Interaction between dopamine and its transporter: role of intracellular sodium ions and membrane potential

The present study addresses the effect of intracellular Na(+) and membrane potential on the binding of dopamine (DA) to the dopamine transporter (DAT). Perforation of plasma membranes of DAT-expressing cells with gramicidin diminished DA uptake and decreased the potency (increases K(i)) of DA in inhibiting the binding of cocaine analog [(3)H]2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane (CFT). It also compromised the ability of external Na(+) to reduce DA K(i). No substantial effect on DA K(i) was observed upon gramicidin treatment in Na(+)-free buffer, membrane depolarization with high [K(+)](o), or elevation of [Na(+)](i) with monensin under non-depolarizing conditions. Elevation of DA K(i) was greater at more positive potentials when [Na(+)](i) was raised to a similar level, or at higher [Na(+)](i) when the membrane was depolarized to a similar level. In cells expressing D313N DAT, DA K(i) was significantly higher but less sensitive to gramicidin than that in wild-type (WT) cells. In contrast, DA K(i) in cell-free membranes was insensitive to Na(+), gramicidin, and D313N mutation. The data suggest that (i) intracellular Na(+) plays a role in affecting the external access to DA binding sites at DAT on depolarized plasma membranes of cells, and (ii) access to DA binding sites in cell-free membranes may occur from the intracellular side of the membrane. Unlike DA binding, CFT binding to both cells and membranes was sensitive to Na(+) and D313N mutation but insensitive to gramicidin, consistent with exclusively external access to sites that are different from but conformationally linked to those for DA.     

Substrates and inhibitors display different sensitivity to expression level of the dopamine transporter in heterologously expressing cells

The use of heterologous expression systems for studying dopamine (DA) transporter (DAT) function has provided important information corroborating and complementing in situ obtained knowledge. Preliminary experiments with human embryonic kidney cells (HEK293) heterologously expressing varying amounts of DAT suggested fluctuations in the potency of cocaine in inhibiting DA uptake and led to the present systematic assessment of the impact of the density of DAT on its function. Transiently expressing intact HEK293 cells, transfected with increasing amounts of DAT cDNA, displayed increasing levels of surface DAT, binding of the cocaine analog [(3)H]2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane ([(3)H]CFT), and uptake of [(3)H]DA, [(3)H]N-methyl-4-phenylpyridinium ([(3)H]MPP(+)), [(3)H]norepinephrine, and [(3)H]serotonin. However, the amount of DAT cDNA and the DAT expression level required to produce 50% of maximal activity was threefold higher for CFT binding than for DA uptake. Increased DAT expression was accompanied by weakened potency in inhibiting [(3)H]DA uptake for cocaine, CFT, benztropine, and its analog JHW025, GBR 12909 and mazindol; their potency in inhibiting [(3)H]CFT binding was unaffected. Inhibition of uptake by the substrates DA, m-tyramine, d-amphetamine, or MPP(+) was also unaffected. Increasing DAT in stably expressing HEK293 cells by stimulation of gene expression with sodium butyrate also decreased the uptake inhibitory potency of a number of the above blockers without affecting the interaction between substrates and DAT. The present results prompt discussion of models explaining how factors regulating DAT expression at the plasma membrane can regulate DAT function and pharmacology.     

Cationic interactions at the human dopamine transporter reveal binding conformations for dopamine distinguishable from those for the cocaine analog 2 alpha-carbomethoxy-3 alpha-(4-fluorophenyl)tropane

In membrane preparations, CFT, a phenyltropane cocaine analog, and dopamine (DA) interact with the recombinant human dopamine transporter (hDAT) in Na+ -free medium. Na+ markedly increased the transporter's affinity for CFT, but had little or no effect on DA potency for inhibiting CFT binding. Raising [Na+ ] from 20 to 155 mm reduced Li+ -induced increase in DA K (i), but not CFT K (d). The presence of 155 mm Na+ enhanced the tolerance to low pH of CFT Kd but not DA Ki. Leucine substitution for tryptophan 84 (W84L) in transmembrane domain (TM) 1 or asparagine substitution for aspartate 313 (D313N) in TM 6 did not or only modestly enhance the affinity of Na+ -independent CFT binding, and retained the near normal ability of DA, Li+, K+, or H+ to inhibit this binding. However, the mutations significantly enhanced the Na+ stimulation of CFT binding as well as the Na+ antagonism against Li+ and H+ inhibition of CFT binding. In contrast, the mutations neither changed the Na+ -insensitive feature of DA Ki nor enhanced the Na+ protection of DA Ki against Li+ 's inhibitory effect, though they caused Na+ protection of DA Ki against H+ 's inhibitory action. These results are consistent with the existence of binding conformations for DA that are distinguishable from those for CFT, and with a differential association of cation interactions with DA and CFT binding. The mutations likely alter Na+ -bound state(s) of hDAT, preferentially strengthening the positive allosteric coupling between Na+ and CFT binding, and reducing the impact of Li+ or H+ on the CFT binding.     

Interaction of catechol and non-catechol substrates with externally or internally facing dopamine transporters

Our previous work suggested that collapsing the Na⁺ gradient and membrane potential converts the dopamine (DA) transporter (DAT) to an inward-facing conformation with a different substrate binding profile. Here, DAT expressing human embryonic kidney 293 cells were permeabilized with digitonin, disrupting ion/voltage gradients and allowing passage of DAT substrates. The potency of p-tyramine and other non-catechols ( d -amphetamine, β-phenethylamine, MPP⁺) in inhibiting cocaine analog binding to DAT in digitonin-treated cells was markedly weakened to a level similar to that observed in cell-free membranes. In contrast, the potency of DA and another catechol, norepinephrine, was not significantly changed by the same treatment, whereas epinephrine showed only a modest reduction. These findings suggest that catechol substrates interact symmetrically with both sides of DAT and non-catechol substrates, favoring binding to outward-facing transporter. In the cocaine analog binding assay, the mutant W84L displayed enhanced intrinsic binding affinity for substrates in interacting with both outward- and inward-facing states; D313N showed wild-type-like symmetric binding; but D267L and E428Q showed an apparent improvement in the permeation pathway from the external face towards the substrate site. Thus, the structure of both substrate and transporter play a role in the sidedness and mode of interaction between them.     

High affinity stereospecific binding of [3H] cocaine in striatum and its relationship to the dopamine transporter

A high affinity (KD 35 nM) binding site for [3H]cocaine is detected in rat brain striatum present at 2-3 pmol/mg protein of synaptic membranes. This binding is displaced by cocaine analogues with the same rank order as their inhibition of [3H]dopamine ([3H]DA) uptake into striatal synaptosomes (r = 0.99), paralleling the order of their central stimulant activity. The potent DA uptake inhibitors nomifensine, mazindol, and benztropine are more potent inhibitors of this high affinity [3H]cocaine binding than desipramine and imipramine. Cathinone and amphetamine, which are more potent central stimulants than cocaine, displace the high affinity [3H]cocaine binding stereospecifically, but with lower potency (IC50 approximately equal to 1 microM) than does cocaine. It is suggested that the DA transporter in striatum is the putative "cocaine receptor." Binding of [3H]cocaine, measured in 10 mM Na2HPO4-0.32 M sucrose, pH 7.4 buffer, is inhibited by physiologic concentrations of Na+ and K+ and by biogenic amines. DA and Na+ reduce the affinity of the putative "cocaine receptor" for [3H]cocaine without changing the Bmax, suggesting that inhibition may be competitive. However, TRIS reduces [3H]cocaine binding noncompetitively while Na+ potentiates it in TRIS buffer. Binding of [3H]mazindol is inhibited competitively by cocaine. In phosphate-sucrose buffer, cocaine and mazindol are equally potent in inhibiting [3H]mazindol binding, but in TRIS-NaCl buffer cocaine has 10 times lower potency. It is suggested that the cocaine receptor in the striatum may be an allosteric protein with mazindol and cocaine binding to overlapping sites, while Na+ and DA are allosteric modulators, which stabilize a lower affinity state for cocaine.     

Amphetamine-induced dopamine efflux. A voltage-sensitive and intracellular Na+-dependent mechanism

Amphetamine (AMPH) elicits its behavioral effects by acting on the dopamine (DA) transporter (DAT) to induce DA overflow into the synaptic cleft. Facilitated exchange diffusion is the classical model used to describe AMPH-induced DA efflux. This model hypothesizes that AMPH-induced DA efflux is mediated by DAT and results from the transport of AMPH into the cell followed by a counter movement of DA out to the extracellular compartment. To further characterize the action of AMPH, we used the patch clamp technique in the whole-cell configuration combined with amperometry on human embryonic kidney HEK-293 cells stably transfected with the human DAT (DAT cells). In DAT cells, AMPH-induced DAT-mediated currents were blocked by cocaine. We demonstrate that DA efflux mediated by DAT is voltage-dependent, electrogenic, and dependent on intracellular Na(+) concentration in the recording electrode. Intracellular Na(+) fluorescence, as measured by confocal microscopy using a Na(+)-sensitive dye, was enhanced by AMPH application. Furthermore, the ability of AMPH to induce DA efflux was regulated by intracellular Na(+) concentration and correlated with the size of the DAT-mediated, AMPH-induced ion flux across the plasma membrane. In the absence of intracellular Na(+) but the presence of high intracellular Cl(-), AMPH-induced inward currents elicited DA efflux proportionally to their dimension and duration. Thus, we propose that AMPH-induced DA efflux depends on two correlated transporter processes. First, AMPH binds to the DAT and is transported, thereby causing an inward current. Second, because of this AMPH-induced inward current, Na(+) becomes more available intracellularly to the DAT, thereby enhancing DAT-mediated reverse transport of DA.     

Aspartate 345 of the dopamine transporter is critical for conformational changes in substrate translocation and cocaine binding

The present study elucidated the role of aspartate 345, a residue conserved in the third intracellular loop of all Na+/Cl(-)-dependent neurotransmitter transporters, in conformational changes of the dopamine (DA) transporter. Asparagine substitution (D345N) resulted in near normal transporter expression on the cell surface but caused extremely low Vmax and Km values for DA uptake, converted the inhibitory effect of Zn2+ on DA uptake to a stimulatory one, and eliminated reverse transport. The cocaine-like inhibitor 2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane or the selective DA transporter inhibitor GBR12935 bound to D345N with a normal affinity and still inhibited DA uptake potently. However, the mutation reduced the binding capacity of the surface transporter for these two inhibitors by 90% or more. Moreover, the binding activity of D345N can be significantly improved by Zn2+ but not by Na+. These results are consistent with a defect in reorientation of the substrate-binding site to the extracellular side, leading to a loss of the outward-facing conformational state where external DA binds to initiate uptake and the inhibitors bind to initiate uptake inhibition. Alanine or glutamate substitution produced a similar phenotype, suggesting that both the negative charge and the residue volume at position 345 are vital. Furthermore, in intact cells, cocaine potentiated the reaction of the membrane-impermeant sulfhydryl reagent methanethiosulfonate ethyltrimethylammonium with the extracellularly located endogenous cysteines of D345N but not those of wild type, and this potentiation was blocked upon K+ substitution for Na+. Thus, cocaine binding to D345N likely induces a different and Na(+)-dependent conformational change, which may contribute to its Na(+)-dependent uptake inhibitory activity.     

New findings on nuclear gangliosides: overview on metabolism and function

Dopamine (DA) is an important transmitter in both motor and limbic pathways. We sought to investigate the role of D(1)-receptor activation in axonal DA release regulation in dorsal striatum using a D(1)-receptor antagonist, SKF-83566. Evoked DA release was monitored in rat striatal slices using fast-scan cyclic voltammetry. SKF-83566 caused a concentration-dependent increase in peak single-pulse evoked extracellular DA concentration, with a maximum increase of ～ 65% in 5 μM SKF-83566. This was accompanied by a concentration-dependent increase in extracellular DA concentration clearance time. Both effects were occluded by nomifensine (1 μM), a dopamine transporter (DAT) inhibitor, suggesting that SKF-83566 acted via the DAT. We tested this by examining [(3)H]DA uptake into LLc-PK cells expressing rat DAT, and confirmed that SKF-83566 is a competitive DAT inhibitor with an IC(50) of 5.7 μM. Binding studies with [(3)H]CFT, a cocaine analog, showed even more potent action of SKF-83566 at the DAT cocaine binding site (IC(50) = 0.51 μM). Thus, data obtained using SKF-83566 as a D(1) DA-receptor antagonist may be confounded by concurrent DAT inhibition. More positively, however, SKF-83566 might be a candidate to attenuate cocaine effects in vivo because of the greater potency of this drug at the cocaine versus DA binding site of the DAT.     

Mutation of Trp84 and Asp313 of the dopamine transporter reveals similar mode of binding interaction for GBR12909 and benztropine as opposed to cocaine

The different psychomotor-stimulant effects of cocaine, GBR12909, and benztropine may partially stem from their different molecular actions on the dopamine transporter (DAT). To explore this possibility, we examined binding of these inhibitors to mutated DATs with altered Na(+) dependence of DAT activities and with enhanced binding of a cocaine analog, [(3)H]2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane (CFT). In [(3)H]CFT competition assays with intact cells, the mutation-induced change in the ability of Na(+) to enhance the apparent affinity of CFT, cocaine, GBR12909, and benztropine was inhibitor-independent. Thus, for the four inhibitors, the curve of [Na(+)] versus apparent ligand affinity was steeper at W84L compared with wild type, shallower at D313N, and flat at W84LD313N. At each mutant, the apparent affinity of CFT and cocaine was enhanced regardless of whether Na(+) was present. However, the apparent affinity of GBR12909 and benztropine for W84L was reduced in the absence of Na(+) but near normal in the presence of 130 mm Na(+), and that for D313N and W84LD313N was barely changed. At the single mutants, the alterations in Na(+) dependence and apparent affinity of the four inhibitors were comparable between [(3)H]CFT competition assays and [(3)H]dopamine uptake inhibition assays. These results demonstrate that DAT inhibitors producing different behavioral profiles can respond in an opposite way when residues of the DAT protein are mutated. For GBR12909 and benztropine, their cocaine-like changes in Na(+) dependence suggest that they prefer a DAT state similar to that for cocaine. However, their cocaine-unlike changes in apparent affinity argue that they, likely via their diphenylmethoxy moiety, share DAT binding epitopes that are different from those for cocaine.     

Influence of sodium on the alpha 2-adrenergic receptor system of human platelets. Role for intraplatelet sodium in receptor binding

The affinity of many types of membrane receptors for agonists is decreased by Na+ in radioligand binding experiments. We studied the alpha 2-adrenergic receptor of human platelets to determine whether Na+ acts at an intracellular or extracellular location. The Na+ content of intact platelets in an isotonic saline buffer was 38 nmol/10(8) platelets. This increased to 138 nmol/10(8) platelets with the Na+-selective ionophore monensin and decreased to 13 nmol/10(8) platelets with incubation in a Na+-free buffer. Epinephrine-induced platelet aggregation was increased by the addition of monensin and was decreased in the Na+-free buffer, while thrombin-induced aggregation was unaltered by either condition. Monensin, gramicidin, and ouabain (which all increased intraplatelet Na+) caused a 2-3-fold increase in the Kd of epinephrine (in competition with [3H]yohimbine) for alpha 2-adrenergic receptors on intact platelets. Conversely, incubation in a Na+-free buffer (which decreased intraplatelet Na+) decreased the Kd of the receptors for epinephrine 2-3-fold. These experiments suggest that changes in intracellular Na+ alter epinephrine binding. Control studies eliminated several alternative explanations for the effect of monensin on epinephrine binding: 1) monensin altered epinephrine binding only with intact platelets and not with platelet membranes; 2) although monensin depolarized platelets (assessed by [3H]methyltriphenylphosphonium uptake), other depolarizing conditions did not change epinephrine binding; 3) although monensin may increase intracellular pH (by exchanging Na+ for H+) such an increase in pH decreased the Kd of alpha 2-receptors on platelet membranes for epinephrine, an effect opposite to that produced by monensin in intact platelets. We conclude that alterations in the intracellular concentration of Na+ may change the affinity of platelet alpha 2-receptors for epinephrine. These results suggest a key role for intracellular Na+ in modulating binding at cell surface receptors in vivo.     

β-Phenylethylamine requires the dopamine transporter to increase extracellular dopamine in Caenorhabditis elegans dopaminergic neurons

β-Phenylethylamine (βPEA) is an endogenous amine that has been shown to increase the synaptic levels of dopamine (DA). A number of in vitro and behavioral studies suggest the dopamine transporter (DAT) plays a role in the effects generated by βPEA, however the mechanism through which βPEA affects DAT has not yet been elucidated. Here, we used Caenorhabditis (C.) elegans DAT (DAT-1) expressing LLC-pk1 cells and neuronal cultures to investigate whether the βPEA-induced increase of extracellular DA required DAT-1. Our data show that βPEA increases extracellular dopamine both in DAT-1 transfected cells and cultures of differentiated neurons. RTI-55, a cocaine homologue and DAT inhibitor, completely blocked the βPEA-induced effect in transfected cells. However in neuronal cultures, RTI-55 only partly inhibited the increase of extracellular DA generated by βPEA. These results suggest that βPEA requires DAT-1 and other, not yet identified proteins, to increase extracellular DA when tested in a native system. Furthermore, our results suggest that βPEA-induced increase of extracellular DA does not require functional monoamine vesicles as genetic ablation of the C. elegans homologue vesicular monoamine transporter, cat-1, did not compromise the ability of βPEA to increase extracellular DA. Finally, our electrophysiology data show that βPEA caused fast-rising and self-inactivating amperometric currents in a subset of wild-type DA neurons but not in neurons isolated from dat-1 knockout animals. Taken together, these data demonstrate that in both DA neurons and heterogeneous cultures of differentiated C. elegans neurons, βPEA releases cytoplasmic DA through DAT-1 to ultimately increase the extracellular concentration of DA.     

Protein kinase C-mediated functional regulation of dopamine transporter is not achieved by direct phosphorylation of the dopamine transporter protein

Dopaminergic neurotransmission is terminated by the action of the presynaptic dopamine transporter (DAT). It mediates Na(+)/Cl(-) -dependent re-uptake of extracellular dopamine (DA) into the cell, and is regarded as a major regulatory mechanism for synaptic transmission. Previous works have documented that protein kinase C (PKC) activator or inhibitor alters DA uptake by DAT, suggesting that PKC phosphorylation plays an important regulatory mechanism in DAT function. Based on the existence of consensus amino acid sequences for PKC phosphorylation, it has been postulated that PKC regulation of DAT is mediated by the direct phosphorylation of DAT protein. In this study, we try to discover whether the functional regulation of DAT by PKC is due to direct phosphorylation of DAT. The PKC null mutant hDAT, where all putative PKC phosphorylation sites are eliminated, has been constructed by the replacement of serine/threonine residues with glycines. The mutation itself showed no effect on the functional activities of DAT. The DA uptake activity of PKC null mutant was equivalent to those of wild-type hDAT (80-110% of wild-type). Phorbol ester activation of PKC inhibited DA uptake of wild-type hDAT by 35%, and staurosphorine blocked the effect of phorbol ester on DA uptake. The same phenomena was observed in PKC null mutant DAT, although no significant phosphorylation was observed by PKC activation. Confocal microscopic analysis using EGFP-fused DAT revealed that the activation of PKC by phorbol ester elicited fluorescent DAT to be internalized into the intracellular space both in wild-type and PKC null mutant DAT in a similar way. These results suggest that PKC-mediated regulation of DAT function is achieved in an indirect manner, such as phosphorylation of a mediator protein or activation of a clathrin-mediated pathway.     

Amphetamine regulation of dopamine transport. Combined measurements of transporter currents and transporter imaging support the endocytosis of an active carrier

Dopaminergic neurotransmission is fine-tuned by the rate of removal of dopamine (DA) from the extracellular space via the Na(+)/Cl(-)-dependent DA transporter (DAT). DAT is a target of psychostimulants such as amphetamine (AMPH) and cocaine. Previously, we reported that AMPH redistributes the human DAT away from the cell surface. This process was associated with a reduction in transport capacity. This loss of transport capacity may result either from a modification of the function of DAT that is independent of its cell surface redistribution and/or from a reduction in the number of active transporters at the plasma membrane that results from DAT trafficking. To discriminate between these possibilities, we stably transfected HEK-293 cells with a yellow fluorescent protein (YFP)-tagged human DAT (hDAT cells). In hDAT cells, acute exposure to AMPH induced a time-dependent loss of hDAT activity. By coupling confocal imaging with patch-clamp whole-cell recordings, we have demonstrated for the first time that the loss of AMPH-induced hDAT activity temporally parallels the accumulation of intracellular hDAT. In addition, presteady-state current analysis revealed a cocaine-sensitive, voltage-dependent capacitance current that correlated with the level of transporter membrane expression and in turn served to monitor the AMPH-induced trafficking of hDAT. We found that the decrease in hDAT cell surface expression induced by AMPH was not paralleled by changes in the ability of the single transporter to carry charges. Quasi-stationary noise analysis of the AMPH-induced hDAT currents revealed that the unitary transporter current remained unaltered during the loss of hDAT membrane expression. Taken together, these data strongly suggest that the AMPH-induced reduction of hDAT transport capacity results from the removal of active hDAT from the plasma membrane.     

Pharmacological characterisation of (E)-N-(3-iodoprop-2-enyl)-2beta-carbomethoxy-3beta-(4'-methylphenyl)nortropane (PE2I) binding to the rat neuronal dopamine transporter expressed in COS cells.

The interaction of (E)-N-(3-iodoprop-2-enyl)-2beta-Carbomethoxy-3beta-(4'-methylphenyl) nortropane (PE2I) with the rat neuronal dopamine transporter (DAT) was studied in transfected COS cells by measuring its ability to inhibit DA uptake and by measuring its affinity in radioligand binding experiments. Saturable [3H]DA uptake was measured in COS cells transiently transfected with the cDNA sequence encoding the rat DAT. Pharmacological characterisation of this uptake revealed functional properties with a V(max) value of 45.05+/-2.62 pmol/mg protein per min and a K(m) value of 2.86+/-0.28 microM. The specific [3H]DA uptake was fully inhibited by 1 microM PE2I. Concentration response curves revealed the high potency of PE2I in inhibiting DA uptake (pEC(50) value of 8.70+/-0.33), 25 times higher than that observed for the reference DAT inhibitor, GBR 12935. On crude homogenates from transfected COS cells, PE2I displaced the specific binding of [3H]GBR 12935 with a pK(i) value of 7.73+/-0.13. Accordingly, [125I]PE2I was found to specifically recognise a single binding site population which is almost completely displaced by GBR 12935 and nomifensine. Saturation experiments revealed the high affinity of [125I]PE2I (K(D) value of 3.8+/-0.63 nM) that correlates with the high potency of PE2I in inhibiting the [3H]DA uptake. This contrasts with the results obtained with GBR 12935 for which a discrepancy was found between its high affinity in binding assays (K(D) value of 0.43+/-0.04 nM) and its rather low potency in functional assays (pEC(50) value of 7.30+/-0.05). A relatively high level of [3H]GBR 12935 binding was detected in non transfected COS cells. Such nomifensine resistant binding is attributed to the interaction of GBR 12935 with cytochrome P-450 as it was displaced by cis-(Z)-flupentixol (an inhibitor of cytochrome P-450). Such interaction was not observed using PE2I. Taken together, these data demonstrate that PE2I was a highly potent inhibitor of cloned DAT compared with GBR 12935 and provided a useful tool for further investigations in cells transfected with cDNA encoding the DAT.     

Hydrogen peroxide induces loss of dopamine transporter activity: a calcium-dependent oxidative mechanism

H2O2 dose dependently inhibited dopamine uptake in PC12 cells and in striatal synaptosomes. Treatment with H2O2 resulted in a reversible reduction in Vmax, with no effect on its Km value. This suppressive effect of H2O2 could be relieved by reducing agents (dithiothreitol and cysteine). Furthermore, an oxidizer (dithiodipyridine) also markedly suppressed the dopamine transporter (DAT). Oxidative stress therefore might contribute to the action of H2O2. H2O2 appeared to modify DAT at both extracellular and intracellular sites because cumene-H2O2 (a radical generator mostly restricted to plasma membranes) at high concentrations also slightly suppressed DAT activity and the intracellular overexpression of catalase ameliorated the inhibitory effect of H2O2. Internalization was unlikely to be involved because concanavalin A, which blocked endocytosis, did not prevent the H2O2-evoked inhibition of DAT activity. Interestingly, H2O2 treatment evoked a Ca2+ influx in PC12 cells. Moreover, removal of external calcium by EGTA or reduction in the intracellular calcium level using BAPTA-AM reversed the inhibitory effect of H2O2. Conversely, depletion of intracellular calcium stores using thapsigargin did not affect the reduction in DAT activity by H2O2. Collectively, our results indicate that the DAT, one of the most important proteins controlling the dopaminergic system, is also a redox sensor. In addition, H2O2 might suppress the DAT by a Ca2+-dependent oxidative pathway.     

Modeling of the interaction of Na+ and K+ with the binding of dopamine and [3H]WIN 35,428 to the human dopamine transporter

Although much is known about the effects of Na+, K+, and Cl- on the functional activity of the neuronal dopamine transporter, little information is available on their role in the initial event in dopamine uptake, i.e., the recognition step. This was addressed here by studying the inhibition by dopamine of the binding of [3H]WIN 35,428 [2beta-carbomethoxy-3beta-(4-fluorophenyl)[3H]tropane], a phenyltropane analogue of cocaine, to the cloned human dopamine transporter expressed in HEK-293 cells. The decrease in the affinity of dopamine (or WIN 35,428) binding affinity with increasing [K+] could be fitted to a competitive model involving an inhibitory cation site (1) overlapping with the dopamine (or WIN 35,428) domain. The K+ IC50 for inhibiting dopamine or WIN 35,428 binding increased linearly with [Na+], indicating a K(D,Na+) of 30-44 mM and a K(D,K+) of 13-16 mM for this cation site. A second Na+ site (2), distal from the WIN 35,428 domain but linked by positive allosterism, was indicated by model fitting of the WIN 35,428 binding affinities as a function of [Na+]. No strong evidence for this second site was obtained for dopamine binding in the absence or presence of low (20 mM) Cl- and could not be acquired for high [Cl-] because of the lack of a suitable substitute ion for Na+. The K(D) but not Bmax of [3H]WIN 35,428 binding increased as a function of the [K+]/[Na+] ratio regardless of total [Cl-] or ion tonicity. A similar plot was obtained for the Ki of dopamine binding, with Cl- at > or = 140 mM decreasing the Ki. At 290 mM Cl- and 300 mM Na+ the potency of K+ in inhibiting dopamine binding was enhanced as compared with the absence of Cl- in contrast to the lack of effect of Cl- up to 140 mM (Na up to 150 mM). The results indicate that Cl- at its extracellular level enhances dopamine binding through a mechanism not involving site 1. The observed correspondence between the WIN 35,428 and dopamine domains in their inclusion of the inhibitory cation site explains why many of the previously reported interrelated effects of Na+ and K+ on the binding site of radiolabeled blockers to the dopamine transporter are applicable to dopamine uptake in which dopamine recognition is the first step.     

α-Synuclein stimulates a dopamine transporter-dependent chloride current and modulates the activity of the transporter

Dysregulation of dopamine (DA) homeostasis is implicated in neurodegenerative diseases, drug addiction, and neuropsychiatric disorders. The neuronal plasma membrane dopamine transporter (DAT) is essential for the maintenance of DA homeostasis in the brain. α-Synuclein is a 140-amino acid protein that forms a stable complex with DAT and is linked to the pathogenesis of neurodegenerative disease. To elucidate the potential functional consequences of DAT/α-synuclein interaction, we explored α-synuclein modulation of DAT activity in midbrain dopaminergic neurons obtained from TH::RFP mice, immortalized DA neurons, and a heterologous system expressing DAT. We used dual pipette whole cell patch clamp recording to measure the DAT-mediated current before and after dialysis of recombinant α-synuclein into immortalized DA neurons. Our data suggest that intracellular α-synuclein induces a Na+ independent but Cl--sensitive inward current in DAT-expressing cells. This current is blocked by DAT blocker GBR12935 and is absent when heat-inactivated α-synuclein is dialyzed into these cells. The functional consequence of this interaction on DAT activity was further examined with real-time monitoring of transport function using a fluorescent substrate of DAT, 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+). Overexpression of α-synuclein in DAT-positive immortalized DA neurons and CHO cells expressing DAT decreased the magnitude and rate of DAT-mediated substrate uptake without a decrease in the initial binding of the substrate at the plasma membrane. Taken together our findings are consistent with the interpretation that DAT/α-synuclein interaction at the cell surface results in a DAT-dependent, Na+-insensitive, Cl-sensitive inward current with a decrease in substrate uptake, suggesting that DAT/α-synuclein interaction can modulate dopamine transmission and thus neuronal function.     

1-Methyl-4-phenylpyridinium-induced down-regulation of dopamine transporter function correlates with a reduction in dopamine transporter cell surface expression

The mechanisms whereby 1-methyl-4-phenylpyridinium (MPP(+)) mediates cell death and Parkinsonism are still unclear. We have shown that dopamine transporter (DAT) is required for MPP(+)-mediated cytotoxicity in HEK-293 cells stably transfected with human DAT. Furthermore, MPP(+) produced a concentration- and time-dependent reduction in the uptake of [3H]dopamine. We observed a significant decrease in [3H]WIN 35428 binding in the intact cells with MPP(+). The saturation analysis of the [3H]WIN 35428 binding obtained from total membrane fractions revealed a decrease in the transporter density (B(max)) with an increase in the dissociation equilibrium constant (K(d)) after MPP(+) treatment. Furthermore, biotinylation assays confirmed that MPP(+) reduced both plasma membrane and intracellular DAT immunoreactivity. Taken together, these findings suggest that the reduction in cell surface DAT protein expression in response to MPP(+) may be a contributory factor in the down-regulation of DAT function while enhanced lysosomal degradation of DAT may signal events leading to cellular toxicity.     

Substrates dissociate dopamine transporter oligomers

Substrate-induced endocytic trafficking of dopamine transporter (DAT) has been observed, but little is known about the regulation of DAT oligomerization by substrate. The present study investigates the effect on substrates on DAT oligomerization and explores a potential link with the presence of DAT at the cell surface in human embryonic kidney cells transiently or stably expressing N-terminal tagged DAT constructs. Dopamine (100 μM) or amphetamine (2–10 μM) reduced Myc-DAT coimmunoprecipitated along with Flag-DAT (oligomeric DAT) in tandem with a reduction in surface DAT determined by biotinylation. Dopamine (10–1000 μM) and amphetamine (0.2–200 μM) reduced DAT oligomerization as assessed by cross-linking with copper sulfate phenanthroline or Cu²⁺. Inhibition of endocytosis by 10 μM phenylarsine oxide or 450 mM sucrose counteracted the effect of 10 μM DA or 2 μM amphetamine in reducing DAT cross-linking. In addition to overall similarities between the results with the two cross-linking agents and between the results with the two different endocytosis inhibitors, some differences were noted as well, likely related to the efficiency of the cross-linking process and the sulfhydryl-reactive properties of phenylarsine oxide, respectively. The present results are the first to indicate regulation of oligomerization of an solute carrier family 6 transporter, the DAT, by substrates that act at DAT. In addition, the present study opens up the possibility of an important linkage between oligomerization of DAT and endocytic or other modulatory mechanisms impacting surface DAT.     

Dopamine neuronal transport kinetics and effects of amphetamine

The dopamine (DA) transporter (DAT) regulates DA neurotransmission by recycling DA back into neurons. Drugs that interfere with DAT function, e.g., cocaine and amphetamine, can have profound behavioral effects. The kinetics of DA transport by DAT in isolated synaptosomal or single cell preparations have been previously studied. To investigate how DA transport is regulated in intact tissue and to examine how amphetamine affects the DAT, the kinetics of DA uptake by the DAT were examined in tissue slices of the mouse caudate-putamen with fast-scan cyclic voltammetry. The data demonstrate that inward DA transport is saturable and sodium-dependent. Elevated levels of cytoplasmic DA resulting from disruption of vesicular storage by incubation with 10 microM Ro 4-1284 did not generate DA efflux or decrease its uptake rate. However, incubation with 10 microM amphetamine reduced the net DA uptake rate and increased extracellular DA levels due to DA efflux through the DAT. In addition, a new, elevated steady-state level of extracellular DA was established after electrically stimulated DA release in the presence of amphetamine, norepinephrine, and exogenous DA. These results from intact tissue are consistent with a kinetic model of the DAT established in more purified preparations in which amphetamine and other transported substances make the inwardly facing DAT available for outward transport of intracellular DA.