Rat mesencephalic neuronal cells cultured for different periods as a model of dopamine transporter ontogenesis

Ventral mesencephalic neurons contained only low-affinity and sodium-independent binding sites of [³H]WIN 35,428 (marker of dopamine transporter) during the first 10d in primary cultures. These sites were present in cytosol, and they are not very probably related to dopamine transporter. After 12 d in culture, membrane-bound, high-affinity, and sodium-dependent [³H]WIN 35,428 binding sites were detected. In membranes prepared from cells 14 d in culture, cocaine displaced [³H]WIN 35,428 binding with similar potency to that in striatal membranes of adult rat brain. The high-affinity [³H]WIN 35,428 binding sites in mesencephalic neuronal cell cultures are very probably related to dopamine transporter. The development of high-affinity [³H]WIN 35,428 binding sites in neurons cultured for different time periods could be a useful model of dopamine transporter ontogenesis.     

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.     

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.     

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.     

Membrane Cholesterol Modulates the Outward Facing Conformation of the Dopamine Transporter and Alters Cocaine Binding

Clearance of synaptically released dopamine is regulated by the plasmalemmal dopamine transporter (DAT), an integral membrane protein that resides within a complex lipid milieu. Here we demonstrate that cholesterol, a major component of the lipid bilayer, can modulate the conformation of DAT and alter cocaine binding to DAT. In striatal synaptosomes and transfected cells, DAT was in cholesterol-rich membrane fractions after mild detergent extraction. After increasing the membrane cholesterol content by treatment of water-soluble cholesterol (cholesterol mixed with methyl-β-cyclodextrin), we observed an increase in DAT binding B_max values for cocaine analogs [³H]WIN35428 and [¹²⁵I]RTI-55, but similar levels of DAT proteins on the cell surface were shown by surface biotinylation assays. Membrane cholesterol addition also markedly enhanced the accessibility of cysteine sulfhydryl moieties in DAT as probed by a membrane-impermeable maleimide-biotin conjugate. We identified cysteine 306, a juxtamembrane residue on transmembrane domain 6 (TM6) of DAT, as the intrinsic residue exhibiting enhanced reactivity. Similar effects on DAT cysteine accessibility and radioligand binding were observed with addition of zinc, a reagent known to promote the outward facing conformation of DAT. Using substituted cysteine mutants on various positions likely to be extracellular, we identified additional residues located on TM1, TM6, TM7, and TM12 of DAT that are sensitive to alterations in the membrane cholesterol content. Our findings in transfected cells and native tissues support the hypothesis that DAT adopts an outward facing conformation in a cholesterol-rich membrane environment, suggesting a novel modulatory role of the surrounding membrane lipid milieu on DAT function.     

Ethanol potentiates the function of the human dopamine transporter expressed in Xenopus oocytes

Ethanol alters a variety of properties of brain dopaminergic neurons including firing rate, synthesis, release, and metabolism. Recent studies suggest that ethanol's action on central dopamine systems may also involve modulation of dopamine transporter (DAT) activity. The human DAT was expressed in Xenopus oocytes to examine directly the effects of ethanol on transporter function. [3H]Dopamine (100 nM) accumulation into DAT-expressing oocytes increased significantly in response to ethanol (10 min; 10-100 mM). In two-electrode voltage-clamp experiments, DAT-mediated currents were also enhanced significantly by ethanol (10-100 mM). The magnitude of the ethanol-induced potentiation of DAT function depended on ethanol exposure time and substrate concentration. Cell surface DAT binding ([3H]WIN 35,428; 4 nM) also increased as a function of ethanol exposure time. Thus, the increase in dopamine uptake was associated with a parallel increase in the number of DAT molecules expressed at the cell surface. These experiments demonstrate that DAT-mediated substrate translocation and substrate-associated ionic conductances are sensitive to intoxicating concentrations of ethanol and suggest that DAT may represent an important site of action for ethanol's effects on central dopaminergic transmission. A potential mechanism by which ethanol acts to enhance DAT function may involve regulation of DAT expression on the cell surface.     

Environmental enrichment decreases cell surface expression of the dopamine transporter in rat medial prefrontal cortex

Rats raised in an enriched environmental condition (EC) exhibit a decreased (35%) maximal velocity (V(max)) of [3H]dopamine (DA) uptake in medial prefrontal cortex (mPFC) compared with rats raised in an impoverished condition (IC); however, no differences between EC and IC groups in V(max) for [3H]DA uptake were found in nucleus accumbens and striatum. Using biotinylation and immunoblotting techniques, the present study examined whether the brain region-specific decrease in DA transporter (DAT) function is the result of a reduction in DAT cell surface expression. In mPFC, nucleus accumbens and striatum, total DAT immunoreactivity was not different between EC and IC groups. Whereas no differences in cell surface expression of DAT were found in nucleus accumbens and striatum, DAT immunoreactivity in the biotinylated cell surface fraction of mPFC was decreased (39%) in EC compared with IC rats, consistent with the magnitude of the previously observed decrease in V(max) for [3H]DA uptake in mPFC in EC rats. These results suggest that the decrease in DAT cell surface expression in the mPFC may be responsible for decreased DAT function in the mPFC of EC compared with IC rats, and that there is plasticity in the regulatory mechanisms mediating DAT trafficking and function.     

Expression of dopamine receptors and transporter in neuroendocrine gastrointestinal tumor cells

C-11- or F-18-DOPA positron emission tomography (DOPA PET) is a new sensitive imaging technique for small neuroendocrine gastrointestinal tumors which evaluates the decarboxylase activity. To further characterize the dopaminergic system in neuroendocrine gastrointestinal tumor cells, we investigated the expression of both dopamine receptors and the transmembrane dopamine transporter (DAT) in the human neuroendocrine pancreatic cell line BON and in the neuroendocrine gut cell line STC-1. Both BON and STC-1 cells expressed mRNA of the dopamine receptors D2-D5 and DAT. mRNA of the dopamine receptor D1 was detected in BON cells only. Both in BON and STC-1 cells, expression of D2 and D5 receptors and DAT was also demonstrated immunocytochemically. For functional receptor characterization intracellular cAMP levels ([cAMP]i) were determined. Whereas in STC-1 cells dopamine and the D1-like (D1/D5) receptor agonist SKF 38393 increased [cAMP]i, [cAMP]i was decreased by dopamine or the D2-like (D2-D4) receptor agonist quinpirole in BON cells. Functional DAT activity was, however, not detected in either cell line. The presence of both dopamine receptors and of the DAT suggests an autocrine and/or paracrine function of dopamine in neuroendocrine gastrointestinal tumor cells. Yet neither the transmembrane dopamine transporter nor dopamine receptors are likely to contribute to positive DOPA PET imaging of neuroendocrine gastrointestinal tumors. However, these molecules may be of diagnostic importance when applying other dopaminergic system tracers.     

3-Aza-6,8-dioxabicyclo[3.2.1]octanes as new enantiopure heteroatom-rich tropane-like ligands of human dopamine transporter

CNS diseases such as Parkinson, schizophrenia, and attention deficit hyperactivity disorder (ADHD) are characterized by a significant alteration of dopamine transporter (DAT) density. Thus, the development of compounds that are able to selectively interact with DAT is of great interest. Herein we describe the design and synthesis of a new set of 3-aza-6,8-dioxabicyclo[3.2.1]octanes having a tropane-like structure with additional heteroatoms at positions 3 and 6. The compounds were evaluated for their in vitro receptor binding properties toward human dopamine (hDAT) and serotonin (hSERT) transporters using [3H]WIN35,428 and [3H]citalopram as specific radioligands, respectively. Biological assays revealed that some compounds having the N-3 atom substituted with aryl groups possess significant affinity and selectivity for monoamine transporters, and in particular, compound 5d displayed an IC50 of 21 nM toward DAT, and a good selectivity toward SERT (IC50=1042 nM). These results suggest that 3-aryl-3-aza-6,8-dioxabicyclo[3.2.1]octanes may represent a new class of DAT ligands.     

21-Aminosteroids Interact with the Dopamine Transporter to Protect Against 1-Methyl-4-Phenylpyridinium-Induced Neurotoxicity

U-78518F, a 21-aminosteroid from the novel family of lipid peroxidation inhibitors (lazaroids), increased survival of dopamine (DA) neurons in mesencephalic cell cultures incubated with the neurotoxin 1-methyl-4-phenylpyridinium (MPP+). Protection against DA neuron death occurred with increasing concentrations of U-78518F up to 30 microM. Non-specific toxicity produced with higher concentrations of MPP+ was not affected by the lazaroid. U-78518F inhibited cellular uptake of [3H]MPP+ and [3H]DA, but not that of gamma-[3H]aminobutyric acid. In human striatal membrane preparations, U-78518F competed with [3H]mazindol for binding to the DA transporter, with a calculated Ki value of 10 microM. Two of four lazaroids tested inhibited [3H]DA uptake in the cell culture system. The protective effects of 21-aminosteroids in MPP(+)-induced neurotoxicity are, in part, a function of the interaction of these agents with the DA transporter.     

The uptake inhibitors cocaine and benztropine differentially alter the conformation of the human dopamine transporter

The binding affinity of the cocaine analog [(3)H]2 beta-carbomethoxy-3beta-(4-fluorophenyl) tropane (WIN) for the dopamine transporter (DAT) is increased by the reaction of Cys-90, at the extracellular end of the first transmembrane segment, with methanethiosulfonate (MTS) reagents. Cocaine enhances the reaction of Cys-90 with the sulfhydryl reagents, thereby augmenting the increase in binding. In contrast, cocaine decreases the reaction of Cys-135 and Cys-342, endogenous cysteines in cytoplasmic loops, with MTS reagents. Because this reaction inhibits [(3)H]WIN binding, cocaine protects against the loss of binding caused by reaction of these cysteines. In the present work, we compare the abilities of DAT inhibitors and substrates to affect the reaction of Cys-90, Cys-135, and Cys-342 with MTS ethyltrimethylammonium (MTSET). The results indicate that the different abilities of compounds to protect against the MTSET-induced inhibition of binding are attributable to differences in their abilities to attenuate the inhibitory effects of modification of Cys-135 and Cys-342 as well as to enhance the reaction with Cys-90 and the resulting potentiation of binding. The inhibitor benztropine was unique in its inability to protect Cys-135. Moreover, whereas cocaine, WIN, mazindol, and dopamine enhanced the reaction of Cys-90 with MTSET, benztropine had no effect on this reaction. These two features combine to give benztropine its weak potency in protecting ligand binding to wild-type DAT from MTSET. These results indicate that different inhibitors of DAT, such as cocaine and benztropine, produce different conformational changes in the transporter. There are differences in the psychomotor stimulant-like effects of these compounds, and it is possible that the different behavioral effects of these DAT inhibitors stem from their different molecular actions on 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.     

Dopamine transporter-mediated cytotoxicity of beta-carbolinium derivatives related to Parkinson's disease: relationship to transporter-dependent uptake

Endogenous or exogenous beta-carboline (betaC) derivatives structurally related to the selective dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite 1-methyl-4-phenylpyridinium (MPP(+)) may contribute to dopaminergic neurodegeneration in Parkinson's disease (PD). We addressed the importance of the dopamine transporter (DAT) for selective dopaminergic toxicity by testing the differential cytotoxicity and cellular uptake of 12 betaCs in human embryonic kidney HEK-293 cells ectopically expressing the DAT gene. Cell death was measured using [4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and trypan blue exclusion assays, and uptake by a fluorescence-based uptake assay. All betaCs and MPP(+) showed general cytotoxicity in parental HEK-293 cells after 72 h with half-maximal toxic concentrations (TC(50) values) in the upper micromolar range. Besides MPP(+), only 2[N]-methylated compounds showed enhanced cytotoxicity in DAT expressing HEK-293 cells with 1.3- to 4.5-fold reduction of TC(50) values compared with parental cell line. The rank order of selectivity was: MPP(+) > 2[N],9[N]-dimethyl-harminium > 2[N]-methyl-harminium > 2[N],9[N]-dimethyl-harmanium = 2[N]-methyl-norharmanium > 2[N]-methyl-harmanium > 2[N],9[N]-dimethyl-norharminium. Consistently, only 2[N]-methylated betaCs were transported into the cell through the DAT with up to five times greater K(m) and 12-220 times smaller V(max) values compared with dopamine and MPP(+). There was a weak relation of DAT-mediated selectivity with the affinity of betaCs at the DAT (K(m)), but not with V(max). Our data suggest that DAT-mediated cellular uptake of 2[N]-methylated betaCs represents a potential mechanism for selective toxicity towards dopaminergic neurons and may be relevant for the pathogenesis of Parkinson's disease.     

Pharmacophore-based discovery of substituted pyridines as novel dopamine transporter inhibitors

Abnormal dopamine signaling in brain has been implicated in several conditions such as cocaine abuse, Parkinson's disease and depression. Potent and selective dopamine transporter inhibitors may be useful as pharmacological tools and therapeutic agents. Simple substituted pyridines were discovered as novel dopamine transporter (DAT) inhibitors through pharmacophore-based 3D-database search. The most potent compound 18 has a K(i) value of 79 nM in inhibition of WIN35,248 binding to dopamine transporter and 255 nM in inhibition of dopamine reuptake, respectively, as potent as cocaine. Preliminary structure-activity relationship studies show that the geometry and the nature of the substituents on the pyridine ring determine the inhibitory activity and selectivity toward the three monoamine transporters. The substituted pyridines described herein represent a class of novel DAT inhibitors with simple chemical structures and their discovery provides additional insights into the binding site of DAT.     

Interaction of Na+, K+, and Cl- with the binding of amphetamine, octopamine, and tyramine to the human dopamine transporter

Little information is available on the role of Na+, K+, and Cl- in the initial event of uptake of substrates by the dopamine transporter, i.e., the recognition step. In this study, substrate recognition was studied via the inhibition of binding of [3H]WIN 35,428 [2beta-carbomethoxy-3beta-(4-fluorophenyl)[3H]tropane], a cocaine analogue, to the human dopamine transporter in human embryonic kidney 293 cells. D-Amphetamine was the most potent inhibitor, followed by p-tyramine and, finally, dl-octopamine; respective affinities at 150 mM Na+ and 140 mM Cl- were 5.5, 26, and 220 microM. For each substrate, the decrease in the affinity with increasing [K+] could be fitted to a competitive model involving the same inhibitory cation site (site 1) overlapping with the substrate domain as reported by us previously for dopamine. K+ binds to this site with an apparent affinity, averaged across substrates, of 9, 24, 66, 99, and 134 mM at 2, 10, 60, 150, and 300 mM Na+, respectively. In general, increasing [Na+] attenuated the inhibitory effect of K+ in a manner that deviated from linearity, which could be modeled by a distal site for Na+, linked to site 1 by negative allosterism. The presence of Cl- did not affect the binding of K+ to site 1. Models assuming low binding of substrate in the absence of Na+ did not provide fits as good as models in which substrate binds in the absence of Na+ with appreciable affinity. The binding of dl-octopamine and p-tyramine was strongly inhibited by Na+, and stimulated by Cl- only at high [Na+] (300 mM), consonant with a stimulatory action of Cl- occurring through Na+ disinhibition.     

Relevance of different striatal markers in assessment of the MPP+-induced dopaminergic nigrostriatal injury in rat

Many striatal dopaminergic markers are available for estimating the degree of the nigrostriatal lesion by MPTP/MPP+, but the changes of these markers are not perfectly matched. In this study we investigated different striatal markers and determined which ones closely reflected the nigrostriatal alteration. The in vivo binding of (E)-N-(3-iodoprop-2-enyl)-2-beta-carbomethoxy-3beta-(4'-methylphenyl)nortropane (PE2I), a selective and potent inhibitor of the neuronal dopamine transporter (DAT) was considered as the reference index of injury of striatal dopaminergic nerve-endings. Rats received a 10-microg MPP+ injection in the right substantia nigra and were killed at 7 days after lesion. The results were as follows: (i) a decrease (66%) of the biodistribution of [125I]PE2I; (ii) a great reduction of the DAT expression measured by the binding of [125I]PE2I in striatal membranes (Bmax decreased by 54%) and in cerebral slices (88%); (iii) an 80% inhibition of the vesicular monoamine transporter expression revealed by the binding of [3H]dihydrotetrabenazine in cerebral slices; (iv) a robust decrease in the quantity of DA and its metabolites (about 50-60%); (v) a slight modification of the DAT activity with a decreased number of functional sites (Vmax decreased by 12%, p < 0.05) without change of the affinity in striatal synaptosomes. Among these markers the binding of [125I]PE2I in membrane homogenates and the content of DA, and its metabolites, in striatum could be the most relevant in vitro indexes of the degenerative state of the nigrostriatal pathway after MPP+ lesion.     

[3H]WIN 35,065–2: A Ligand for Cocaine Receptors in Striatum

[3H]WIN 35,065-2 binding to striatal membranes was characterized, primarily by centrifugation assay. Like [3H]cocaine, [3H]WIN 35,065-2 binds to both high- and low-affinity sites. [3H]WIN 35,065-2, however, exhibits consistently higher affinities than [3H]cocaine. Saturation experiments indicate a low-affinity binding site with an apparent KD of approximately 160 nM and a Bmax of 135 fmol/mg of tissue. A high-affinity site has also been identified with an apparent KD of 5.6 nM and a Bmax of 5.2 fmol/mg of tissue. The specific-to-nonspecific binding ratios with [3H]WIN 35,065-2 were higher than with [3H]cocaine in both centrifugation and filtration assays. Pharmacological characterization suggests that [3H]WIN 35,065-2 binds to the dopamine transporter. Mazindol, GBR 12909, nomifensine, and (-)-cocaine are potent inhibitors of [3H]WIN 35,065-2 binding. In contrast, the norepinephrine transporter ligand desipramine is a weak inhibitor, and the serotonin transporter ligand citalopram does not inhibit binding. The effect of sodium on binding was examined under conditions in which (a) the low-affinity site was primarily (87%) occupied and (b) approximately 50% of both sites were occupied. The results indicate that both sites are sodium dependent. Injection of 6-hydroxydopamine into the striatum results in a significant loss of both high- and low-affinity sites, a finding suggesting that both sites are on dopaminergic nerve terminals. Taken together, these data are consistent with the presence of multiple cocaine binding sites associated with the dopamine transporter.     

Regulation of dopamine transporter function and cell surface expression by D3 dopamine receptors

D(3) dopamine receptors are expressed by dopamine neurons and are implicated in the modulation of presynaptic dopamine neurotransmission. The mechanisms underlying this modulation remain ill defined. The dopamine transporter, which terminates dopamine transmission via reuptake of released neurotransmitter, is regulated by receptor- and second messenger-linked signaling pathways. Whether D3 receptors regulate dopamine transporter function is unknown. We addressed this issue using a fluorescent imaging technique that permits real time quantification of dopamine transporter function in living single cells. Accumulation of the fluorescent dopamine transporter substrate trans-4-[4-(dimethylamino)styryl]-1-methylpyridinium (ASP(+)) in human embryonic kidney cells expressing human dopamine transporter was saturable and temperature-dependent. In cells co-expressing dopamine transporter and D3 receptors, the D2/D3 agonist quinpirole produced a rapid, concentration-dependent, and pertussis toxin-sensitive increase of ASP(+) uptake. Similar agonist effects were observed in Neuro2A cells and replicated in human embryonic kidney cells using a radioligand uptake assay in which binding to and activation of D3 receptors by [(3)H]dopamine was prevented. D3 receptor stimulation activated phosphoinositide 3-kinase and MAPK. Inhibition of either kinase prevented the quinpirole-induced increase in uptake. D3 receptor activation differentially affected dopamine transporter function and subcellular distribution depending on the duration of agonist exposure. Biotinylation experiments revealed that the rapid increase of uptake was associated with increased cell surface and decreased intracellular expression and increased dopamine transporter exocytosis. In contrast, prolonged agonist exposure reduced uptake and transporter cell surface expression. These results demonstrate that D3 receptors regulate dopamine transporter function and identify a novel mechanism by which D3 receptors regulate extracellular dopamine concentrations.     

Affinities of methylphenidate derivatives for dopamine,norepinephrine and serotonin transporters

We have synthesized several derivatives of dl-threo-methylphenidate (Ritalin) bearing substituents on the phenyl ring. IC₅₀ values for binding of these compounds to rat brain monoamine transporters were assessed using [³H]WIN 35,428 (striatal membranes, dopamine transporters, DAT), [³H]nisoxetine (frontal cortex membranes, norepinephrine transporters, NET) and [³H]paroxetine (brain stem membranes, 5HT transporters, 5HTT). Affinities (1/Ki) decreased in the order: DAT > NET ⪢ 5HTT. Substitution at the para position of dl-threo-methylphenidate generally led to retained or increased affinity for the dopamine transporter (bromo > iodo > methoxy > hydroxy). Substitution at the meta position also increased affinity for the DAT (m-bromo > methylphenidate; m-iodo-p-hydroxy > p-hydroxy). Substitution at the ortho position with bromine considerably decreased affinity. Similar IC₅₀ values for binding of o-bromomethylphenidate to the dopamine transporter were measured at 0, 22 and 37 degrees. N-Methylation of the piperidine ring of methylphenidate also considerably reduced affinity. The dl-erythro isomer of obromomethylphenidate did not bind to the DAT (IC₅₀ > 50,000 nM). Affinities at the dopamine and norepinephrine transporters for substituted methylphenidate derivatives were well correlated (r² = 0.90). Abilities of several methylphenidate derivatives to inhibit [³H]dopamine uptake in striatal synaptosomes corresponded well with inhibition of [³H]WIN 35, 428 binding. None of the compounds examined exhibited significant affinity to dopamine D1 or D2 receptors (IC₅₀ > 500 or 5,000 nM, respectively), as assessed by inhibition of binding of [³H]SCH 23390 or [¹²³I]epidepride, respectively, to striatal membranes.     

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.