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The Journal of Biological Chemistry Feb 2023Extracellular dopamine (DA) levels are constrained by the presynaptic DA transporter (DAT), a major psychostimulant target. Despite its necessity for DA...
Extracellular dopamine (DA) levels are constrained by the presynaptic DA transporter (DAT), a major psychostimulant target. Despite its necessity for DA neurotransmission, DAT regulation in situ is poorly understood, and it is unknown whether regulated DAT trafficking impacts dopaminergic signaling and/or behaviors. Leveraging chemogenetics and conditional gene silencing, we found that activating presynaptic Gq-coupled receptors, either hM3Dq or mGlu5, drove rapid biphasic DAT membrane trafficking in ex vivo striatal slices, with region-specific differences between ventral and dorsal striata. DAT insertion required D2 DA autoreceptors and intact retromer, whereas DAT retrieval required PKC activation and Rit2. Ex vivo voltammetric studies revealed that DAT trafficking impacts DA clearance. Furthermore, dopaminergic mGlu5 silencing elevated DAT surface expression and abolished motor learning, which was rescued by inhibiting DAT with a subthreshold CE-158 dose. We discovered that presynaptic DAT trafficking is complex, multimodal, and region specific, and for the first time, we identified cell autonomous mechanisms that govern presynaptic DAT tone. Importantly, the findings are consistent with a role for regulated DAT trafficking in DA clearance and motor function.
Topics: Dopamine; Dopamine Plasma Membrane Transport Proteins; Receptors, Presynaptic; Animals; Mice; Corpus Striatum
PubMed: 36640864
DOI: 10.1016/j.jbc.2023.102900 -
The Journal of Biological Chemistry Aug 2023Amphetamines (AMPHs) are substrates of the dopamine transporter (DAT) and reverse the direction of dopamine (DA) transport. This has been suggested to depend on...
Amphetamines (AMPHs) are substrates of the dopamine transporter (DAT) and reverse the direction of dopamine (DA) transport. This has been suggested to depend on activation of Ca-dependent pathways, but the mechanism underlying reverse transport via endogenously expressed DAT is still unclear. Here, to enable concurrent visualization by live imaging of extracellular DA dynamics and cytosolic Ca levels, we employ the fluorescent Ca sensor jRGECO1a expressed in cultured dopaminergic neurons together with the fluorescent DA sensor GRAB expressed in cocultured "sniffer" cells. In the presence of the Na-channel blocker tetrodotoxin to prevent exocytotic DA release, AMPH induced in the cultured neurons a profound dose-dependent efflux of DA that was blocked both by inhibition of DAT with cocaine and by inhibition of the vesicular monoamine transporter-2 with Ro-4-1284 or reserpine. However, the AMPH-induced DA efflux was not accompanied by an increase in cytosolic Ca and was unaffected by blockade of voltage-gated calcium channels or chelation of cytosolic Ca. The independence of cytosolic Ca was further supported by activation of N-methyl-D-aspartate-type ionotropic glutamate receptors leading to a marked increase in cytosolic Ca without affecting AMPH-induced DA efflux. Curiously, AMPH elicited spontaneous Ca spikes upon blockade of the D2 receptor, suggesting that AMPH can regulate intracellular Ca in an autoreceptor-dependent manner regardless of the apparent independence of Ca for AMPH-induced efflux. We conclude that AMPH-induced DA efflux in dopaminergic neurons does not require cytosolic Ca but is strictly dependent on the concerted action of AMPH on both vesicular monoamine transporter-2 and DAT.
Topics: Amphetamine; Cocaine; Dopamine; Dopamine Plasma Membrane Transport Proteins; Dopaminergic Neurons; Vesicular Monoamine Transport Proteins; Humans; Cell Line, Tumor
PubMed: 37468107
DOI: 10.1016/j.jbc.2023.105063 -
Biomolecules May 2020The activity of the hypothalamus-pituitary-adrenal (HPA) axis is pivotal in homeostasis and presides the adaptative response to stress. Dopamine Transporter (DAT) plays...
The activity of the hypothalamus-pituitary-adrenal (HPA) axis is pivotal in homeostasis and presides the adaptative response to stress. Dopamine Transporter (DAT) plays a key role in the regulation of the HPA axis. We used young adult female DAT Knockout (KO) rats to assess the effects of DAT ablation (partial, heterozygous DAT+/-, or total, homozygous DAT-/-) on vulnerability to stress. DAT-/- rats show profound dysregulation of pituitary homeostasis, in the presence of elevated peripheral corticosterone, before and after acute restraint stress. During stress, DAT-/- rats show abnormal autonomic response at either respiratory and cardiovascular level, and delayed body temperature increase. DAT+/- rats display minor changes of hypophyseal homeostatic mechanisms. These rats display a similar pituitary activation to that of the control animals, albeit in the presence of higher release of peripheral corticosterone than DAT-/- after stress, and reduced temperature during stress. Our data indicate that DAT regulates the HPA axis at both the central and peripheral level, including autonomic function during stress. In particular, the partial deletion of DAT results in increased vulnerability to stress in female rats, which display central and peripheral alterations that are reminiscent of PTSD, and they might provide new insights in the pathophysiology of this disorder.
Topics: Animals; Dopamine Plasma Membrane Transport Proteins; Female; Locomotion; Male; Pituitary Gland; Rats; Rats, Wistar; Stress, Psychological
PubMed: 32486390
DOI: 10.3390/biom10060842 -
Journal of Neurochemistry Apr 2006Dopamine (DA) uptake through the neuronal plasma membrane DA transporter (DAT) is essential for the maintenance of normal DA homeostasis in the brain. The DAT-mediated... (Review)
Review
Dopamine (DA) uptake through the neuronal plasma membrane DA transporter (DAT) is essential for the maintenance of normal DA homeostasis in the brain. The DAT-mediated re-uptake system limits not only the intensity but also the duration of DA actions at presynaptic and postsynaptic receptors. This protein is the primary target for cocaine and amphetamine, both highly addictive and major substances of abuse worldwide. DAT is also the molecular target for therapeutic agents used in the treatment of mental disorders, such as attention deficit hyperactivity disorder and depression. Given the role played by the DAT in regulation of DA neurotransmission and its contribution to the abuse potential of psychostimulants, it becomes not only important but also necessary to understand the functional regulation of this protein. To investigate the cellular and molecular mechanisms associated with DAT function and regulation, our laboratory and others have embarked on a systematic search for DAT protein-protein interactions. Recently, a growing number of proteins have been shown to interact with DAT. These novel interactions might be important in the assembly, targeting, trafficking and/or regulation of transporter function. In this review, I summarize the main findings obtained from the characterization of DAT-interacting proteins and discuss the functional implications of these novel interactions. Based on these new data, I propose to use the term DAT proteome to explain how interacting proteins regulate DAT function. These novel interactions might help define new mechanisms associated with the function of the transporter.
Topics: Animals; Carrier Proteins; Dopamine; Dopamine Plasma Membrane Transport Proteins; GTP-Binding Proteins; Humans; Nuclear Proteins; Protein Kinase C; Proteome; Qa-SNARE Proteins; Synucleins
PubMed: 16635244
DOI: 10.1111/j.1471-4159.2006.03719.x -
Psychopharmacology Aug 2012Δ(9)-tetrahydrocannabinol (Δ(9)-THC) modifies dopamine efflux. However, the extent to which cannabinoid and dopamine drugs modify each other's behavioral effects has...
RATIONALE
Δ(9)-tetrahydrocannabinol (Δ(9)-THC) modifies dopamine efflux. However, the extent to which cannabinoid and dopamine drugs modify each other's behavioral effects has not been fully established.
OBJECTIVES
This study examined dopamine releasers and/or transport inhibitors alone and in combination with cannabinoids in two drug discrimination assays.
METHODS
Experimentally and pharmacologically experienced rhesus monkeys (n = 5) discriminated Δ(9)-THC (0.1 mg/kg i.v.) from vehicle while responding under a fixed ratio 5 schedule of stimulus-shock termination. A separate group (n = 6) of monkeys responded under the same schedule, received daily Δ(9)-THC (1 mg/kg/12 h s.c.), and discriminated the cannabinoid antagonist rimonabant (1 mg/kg i.v.), i.e., cannabinoid withdrawal, from vehicle. A sign of withdrawal sign (head shaking) was examined in monkeys receiving Δ(9)-THC daily.
RESULTS
Rimonabant antagonized the Δ(9)-THC discriminative stimulus and a dose of Δ(9)-THC greater than the daily treatment attenuated the rimonabant discriminative stimulus. In monkeys discriminating Δ(9)-THC, the dopamine transporter ligands cocaine, amphetamine, bupropion, RTI 113, and RTI 177 produced a maximum of 2% responding on the drug lever and blocked the discriminative stimulus effects of Δ(9)-THC. In Δ(9)-THC treated monkeys discriminating rimonabant, the dopamine transporter ligands partially substituted for and increased the potency of rimonabant to produce discriminative stimulus effects. The dopamine antagonist haloperidol enhanced the Δ(9)-THC discriminative stimulus without significantly modifying the rimonabant discriminative stimulus. Imipramine and desipramine, which have low affinity for dopamine transporters, were less effective in modifying either the Δ(9)-THC or rimonabant discriminations. The dopamine transporter ligands and haloperidol attenuated head shaking, whereas imipramine and desipramine did not.
CONCLUSIONS
Dopamine release and/or inhibition of dopamine transport blocks detection of Δ(9)-THC and is potentially the mechanism by which some therapeutics (e.g., bupropion) reduce the subjective effects of marijuana and enhance the subjective effects of marijuana withdrawal.
Topics: Amphetamine; Animals; Cocaine; Discrimination Learning; Dopamine Plasma Membrane Transport Proteins; Dose-Response Relationship, Drug; Dronabinol; Female; Haloperidol; Imipramine; Macaca mulatta; Male; Piperidines; Pyrazoles; Receptors, Cannabinoid; Rimonabant; Tropanes
PubMed: 22374253
DOI: 10.1007/s00213-012-2661-9 -
European Journal of Pharmacology Sep 2012The dopamine transporter removes the neurotransmitter from the synapse, regulating dopamine availability. The transporter can be internalized and its function is blocked...
The dopamine transporter removes the neurotransmitter from the synapse, regulating dopamine availability. The transporter can be internalized and its function is blocked by cocaine and other ligands. Melittin inhibits dopamine transporter function and causes internalization of the recombinant transporter in stably transfected HEK-293 cells, but the specific pathways for internalization and disposition of the transporter are unknown. Here we report that melittin treatment increased both transporter internalization and colocalization with clathrin, effects that were blocked by pretreatment with cocaine. Density gradient centrifugation revealed that melittin treatment caused the dopamine transporter to associate with a density fraction containing the early endosome marker Rab 5A. Confocal microscopy revealed that melittin treatment also increased transporter colocalization with Rab 5A and decreased colocalization with the late endosome marker Rab 7 and the recycling endosome marker Rab 11. Following 60 min of melittin treatment, the transporter was trafficked back to the membrane. By comparison, phorbol ester treatment increased transporter colocalization with early endosome antigen 1 and Rab 7 in a time-dependent manner. Cocaine treatment alone does not affect transporter trafficking in these cells. Results indicate multiple dopamine transporter internalization and recycling pathways that depend on transporter-ligand interactions and post-translational modifications.
Topics: Biotinylation; Dopamine Plasma Membrane Transport Proteins; HEK293 Cells; Humans; Intracellular Space; Melitten; Protein Transport; Tetradecanoylphorbol Acetate; Transfection
PubMed: 22683840
DOI: 10.1016/j.ejphar.2012.05.020 -
International Journal of Molecular... Nov 2020Dopamine transporter knockout (DATk) mice are known to demonstrate profound hyperactivity concurrent with elevated (5-fold) extracellular dopamine in the basal ganglia....
Dopamine transporter knockout (DATk) mice are known to demonstrate profound hyperactivity concurrent with elevated (5-fold) extracellular dopamine in the basal ganglia. At the same time, heterozygous DAT mice (DATh) demonstrate a 2-fold increase in dopamine levels yet only a marginal elevation in locomotor activity level. Another model of dopaminergic hyperactivity is the D3 dopamine receptor knockout (D3k) mice, which present only a modest hyperactivity phenotype, predominately manifested as stereotypical behaviors. In the D3k mice, the hyperactivity is also correlated with elevated extracellular dopamine levels (2-fold) in the basal ganglia. Cross-breeding was used to evaluate the functional consequences of the deletion of both genes. In the heterozygous DAT mice, inactivation of the D3R gene (DATh/D3k) resulted in significant hyperactivity and further elevation of striatal extracellular dopamine above levels observed in respective single mutant mice. The decreased weight of DATk mice was evident regardless of the D3 dopamine receptor genotype. In contrast, measures of thermoregulation revealed that the marked hypothermia of DATk mice (-2 °C) was reversed in double knockout mice. Thus, the extracellular dopamine levels elevated by prolonging uptake could be elevated even further by eliminating the D3 receptor. These data also suggest that the hypothermia observed in DATk mice may be mediated through D3 receptors.
Topics: Animals; Basal Ganglia; Dopamine; Dopamine Plasma Membrane Transport Proteins; Female; Heterozygote; Male; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; Psychomotor Agitation; Receptors, Dopamine D3; Synaptic Transmission; Up-Regulation
PubMed: 33153031
DOI: 10.3390/ijms21218216 -
Scientific Reports Oct 2019The aim of this study was to evaluate associations of motor and non-motor symptoms with dopamine transporter binding in prodromal stage of synucleinopathies. We examined...
The aim of this study was to evaluate associations of motor and non-motor symptoms with dopamine transporter binding in prodromal stage of synucleinopathies. We examined 74 patients with idiopathic REM sleep behavior disorder (RBD), which is a prodromal synucleinopathy, and 39 controls using Movement Disorders Society-Unified Parkinson's Disease Rating Scale (MDS-UPDRS), Montreal Cognitive Assessment, University of Pennsylvania Smell Identification Test (UPSIT), Farnsworth-Munsell 100 hue test, orthostatic test, Scales for Outcomes in PD-Autonomic, Beck depression inventory-II, State-Trait Anxiety Inventory, and video-polysomnography. Electromyographic muscle activity during REM sleep was quantified according to Sleep Innsbruck-Barcelona criteria. In 65 patients, dopamine transporter single-photon emission computed tomography (DAT-SPECT) imaging was performed, putaminal binding ratio was calculated and scans were classified as normal, borderline, or abnormal. Compared to controls, RBD patients had significantly more severe scores in all examined tests. Patients with abnormal DAT-SPECT had higher MDS-UPDRS motor score (p = 0.006) and higher prevalence of orthostatic hypotension (p = 0.008). Putaminal binding ratio was positively associated with UPSIT score (p = 0.03) and negatively associated with tonic (p = 0.003) and phasic (p = 0.01) muscle activity during REM sleep. These associations likely reflect simultaneous advancement of underlying pathology in substantia nigra and susceptible brainstem and olfactory nuclei in prodromal synucleinopathy.
Topics: Aged; Case-Control Studies; Dopamine Plasma Membrane Transport Proteins; Female; Humans; Male; Mental Status and Dementia Tests; Middle Aged; Polysomnography; Protein Binding; REM Sleep Behavior Disorder
PubMed: 31664065
DOI: 10.1038/s41598-019-51710-y -
Journal of Nuclear Medicine : Official... Sep 2010The dopamine transporter (DAT) is a transmembrane protein responsible for reuptake of dopamine from the synaptic cleft and termination of dopaminergic transmission.... (Review)
Review
The dopamine transporter (DAT) is a transmembrane protein responsible for reuptake of dopamine from the synaptic cleft and termination of dopaminergic transmission. Several radioligands are available for DAT imaging with SPECT and PET. This review summarizes the main SPECT and PET radioligands and the main applications of DAT imaging in neuropsychiatric disorders.
Topics: Attention Deficit Disorder with Hyperactivity; Biomarkers; Dopamine; Dopamine Plasma Membrane Transport Proteins; Humans; Molecular Imaging; Parkinson Disease
PubMed: 20720060
DOI: 10.2967/jnumed.109.065656 -
The Journal of Biological Chemistry 2021Dopamine transporter (DAT) mediates the reuptake of synaptically released dopamine, and thus controls the duration and intensity of dopamine neurotransmission. Mammalian...
Dopamine transporter (DAT) mediates the reuptake of synaptically released dopamine, and thus controls the duration and intensity of dopamine neurotransmission. Mammalian DAT has been observed to form oligomers, although the mechanisms of oligomerization and its role in DAT activity and trafficking remain largely unknown. We discovered a series of small molecule compounds that stabilize trimers and induce high-order oligomers of DAT and concomitantly promote its clathrin-independent endocytosis. Using a combination of chemical cross-linking, fluorescence resonance energy transfer microscopy, antibody-uptake endocytosis assay, live-cell lattice light sheet microscopy, ligand binding and substrate transport kinetics analyses, and molecular modeling and simulations, we investigated molecular basis of DAT oligomerization and endocytosis induced by these compounds. Our study showed that small molecule-induced DAT oligomerization and endocytosis are favored by the inward-facing DAT conformation and involve interactions of four hydrophobic residues at the interface between transmembrane (TM) helices TM4 and TM9. Surprisingly, a corresponding quadruple DAT mutant displays altered dopamine transport kinetics and increased cocaine-analog binding. The latter is shown to originate from an increased preference for outward-facing conformation and inward-to-outward transition. Taken together, our results demonstrate a direct coupling between conformational dynamics of DAT, functional activity of the transporter, and its oligomerization leading to endocytosis. The high specificity of such coupling for DAT makes the TM4-9 hub a new target for pharmacological modulation of DAT activity and subcellular localization.
Topics: Animals; Cell Line; Clathrin; Dopamine; Dopamine Plasma Membrane Transport Proteins; Endocytosis; Endothelial Cells; Fluorescence Resonance Energy Transfer; Humans; Models, Molecular; Protein Binding; Protein Conformation; Small Molecule Libraries; Swine
PubMed: 33610553
DOI: 10.1016/j.jbc.2021.100430