Multiple ionic conductances of the human dopamine transporter: the actions of dopamine and psychostimulants

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.

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 μmcocaine (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 I_Drug− I_Control convention. Comparison of the DAI–V with the DA + Cocaine I–V reveals the transport-associated component that is susceptible to blockade by cocaine.

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 mmNa⁺) Ringer’s buffer, of 10 μm DA (▵) and 3 μm cocaine (□). Subtractive currents (I_Control −I_Drug) determined during voltage jump protocols represent the I–V of the leak conductance, which is blocked by all three drug treatments. CocaineI–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.

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.

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 (I_Control −I_Cocaine) 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 (I_DA −I_Control) plotted inC.

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, I_DA −I_Control 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 K_0.5 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 ofK_0.5 and CI₉₅ values (error bars) were determined from affinity values derived individually for each of six oocytes (see Materials and Methods). The apparent affinities (K_0.5) of DA for eliciting transport-associated current (•) and for blocking a leak current (▪) are displayed. The K_0.5 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 (K_T, ——; 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.

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. Thedashed line at 2.0 represents the net charge:DA flux ratio predicted for a fixed transport stoichiometry of 1 DA⁺/2 Na⁺/1 Cl⁻.

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 asI_Drug −I_Control. These and other compounds (listedbelow 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 asI_Control −I_Drug. The reversal potential of the conductance blocked by these drugs was approximately −20 mV and was independent of drug concentration (≤30 μm). TheI–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.