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Previous Article | Next Article
Volume 17, Number 3, Issue of February 1, 1997 pp. 960-974
Copyright ©1997 Society for NeuroscienceMultiple 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
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
Key words: Na+/Clper 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
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
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
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
MATERIALS AND METHODS
hDAT cloning and expression. Total RNA was extracted according to the method of Chomczynski and Sacchi (1987)and 3
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
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
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
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
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 
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 (
[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
) and 10 µM cocaine (
) reveal that both drugs block a conductance with the same reversal potential (
[View Larger Version of this Image (15K GIF file)]
Concentration-response data were collected by measuring subtracted currents (IDA
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 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
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
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 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
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) (sec 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
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
[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 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 
Fig. 2. Membrane conductance changes evoked by DA and cocaine. Currents measured in a single voltage-clamped oocyte during voltage ramps (
[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
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
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)
From these data, it appears that the electrophysiological action of cocaine on hDAT is limited to blocking ionic conductances 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
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, 3.0 µM;
, 30 µM). Over the potential range
) was well fit by a simple Michaelis-Menten equation for individual potentials over the range
[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
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
Although the Michaelis-Menten function provided a less satisfactory fit to the DA concentration dependence of current at voltages more positive than 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)
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 ( 
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
[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
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 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-phenethylamine, and (
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 (
[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 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 abscissa
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
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
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
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
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., 1992DA 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 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
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 Two components of transport-associated currents
Despite the numerous common properties that appear to unite transport-associated currents and substrate translocation in hDAT-expressing oocytes, clamped uptake experiments suggested that the transport-associated currents may be comprised of two components, only one of which is stoichiometrically tied to substrate translocation. Concurrent measurement of DA-elicited currents and DA uptake in voltage-clamped oocytes provides strong evidence that not all of the DA-elicited transport current arises from stoichiometric coupling of 2 Na+ and 1 Cl
Two different mechanistic hypotheses might account for the disparities between DA-elicited currents and uptake velocities. One is that the DA transporter operates with variable coupling of substrate and driving ions, and that hyperpolarization from
If a portion of the hDAT transport-associated current is indeed stoichiometrically uncoupled from substrate movement, this would suggest that hDAT mediates three discernible ionic conductances: the putative stoichiometrically coupled movement of DA+/2 Na+/Cl Characterization of the leak conductance
The action of DA on the tonic leak conductance at hyperpolarized potentials is stated in a tentative manner, because it is ordinarily masked by the transport-associated current under those ionic conditions that permit substrate translocation. It was possible, however, to isolate the action of DA on the tonic leak conductance from its action on transport-associated conductances by replacing external Na+ in the superfusing Ringer's buffer. When K+, Li+, or choline was substituted for Na+, both specific [3H]DA uptake into oocytes and the transport-associated inward current elicited by DA were abolished (Fig. 7). Nevertheless, the ability of drugs, including DA and cocaine, to block the leak conductance in these buffers remained intact, and they did so at concentrations essentially the same as those effective in normal Na+ Ringer's. In contrast to normal Na+ conditions, however, the I-V plots of DA and cocaine were comparable across the entire voltage range; furthermore, the I-V values reversed at the same potentials (Fig. 7), indicating that it is highly probable that both substrate and nonsubstrate translocation inhibitors block an identical hDAT leak conductance. Because ion substitution experiments revealed that the leak conductance blockade by DA (and other substrates) occurred at negative as well as positive potentials, it seems reasonable to infer that substrates also block the leak conductance at negative potentials in Na+ Ringer's despite the overwhelming effect of the transport-associated currents they evoke.
The tonic leak conductance blocked by both DA and cocaine also appears to be susceptible to inhibition by virtually all DAT ligands tested regardless of their effect on the transport-associated current. The inhibition can be seen in subtractive currents for all drugs in the voltage range above
The leak conductances of hDAT and rSERT also share a rather uncommon ionic selectivity. Analysis of the shifts in the cocaine reversal potential attributable to ion substitution for Na+ indicate that Li+ and K+ are, respectively, 2.4-fold and 0.96-fold as permeant as Na+ through the hDAT leak conductance. These values are reasonably close to the estimates of Mager and colleagues (1994), who report that the magnitude of the rSERT leak current is increased threefold by substitution of Li+ for Na+, but reduced by 27% by K+ substitution. We found no evidence suggesting that Cl
Similarly, it is not currently known whether the site at which substrates bind to block the leak conductance is in the permeation pathway or outside of it. Our ion substitution results indicate, however, that the binding of cosubstrate ions is not a prerequisite for substrate binding and leak blockade, because DA readily blocks the leak conductance in the complete absence of external Na+ and Cl hDAT currents as a pharmacological tool: implications for DAT-mediated DA release
The demonstration that the transport-associated and leak currents are intrinsic to hDAT function helps to highlight the possibility that the psychomotor stimulants, a class of drugs with broad societal relevance, may influence the electrical properties of neurons directly as well as indirectly through the regulation of extracellular DA levels. For instance, the observation that hDAT leak conductance can be inhibited by the same pharmacological agents and concentrations as those affecting DA translocation suggests that drugs such as cocaine, methylphenidate, pemoline, and mazindol modulate ionic flux as well as substrate uptake into dopaminergic cells in vivo. If protons are indeed carried by the hDAT leak conductance, our data suggest that the selectivity and voltage dependence of the leak is comparable to that of voltage-activated proton-selective conductances (DeCoursey and Cherny, 1994
Apart from the physiological consequences of the ionic currents of hDAT, the transport-associated and leak currents provide new tools with which to elucidate hDAT pharmacology and the mechanisms of action of psychomotor stimulants. Using the criterion of whether drugs elicited transport-associated currents, it was easy to discriminate hDAT substrates from nonsubstrate translocation inhibitors (Fig. 6). Our categorization accords well with and extends the current understanding of how drugs act on hDAT. Because both substrate and nonsubstrate ligands inhibit uptake of radiolabeled substrates by DAT, differentiation of the two classes of drugs has relied on two types of biochemical assays. By one approach, striatal preparations or transfected cells are used to measure the sequestration of putative substrates that have been radiolabeled or are detectable by other methods (e.g., electrochemistry, chromatography, spectrophotometry, or a combination). The second approach assesses the ability of putative substrates to promote the efflux of an authentic preloaded substrate, presumably through a mechanism akin to carrier-mediated facilitated diffusion (Stein, 1986
Although assays of uptake and efflux have formed the basis of the contemporary classification of hDAT substrates and nonsubstrate inhibitors, they have not provided altogether unambiguous results, because the endpoints measured in such assays do not measure the activity of DAT alone but can also be influenced by agents that modify DA synthesis, degradation, and vesicular sequestration, as well as those that alter membrane potential and spontaneous release processes. Indeed, although it is well accepted that amphetamine, methamphetamine, and several Conclusion
Data have been presented that indicate that hDAT mediates both a transport-associated current and a constitutive leak current in Xenopus oocytes, and that these currents can be distinguished from each other by the criteria of ionic dependence and selectivity, voltage dependence, and the opposing actions substrates have on them. Furthermore, our evidence suggests that the transport-associated current itself is complex and may arise from two ionic conductances, both of which are activated by DA under conditions that permit substrate translocation (or a single transport conductance that operates with variable coupling). In contrast, the tonic leak conductance is inhibited by hDAT substrates and nontranslocated uptake blockers alike. All three putative conductances appear to be sensitive to individual hDAT ligands in the same concentration range, and therefore, all may be of potential relevance to the actions of hDAT agents. We do not know whether the three conductances are all intrinsic to a single transporter molecule, to hDAT oligomers, or to distinct subpopulations of modified hDAT molecules, or whether some ionic conductance attributed to hDAT is indirectly mediated through an extrinsic protein (Sonders and Amara, 1996
Although our results predict that neuronal membrane potential will directly modulate hDAT uptake activity, at this stage it is not known whether hDAT-mediated currents contribute significantly to the electrical properties of dopaminergic neurons in vivo. Whole-cell currents attributable to neurotransmitter uptake have been demonstrated in a variety of neuronal and glial cells (for review, see Lester et al., 1994
FOOTNOTES
Received Aug. 29, 1996; revised Nov. 12, 1996; accepted Nov. 18, 1996.
These investigations have been supported by 5T32DA07262, DA07595, GM48709, DA04216, K02DA00174, F32DA05706, and HHMI. Tissue was obtained from the Oregon Brain Bank, which is partially supported by Grant P30A908017. We thank Geoffrey Murdoch, Wendy Fairman, and Weibin Zhang for multiple contributions to this work, and Jonathan Javitch for helpful editorial comments.
Correspondence should be addressed to Dr. Susan G. Amara, Vollum Institute, L474, Oregon Health Sciences University, Portland, OR 97201 (amaras{at}ohsu.edu) or to Dr. Mark S. Sonders (sondersm{at}ohsu.edu).
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