Dopamine D2 Receptor-Deficient Mice Exhibit Decreased Dopamine Transporter Function but No Changes in Dopamine Release in Dorsal Striatum

Abstract : Presynaptic D₂ dopamine (DA) autoreceptors, which are well known to modulate DA release, have recently been shown to regulate DA transporter (DAT) activity. To examine the effects of D₂ DA receptor deficiency on DA release and DAT activity in dorsal striatum, we used mice genetically engineered to have two (D₂^+/+), one (D₂^+/-), or no (D₂^-/-) functional copies of the gene coding for the D₂ DA receptor. In vivo microdialysis studies demonstrated that basal and K⁺-evoked extracellular DA concentrations were similar in all three genotypes. However, using in vivo electrochemistry, the D₂^-/- mice were found to have decreased DAT function, i.e., clearance of locally applied DA was decreased by 50% relative to that in D₂^+/+ mice. In D₂^+/+ mice, but not D₂^-/- mice, local application of the D₂-like receptor antagonist raclopride increased DA signal amplitude, indicating decreased DA clearance. Binding assays with the cocaine analogue [³H]WIN 35,428 showed no genotypic differences in either density or affinity of DAT binding sites in striatum or substantia nigra, indicating that the differences seen in DAT activity were not a result of decreased DAT expression. These results further strengthen the idea that the D₂ DA receptor subtype modulates activity of the striatal DAT.     

Carrier-Mediated Release, Transport Rates, and Charge Transfer Induced by Amphetamine, Tyramine, and Dopamine in Mammalian Cells Transfected with the Human Dopamine Transporter

Abstract: Amphetamine and related substances induce dopamine release. According to a traditional explanation, this dopamine release occurs in exchange for amphetamine by means of the dopamine transporter (DAT). We tested this hypothesis in human embryonic kidney 293 cells stably transfected with the human DAT by measuring the uptake of dopamine, tyramine, and d - and l -amphetamine as well as substrate-induced release of preloaded N -methyl-4-[³H]phenylpyridinium ([³H]MPP⁺). The uptake of substrates was sodium-dependent and was inhibited by ouabain and cocaine, which also prevented substrate-induced release of MPP⁺. Patch-clamp recordings revealed that all four substrates elicited voltage-dependent inward currents (on top of constitutive leak currents) that were prevented by cocaine. Whereas individual substrates had similar affinities in release, uptake, and patch-clamp experiments, maximal effects displayed remarkable differences. Hence, maximal effects in release and current induction were ∼25% higher for d -amphetamine as compared with the other substrates. By contrast, dopamine was the most efficacious substrate in uptake experiments, with its maximal initial uptake rate exceeding those of amphetamine and tyramine by factors of 20 and 4, respectively. Our experiments indicate a poor correlation between substrate-induced release and the transport of substrates, whereas the ability of substrates to induce currents correlates well with their releasing action.     

Glycosylation of a Vesicular Monoamine Transporter: A Mutation in a Conserved Proline Residue Affects the Activity, Glycosylation, and Localization of the Transporter

Abstract: The role of N -glycosylation in the expression, ligand recognition, activity, and intracellular localization of a rat vesicular monoamine transporter (rVMAT1) was investigated. The glycosylation inhibitor tunicamycin induced a dose-dependent decrease in the rVMAT1-mediated uptake of [³H]serotonin. Part of this effect was due to a general toxic effect of the drug. Therefore, to assess the contribution of each of the glycosylation sites to the transporter activity, the three putative N -glycosylation sites were mutated individually, in combination, and in toto ("triple" mutant). Mutation of each glycosylation site caused a minor and additive decrease in activity, up to the triple mutant, which retained at least 50% of the wild-type activity. No significant differences were found either in the time dependence of uptake or the apparent affinity for ligands of the triple mutant compared with the wild-type protein. It is interesting that in contrast to plasma-membrane neurotransmitter transporters, the unglycosylated form of rVMAT1 distributed in the cell as the wild-type protein. Pro⁴³ is a highly conserved residue located at the beginning of the large loop in which all the potential glycosylation sites are found. A Pro⁴³Leu mutant transporter was inactive. It is remarkable that despite the presence of glycosylation sites, the mutant transporter was not glycosylated. Moreover, the distribution pattern of the Pro⁴³Leu mutant clearly differed from that of the wild type. In contrast, a Pro⁴³Gly mutant displayed an activity practically identical to the wild-type protein. As this replacement generated a protein with wild-type characteristics, we suggest that the conformation conferred by the amino acid at this position is essential for activity.     

Direct evidence that two cysteines in the dopamine transporter form a disulfide bond

We have generated a fully functional dopamine transporter (DAT) mutant (dmDATx7) with all cysteines removed except the two cysteines in extracellular loop 2 (EL2). Random mutagenesis at either or both EL2 cysteines did not produce any functional transporter mutants, suggesting that the two cysteines cannot be replaced by any other amino acids. The cysteine-specific reagent MTSEA-biotin labeled dmDATx7 only after a DTT treatment which reduces disulfide bond. Since there are no other cysteines in dmDATx7, the MTSEA-biotin labeling must be on the EL2 cysteines made available by the DTT treatment. This result provides the first direct evidence that the EL2 cysteines form a disulfide bond. Interestingly, the DTT treatment had little effect on transport activity suggesting that the disulfide bond is not necessary for the uptake function of DAT. Our results and previous results are consistent with the notion that the disulfide bond between EL2 cysteines is required for DAT biosynthesis and/or its delivery to the cell surface.     

Cations Affect [3H]Mazindol and [3H]WIN 35,428 Binding to the Human Dopamine Transporter in a Similar Fashion

Abstract: The present study addresses the possibility that there are different cocaine-related and mazindol-related binding domains on the dopamine transporter (DAT) that show differential sensitivity to cations. The effects of Zn²⁺, Mg²⁺, Hg²⁺, Li⁺, K⁺, and Na⁺ were assessed on the binding of [³H]mazindol and [³H]WIN 35,428 to the human (h) DAT expressed in C6 glioma cells under identical conditions for intact cell and membrane assays. The latter were performed at both 0 and 21°C. Zn²⁺ (30–100 µ M ) stimulated binding of both radioligands to membranes, with a relatively smaller effect for [³H]mazindol; Mg²⁺ (0.1–100 µ M ) had no effect; Hg²⁺ at ∼3 µ M stimulated binding to membranes, with a relatively smaller effect for [³H]mazindol than [³H]WIN 35,428 at 0°C, and at 30–100 µ M inhibited both intact cell and membrane binding; Li⁺ and K⁺ substitution (30–100 m M ) inhibited binding to membranes more severely than to intact cells; and Na⁺ substitution was strongly stimulatory. With only a few exceptions, the patterns of ion effects were remarkably similar for both radioligands at both 0 and 21°C, suggesting the involvement of common binding domains on the hDAT impacted similarly by cations. Therefore, if there are different binding domains for WIN 35,428 and mazindol, these are not affected differentially by the cations studied in the present experiments, except for the stimulatory effect of Zn²⁺ at 0 and 21°C and Hg²⁺ at 0°C.     

Modulation of Human Glutamate Transporter Activity by Phorbol Ester

Abstract: Termination of synaptic glutamate transmission depends on rapid removal of glutamate by neuronal and glial high-affinity transporters. Molecular biological and pharmacological studies have demonstrated that at least five subtypes of Na⁺-dependent mammalian glutamate transporters exist. Our study demonstrates that Y-79 human retinoblastoma cells express a single Na⁺-dependent glutamate uptake system with a K _m of 1.7 ± 0.42 µ M that is inhibited by dihydrokainate and dl - threo -β-hydroxyaspartate (IC₅₀ = 0.29 ± 0.17 µ M and 2.0 ± 0.43 µ M , respectively). The protein kinase C activator phorbol 12-myristate 13-acetate caused a concentration-dependent inhibition of glutamate uptake (IC₅₀ = 0.56 ± 0.05 n M ), but did not affect Na⁺-dependent glycine uptake significantly. This inhibition of glutamate uptake resulted from a fivefold decrease in the transporter's affinity for glutamate, without significantly altering the V _max. 4α-Phorbol 12,13-didecanoate, a phorbol ester that does not activate protein kinase C, did not alter glutamate uptake significantly. The phorbol 12-myristate 13-acetate-induced inhibition of glutamate uptake was reversed by preincubation with staurosporine. The biophysical and pharmacological profile of the human glutamate transporter expressed by the Y-79 cell line indicates that it belongs to the dihydrokainate-sensitive EAAT2/GLT-1 subtype. This conclusion was confirmed by western blot analysis. Protein kinase C modulation of glutamate transporter activity may represent a mechanism to modulate extracellular glutamate and shape postsynaptic responses.     

Metabolism of Catecholamines by Catechol-O-Methyltransferase in Cells Expressing Recombinant Catecholamine Transporters

Abstract: To determine if catechol- O -methyltransferase (COMT) metabolizes catecholamines within cell lines used for heterologous expression of plasmalemmal transporters and alters the measured characteristics of ³H-substrate transport, the uptake of monoamine transporter substrates was assessed in three cell lines (C6 glioma, L-M fibroblast, and HEK293 cells) that had been transfected with the recombinant human transporters. Uptake and cellular retention of ³H-catecholamines was increased by up to fourfold by two COMT inhibitors, tropolone and Ro 41-0960, with potencies similar to those for inhibition of COMT activity, whereas the uptake of two transporter substrates that are not substrates for COMT, [³H]serotonin and [³H]MPP⁺, was unaffected. Direct measurement of monoamine substrates by HPLC confirmed that tropolone (1 m M ) increased the retention of the catecholamines dopamine and norepinephrine, but not the retention of serotonin in HEK293 cells. Saturation analysis of the uptake of [³H]dopamine by C6 cells expressing the dopamine transporter demonstrated that tropolone (1 m M ) decreased the apparent K _m of transport from 0.61 µ M to 0.34 µ M without significantly altering the maximal velocity of transport. These data suggest that endogenous COMT activity in mammalian cells may alter neurotransmitter deposition and thus the apparent kinetic characteristics of transport.     

Regulation of Extracellular Dopamine by the Norepinephrine Transporter

Abstract: There is growing evidence of an interaction between dopamine and norepinephrine. To test the hypothesis that norepinephrine terminals are involved in the uptake and removal of dopamine from the extracellular space, the norepinephrine uptake blocker desmethylimipramine (DMI) was infused locally while the extracellular concentrations of dopamine were simultaneously monitored. DMI increased the extracellular concentrations of dopamine in the medial prefrontal cortex and nucleus accumbens shell but had no effect in the striatum. The combined systemic administration of haloperidol and the local infusion of DMI produced an augmented increase in extracellular dopamine in the cortex compared with the increase produced by either drug alone. This synergistic increase in dopamine overflow is likely due to the combination of impulse-mediated dopamine release produced by haloperidol and blockade of the norepinephrine transporter. No such synergistic effects were observed in the nucleus accumbens and striatum. Local perfusion of the α₂-antagonist idazoxan also increased the extracellular concentrations of dopamine in the cortex. Although the stimulation of extracellular dopamine by idazoxan and DMI could be due to the increased extracellular concentrations of norepinephrine produced by these drugs, an increase in dopamine also was observed in lesioned rats that were depleted of norepinephrine and challenged with haloperidol. This contrasted with the lack of an effect of haloperidol on cortical dopamine in unlesioned controls. These results suggest that norepinephrine terminals regulate extracellular dopamine concentrations in the medial prefrontal cortex and to a lesser extent in the nucleus accumbens shell through the uptake of dopamine by the norepinephrine transporter.     

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.     

Insertion of Tetracysteine Motifs into Dopamine Transporter Extracellular Domains

The neuronal dopamine transporter (DAT) is a major determinant of extracellular dopamine (DA) levels and is the primary target for a variety of addictive and therapeutic psychoactive drugs. DAT is acutely regulated by protein kinase C (PKC) activation and amphetamine exposure, both of which modulate DAT surface expression by endocytic trafficking. In order to use live imaging approaches to study DAT endocytosis, methods are needed to exclusively label the DAT surface pool. The use of membrane impermeant, sulfonated biarsenic dyes holds potential as one such approach, and requires introduction of an extracellular tetracysteine motif (tetraCys; CCPGCC) to facilitate dye binding. In the current study, we took advantage of intrinsic proline-glycine (Pro-Gly) dipeptides encoded in predicted DAT extracellular domains to introduce tetraCys motifs into DAT extracellular loops 2, 3, and 4. [³H]DA uptake studies, surface biotinylation and fluorescence microscopy in PC12 cells indicate that tetraCys insertion into the DAT second extracellular loop results in a functional transporter that maintains PKC-mediated downregulation. Introduction of tetraCys into extracellular loops 3 and 4 yielded DATs with severely compromised function that failed to mature and traffic to the cell surface. This is the first demonstration of successful introduction of a tetracysteine motif into a DAT extracellular domain, and may hold promise for use of biarsenic dyes in live DAT imaging studies.     

D2 Receptors May Modulate the Function of the Striatal Transporter for Dopamine: Kinetic Evidence from Studies In Vitro and In Vivo

Abstract: Recently it was hypothesized by others that the D₂dopamine receptor can regulate the uptake of dopamine. However, the evidence in support of this hypothesis, although compelling, was not based on observations related to direct measures of the kinetic activity of the transporter itself. Here kinetic evidence in support of this hypothesis is shown. The apparent time-resolved initial velocity of the transport of 1.0 μ M dopamine into striatal suspensions, measured using rotating disk electrode voltammetry, was found to increase in the presence of the D₂ receptor agonist, quinpirole, at 100 μ M . This effect was reversed by sulpiride. In separate studies it was shown that acute and chronic treatments with haloperidol at 0.5 mg/kg, i.p., reduced the reuptake transport of dopamine in vivo following intrastriatal stimulation of its release by K⁺. Thus, it appears that D₂ receptors may influence the functioning of the striatal transporter for dopamine. These results are consistent with a model in which presynaptically released dopamine may feed back onto the function of its transporter to increase the velocity of the clearance of synaptic dopamine following an action potential, suggesting the existence of a mechanism, in addition to release and synthesis modulation, for fine-tuning dopaminergic chemical signaling.     

Inhibition of the High-Affinity Brain Glutamate Transporter GLAST-1 via Direct Phosphorylation

Abstract: Neurotransmission at excitatory glutamatergic synapses is terminated by the reuptake of the neurotransmitter by high-affinity transporters, which keep the extracellular glutamate concentration below excitotoxic levels. The amino acid sequence of the recently isolated and cloned brain-specific glutamate/aspartate transporter (GLAST-1) of the rat reveals three consensus sequences of putative phosphorylation sites for protein kinase C (PKC). The PKC activator phorbol 12-myristate 13-acetate (PMA) decreased glutamate transport activity in Xenopus oocytes and human embryonic kidney cells (HEK293) expressing the cloned GLAST-1 cDNA, within 20 min, to 25% of the initial transport activity. This down-regulation was blocked by the PKC inhibitor staurosporine. GLAST-1 transport activity remains unimpaired by phorbol 12-monomyristate. Removal of all putative PKC sites of wild-type GLAST-1 by site-directed mutagenesis did not abolish inhibition of glutamate transport. [³²P]Phosphate-labeled wild-type and mutant transport proteins devoid of all predicted PKC sites were detected by immunoprecipitation after stimulation with PMA. Immunoprecipitation of [³⁵S]methionine-labeled transporter molecules indicates a similar stability of phosphorylated and nonphosphorylated GLAST-1 protein. Immunofluorescence staining did not differentiate surface staining of HEK293 cells expressing GLAST-1 with and without PMA treatment. These data suggest that the neurotransmitter transporter activity of GLAST-1 is inhibited by phosphorylation at a non-PKC consensus site.     

Expression and purification of cysteine mutation isoforms of rat lipocalin-type prostaglandin D synthase for nuclear magnetic resonance study

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is the only member of the lipocalin superfamily that displays enzymatic activity. It binds lipophilic ligands with high affinity and also can catalyze PGH₂ to produce PGD₂. Three cysteine residues, Cys⁶⁵, Cys⁸⁹, and Cys¹⁸⁶ in L-PGDS, are conserved among all species, of which Cys⁸⁹ and Cys¹⁸⁶ residues form a disulfide bridge. In this study, we clarified the effects of thiol groups on the structure of the protein and investigated the structural significance of Cys residues of rat L-PGDS by site-directed mutagenesis. Four mutants were constructed by substituting Cys residues with alanine to identify the correct formation of disulfide bonds among these three residues. The effects of thiol groups on the structure of rat L-PGDS were also identified by these mutants. Analysis of HSQC experiments indicated that these enzymes were all properly folded with well defined tertiary structures. As the first step towards the 3-D nuclear magnetic resonance solution structure, we optimized expression of recombinant rat L-PGDS in Escherichia coli and established an efficient and economic purification protocol yielding large amounts of pure isotopically labeled rat L-PGDS. The results of assignments indicated that the wild-type rat L-PGDS obtained using this expression system was suitable for determination of 3-D nuclear magnetic resonance solution structure.     

Rapid substrate-induced down-regulation in function and surface localization of dopamine transporters: rat dorsal striatum versus nucleus accumbens

The dopamine transporter (DAT) substrates dopamine, d-amphetamine (AMPH), and methamphetamine are known to rapidly and transiently reduce DAT activity and/or surface expression in dorsal striatum and heterologous expression systems. We sought to determine if similar substrate-induced regulation of DATs occurs in rat nucleus accumbens. In dorsal striatum synaptosomes, brief (15-min) in vitro substrate pre-exposure markedly decreased maximal [³H]dopamine uptake velocity whereas identical substrate pre-exposure in nucleus accumbens synaptosomes produced a smaller, non-significant reduction. However, 45 min after systemic AMPH administration, maximal ex vivo [³H]dopamine uptake velocity was significantly reduced in both brain regions. Protein kinase C inhibition blocked AMPH's down-regulation of DAT activity. DAT synaptosomal surface expression was not modified following either the brief in vitro or in vivo AMPH pre-exposure but was reduced after a longer (1-h) in vitro pre-exposure in both brain regions. Together, our findings suggest that relatively brief substrate exposure results in greater down-regulation of DAT activity in dorsal striatum than in nucleus accumbens. Moreover, exposure to AMPH appears to regulate striatal DATs in a biphasic manner, with an initial protein kinase C-dependent decrease in DAT-mediated uptake velocity and then, with longer exposure, a reduction in DAT surface expression.     

Characterization of the Palmitoylation Domain of SNAP-25

Abstract: SNAP-25 (synaptosomal associated protein of 25 kDa) is a neural specific protein that has been implicated in the synaptic vesicle docking and fusion process. It is tightly associated with membranes, and it is one of the major palmitoylated proteins found in neurons. The functional role of palmitoylation for SNAP-25 is unclear. In this report, we show that the palmitate of SNAP-25 is rapidly turned over in PC12 cells, with a half-life of ∼3 h, and the half-life for the protein is 8 h. Mutation of Cys to Ser at positions 85, 88, 90, and 92 reduced the palmitoylation to 9, 21, 42, and 35% of the wild-type protein, respectively. Additional mutations of either Cys^85,88 or Cys^90,92 nearly abolished palmitoylation of the protein. A similar effect on membrane binding for the mutant SNAP-25 was observed, which correlated with the degree of palmitoylation. These results suggest that all four Cys residues are involved in palmitoylation and that membrane association of SNAP-25 may be regulated through dynamic palmitoylation.     

Presence and Function of Dopamine Transporter (DAT) in Stallion Sperm: Dopamine Modulates Sperm Motility and Acrosomal Integrity

Dopamine is a catecholamine with multiple physiological functions, playing a key role in nervous system; however its participation in reproductive processes and sperm physiology is controversial. High dopamine concentrations have been reported in different portions of the feminine and masculine reproductive tract, although the role fulfilled by this catecholamine in reproductive physiology is as yet unknown. We have previously shown that dopamine type 2 receptor is functional in boar sperm, suggesting that dopamine acts as a physiological modulator of sperm viability, capacitation and motility. In the present study, using immunodetection methods, we revealed the presence of several proteins important for the dopamine uptake and signalling in mammalian sperm, specifically monoamine transporters as dopamine (DAT), serotonin (SERT) and norepinephrine (NET) transporters in equine sperm. We also demonstrated for the first time in equine sperm a functional dopamine transporter using 4-[4-(Dimethylamino)styryl]-N-methylpyridinium iodide (ASP⁺), as substrate. In addition, we also showed that dopamine (1 mM) treatment in vitro, does not affect sperm viability but decreases total and progressive sperm motility. This effect is reversed by blocking the dopamine transporter with the selective inhibitor vanoxerine (GBR12909) and non-selective inhibitors of dopamine reuptake such as nomifensine and bupropion. The effect of dopamine in sperm physiology was evaluated and we demonstrated that acrosome integrity and thyrosine phosphorylation in equine sperm is significantly reduced at high concentrations of this catecholamine. In summary, our results revealed the presence of monoamine transporter DAT, NET and SERT in equine sperm, and that the dopamine uptake by DAT can regulate sperm function, specifically acrosomal integrity and sperm motility.     

Formation of a Disulfide Bond in the Immunoglobulin Domain of the Myelin P0 Protein Is Essential for Its Adhesion

Abstract: It is widely accepted, although never demonstrated, that the formation of a disulfide bond in the majority of immunoglobulin (Ig)-like domains stabilizes their final conformation and thus is essential to their functioning as adhesion/recognition molecules. The myelin P₀ protein, which has been shown directly to behave as a homophilic adhesion molecule, contains a single Ig-like domain, stabilized by a putative Cys²¹-Cys⁹⁸ disulfide bond. To test if this bond is indeed necessary to the adhesive function of P₀, the nucleotides in the P₀ cDNA coding for Cys²¹ were altered to code for an alanine. The mutated P₀ cDNA was transfected into Chinese hamster ovary cells, expression of the mutated P₀ protein was characterized, and the adhesiveness of Cys²¹-mutated P₀-expressing cells and that of cells expressing equivalent surface amounts of the unmutated protein were compared. It was found, as we previously reported, that incubation of a single cell suspension of the unmutated P₀-expressing cells resulted in the rapid formation of large aggregates. In contrast, after a similar incubation the cells expressing the Cys²¹-mutated P₀ were still mostly single cells, a result indistinguishable from that observed with the control transfected cells. This suggests that the P₀ protein, when mutated at Cys²¹, does not behave as a homophilic adhesion molecule, which in turn implies that the formation of an Ig domain disulfide bond is essential to the functioning of this molecule.