz voltage equation suggests
that if protons do permeate through the leak conductance, their
relative permeability is more than four orders of magnitude greater
than that of alkali cations.
DISCUSSION
The Xenopus oocyte expression system has proven an
exceedingly useful method for investigating the function of cloned
Na+-coupled transport proteins, in part because both
biochemical and electrical indices of transport can be studied. To
characterize the electrogenic properties of a DAT cloned from human
midbrain RNA, we have measured substrate uptake, radioligand binding,
and both ramp and steady-state currents evoked under voltage-clamp conditions in oocytes injected with hDAT cRNA. Expression of the hDAT
in oocytes gives rise to DA transport activity, the pharmacological properties of which are consistent with those displayed in heterologous mammalian cell lines transfected with hDAT; the apparent affinity of DA
uptake in oocytes, the inhibition constants for cocaine and
S(+)amphetamine, as well as the potencies of mazindol, GBR12935, and
RTI-55, were within the ranges determined previously in cell lines
expressing hDAT (Giros et al., 1992; Pifl et al., 1993; Giros et al.,
1994; Pristupa et al., 1994; Eshleman et al., 1995). As would be
expected for a member of the Na+/Cl-dependent
neurotransmitter transporter family, removal of either extracellular
Na+ or Cl ions virtually abolished the
transport of [3H]DA into hDAT-expressing oocytes.
DA uptake and transport-associated current
Using two-electrode voltage-clamp techniques, we have found that
application of substrates to hDAT-expressing oocytes reliably elicited
voltage-dependent steady-state currents that were not observed in
control oocytes. When studied under conditions that precluded substrate
translocation, the electrophysiological actions of substrates and
nonsubstrate transport inhibitors revealed that hDAT mediates a
cationic leak conductance that is constitutive and uncoupled from the
uptake process. The two currents that hDAT expression confers on
oocytes are designated the transport-associated and the leak
currents.
Several lines of evidence suggest that the inward transport-associated
current observed at hyperpolarized potentials after application of DA
(or other hDAT substrates) is closely linked to substrate translocation
by hDAT. The transport-associated current was caused by a conductance
increase (Fig. 2C), consistent with the coupled movement of
a positively charged DA molecule and Na+ ions. Numerous
pharmacological attributes of the transport-associated current were
essentially identical to those of [3H]DA uptake. The
concentration dependence of [3H]DA uptake velocity and of
DA-evoked transport-associated current amplitude were both well fit by
rectangular hyperbolic functions with similar kinetic constants
(KT = 1.7 µM and
K0.5 = 1.9-2.9 µM). The set of
pharmacological agents that evoked transport-associated currents
includes compounds previously determined to be substrates by
biochemical assay (Fig. 6A); conversely, nonsubstrate
uptake blockers (e.g., cocaine and methylphenidate) were unable to
elicit transport-associated currents (Fig. 6B).
Furthermore, the apparent affinities of S(+)amphetamine were nearly
identical for inhibiting [3H]DA uptake
(Ki = 0.3 µM) and for
evoking transport currents (K0.5 = 0.5 µM), as would be expected for a competitive inhibitor of
DA uptake that is an alternative transport substrate (Ross, 1976).
Finally, treatments that prevented uptake of substrate, such as
coincubation with pharmacologically appropriate concentrations of
cocaine or replacement of extracellular Na+ or
Cl, also abolished transport-associated currents evoked
by DA (Figs. 3, 7).
Dependence of DA uptake on membrane potential
The close association of substrate-evoked voltage-dependent
currents with substrate translocation strongly supports the assertion that hDAT is an electrogenic transporter, a hypothesis developed from
studies of the apparent ionic coupling of rodent DATs (Krueger, 1990;
McElvain and Schenk, 1992; Kilty, 1993; Gu et al., 1994). Our results
provide direct evidence of the electrogenic nature of hDAT in their
demonstration that the velocity of DA uptake increased with
hyperpolarization (Fig. 5A). Several studies have demonstrated that the ability of striatal preparations to accumulate [3H]DA uptake could be diminished by agents that alter
neuronal membrane potentials, such as ouabain, elevated external
K+ concentrations, veratridine, batrachotoxin, and
metabolic inhibitors (Baldessarini and Vogt, 1971; Holz and Coyle,
1974; Liang and Rutledge, 1982; Krueger, 1990). One ambiguity in these
results, however, is that the effects of these agents on membrane
potential could not be separated from their effects on the ionic
gradients that drive substrate translocation.
hDAT-expressing oocytes, on the other hand, offered the opportunity to
directly assess the influence of membrane potential on DA uptake
velocity, because the voltage could be manipulated independently of
ionic gradients, and the expression levels were sufficient to allow the
biochemical quantitation of DA accumulated by single cells during
periods of voltage clamp. The increase in uptake velocity observed with
membrane hyperpolarization is consistent with the expectation that the
thermodynamic driving force for an electrogenic transporter includes
transmembrane electrical potential, as well as chemical gradients for
substrates and cotransported ions. Furthermore, the data indicate that
the effect of membrane potential on DA uptake is mediated primarily
through alterations in the turnover rate (Vmax)
of hDAT rather than its apparent affinity for substrate, because DA
concentrations used in the clamped uptake experiments were near
saturating. An effect of voltage on turnover rate, but not on substrate
affinity, is also supported by the finding that the
K0.5 of DA for eliciting transport-associated currents was largely independent of membrane potential (Fig.
4D). The voltage-dependent and rate-limiting step in
the hDAT translocation reaction, therefore, is likely to occur
subsequent to substrate binding.
The clamped uptake experiments, apart from providing some biophysical
insight into hDAT translocation, also suggest a physiological context
in which the voltage dependence of DA uptake may help regulate
intercellular signaling by dopaminergic neurons. Recent in
vitro and in vivo studies have suggested that drugs
acting at D2-like autoreceptors modulate the velocity of DA
uptake (Meiergerd et al., 1993; Parsons et al., 1993; Cass and
Gerhardt, 1994), and the voltage dependence of hDAT function
demonstrated in our studies could provide the mechanism through which
autoreceptor activation influences DA reuptake. In situ
hybridization of rat DAT mRNA indicates that DAT is expressed
exclusively in dopaminergic neurons (Cerruti et al., 1993; Lorang et
al., 1994), and immunocytochemical studies demonstrate that terminal
and somatodendritic zones of rat midbrain DA neurons are rich in both
DAT (Ciliax et al., 1995; Nirenberg et al., 1996) and
D2-like autoreceptors (Sesack et al., 1994; Yung et al.,
1995). Ample electrophysiological evidence shows that activation of
autoreceptors in dopaminergic cells in the substantia nigra and ventral
tegmental area causes the cells to hyperpolarize through the
G-protein-mediated opening of K+ channels (for review, see
Lacey, 1993; White, 1996). Our data predict that in regions of
dopaminergic neurons in which DAT and D2 receptors are
colocalized, hyperpolarization attributable to autoreceptor activation
by DA will increase the DAT turnover rate and thus accelerate the
clearance of extracellular DA. Faster removal of extracellular DA
complements the autoreceptor-mediated inhibition of DA release and may
sharpen the temporal response of DA signaling in these important cell
groups. A second consequence predicted from the colocalization of
autoreceptors and DAT is that the DA-activated K+
conductance would act to offset the depolarizing action of the transport-associated current, and thereby cancel the small, positive feedback effect that DA uptake might contribute toward promoting Ca2+-dependent vesicular DA release.
Two components of transport-associated currents
Despite the numerous common prop
