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Journal of Chemical Neuroanatomy Oct 2017The neurotransmitter dopamine (DA) plays a key role in several biological processes including reward, mood, motor activity and attention. Synaptic DA homeostasis is... (Review)
Review
The neurotransmitter dopamine (DA) plays a key role in several biological processes including reward, mood, motor activity and attention. Synaptic DA homeostasis is controlled by the dopamine transporter (DAT) which transports extracellular DA into the presynaptic neuron after release and regulates its availability to receptors. Many neurological disorders such as schizophrenia, bipolar disorder, Parkinson disease and attention-deficit hyperactivity disorder are associated with imbalances in DA homeostasis that may be related to DAT dysfunction. DAT is also a target of psychostimulant and therapeutic drugs that inhibit DA reuptake and lead to elevated dopaminergic neurotransmission. We have recently demonstrated the acute and chronic modulation of DA reuptake activity and DAT stability through S-palmitoylation, the linkage of a 16-carbon palmitate group to cysteine via a thioester bond. This review summarizes the properties and regulation of DAT palmitoylation and describes how it serves to affect various transporter functions. Better understanding of the role of palmitoylation in regulation of DAT function may lead to identification of therapeutic targets for modulation of DA homeostasis in the treatment of dopaminergic disorders.
Topics: Animals; Dopamine Plasma Membrane Transport Proteins; Humans; Lipoylation; Protein Processing, Post-Translational
PubMed: 28115272
DOI: 10.1016/j.jchemneu.2017.01.002 -
Molecular Psychiatry Apr 2022Silencing of dopamine transporter (DAT), a main controlling factor of dopaminergic signaling, results in biochemical and behavioral features characteristic for...
Silencing of dopamine transporter (DAT), a main controlling factor of dopaminergic signaling, results in biochemical and behavioral features characteristic for neuropsychiatric diseases with presumed hyperdopaminergia including schizophrenia, attention deficit hyperactivity disorder (ADHD), bipolar disorder, and obsessive-compulsive disorder (OCD). Investigation of DAT silencing thus provides a transdiagnostic approach towards a systems-level understanding of common underlying pathways. Using a high-field multimodal imaging approach and a highly sensitive cryogenic coil, we integrated structural, functional and metabolic investigations in tandem with behavioral assessments on a newly developed preclinical rat model, comparing DAT homozygous knockout (DAT-KO, N = 14), heterozygous knockout (N = 8) and wild-type male rats (N = 14). We identified spatially distributed structural and functional brain alterations encompassing motor, limbic and associative loops that demonstrated strong behavioral relevance and were highly consistent across imaging modalities. DAT-KO rats manifested pronounced volume loss in the dorsal striatum, negatively correlating with cerebellar volume increase. These alterations were associated with hyperlocomotion, repetitive behavior and loss of efficient functional small-world organization. Further, prefrontal and midbrain regions manifested opposite changes in functional connectivity and local network topology. These prefrontal disturbances were corroborated by elevated myo-inositol levels and increased volume. To conclude, our imaging genetics approach provides multimodal evidence for prefrontal-midbrain decoupling and striato-cerebellar neuroplastic compensation as two key features of constitutive DAT blockade, proposing them as transdiagnostic mechanisms of hyperdopaminergia. Thus, our study connects developmental DAT blockade to systems-level brain changes, underlying impaired action inhibition control and resulting in motor hyperactivity and compulsive-like features relevant for ADHD, schizophrenia and OCD.
Topics: Animals; Attention Deficit Disorder with Hyperactivity; Brain; Dopamine Plasma Membrane Transport Proteins; Hyperkinesis; Male; Mesencephalon; Rats
PubMed: 35246636
DOI: 10.1038/s41380-022-01471-4 -
NeuroImage. Clinical 2021To evaluate possible differences between brain dopamine transporter (DAT) binding in a group of symptomatic parkinsonism patients without dopaminergic degeneration and...
OBJECTIVE
To evaluate possible differences between brain dopamine transporter (DAT) binding in a group of symptomatic parkinsonism patients without dopaminergic degeneration and healthy individuals.
BACKGROUND
Dopaminergic neuroimaging studies of Parkinson's disease (PD) have often used control groups formed from symptomatic patients with apparently normal striatal dopamine function. We sought to investigate whether symptomatic patients can be used to represent dopaminergically normal healthy controls.
METHODS
Forty healthy elderly individuals were scanned with DAT [I]FP-CIT SPECT and compared to 69 age- and sex-matched symptomatic patients with nondegenerative conditions (including essential tremor, drug-induced parkinsonism and vascular parkinsonism). An automated region-of-interest based analysis of the caudate nucleus and the anterior/posterior putamen was performed. Specific binding ratios (SBR = [ROI-occ]/occ) were compared between the groups.
RESULTS
DAT binding in symptomatic patients was 8.6% higher in the posterior putamen than in healthy controls (p = 0.03). Binding correlated negatively with age in both groups but not with motor symptom severity, cognitive function or depression ratings.
CONCLUSIONS
Putaminal DAT binding, as measured with [I]FP-CIT SPECT, was higher in symptomatic controls than in healthy individuals. The reason for the difference is unclear but can include selection bias when DAT binding is used to aid clinical diagnosis and possible self-selection bias in healthy volunteerism. This effect should be taken into consideration when designing and interpreting neuroimaging trials investigating the dopamine system with [I]FP-CIT SPECT.
Topics: Aged; Corpus Striatum; Dopamine Plasma Membrane Transport Proteins; Humans; Neuroimaging; Parkinson Disease; Tomography, Emission-Computed, Single-Photon
PubMed: 34482280
DOI: 10.1016/j.nicl.2021.102807 -
International Journal of Molecular... Jun 2023Alterations in dopamine neurotransmission are associated with obesity and food preferences. Otsuka Long-Evans Tokushima Fatty (OLETF) rats that lack functional...
Alterations in dopamine neurotransmission are associated with obesity and food preferences. Otsuka Long-Evans Tokushima Fatty (OLETF) rats that lack functional cholecystokinin receptor type-1 (CCK-1R), due to a natural mutation, exhibit impaired satiation, are hyperphagic, and become obese. In addition, compared to lean control Long-Evans Tokushima (LETO) rats, OLETF rats have pronounced avidity for over-consuming palatable sweet solutions, have greater dopamine release to psychostimulants, reduced dopamine 2 receptor (D2R) binding, and exhibit increased sensitivity to sucrose reward. This supports altered dopamine function in this strain and its general preference for palatable solutions such as sucrose. In this study, we examined the relationship between OLETF's hyperphagic behavior and striatal dopamine signaling by investigating basal and amphetamine stimulated motor activity in prediabetic OLETF rats before and after access to sucrose solution (0.3 M) compared to non-mutant control LETO rats, as well as availability of dopamine transporter (DAT) using autoradiography. In the sucrose tests, one group of OLETF rats received ad libitum access to sucrose while the other group received an amount of sucrose equal to that consumed by the LETO. OLETFs with ad libitum access consumed significantly more sucrose than LETOs. Sucrose exerted a biphasic effect on basal activity in both strains, i.e., reduced activity for 1 week followed by increased activity in weeks 2 and 3. Basal locomotor activity was reduced (-17%) in OLETFs prior to sucrose, compared to LETOs. Withdrawal of sucrose resulted in increased locomotor activity in both strains. The magnitude of this effect was greater in OLETFs and the activity was increased in restricted compared to ad-libitum-access OLETFs. Sucrose access augmented AMPH-responses in both strains with a greater sensitization to AMPH during week 1, an effect that was a function of the amount of sucrose consumed. One week of sucrose withdrawal sensitized AMPH-induced ambulatory activity in both strains. In OLETF with restricted access to sucrose, withdrawal resulted in no further sensitization to AMPH. DAT availability in the nucleus accumbens shell was significantly reduced in OLETF compared with aged-matched LETO. Together, these findings show that OLETF rats have reduced basal DA transmission and a heightened response to natural and pharmacological stimulation.
Topics: Animals; Rats; Dopamine; Dopamine Plasma Membrane Transport Proteins; Obesity; Rats, Inbred OLETF; Rats, Long-Evans; Receptors, Cholecystokinin; Sucrose
PubMed: 37298724
DOI: 10.3390/ijms24119773 -
Journal of Neurochemistry Apr 2010The dopamine transporter (DAT) plays a key role in regulating dopaminergic signalling in the brain by mediating rapid clearance of dopamine from the synaptic clefts. The... (Review)
Review
The dopamine transporter (DAT) plays a key role in regulating dopaminergic signalling in the brain by mediating rapid clearance of dopamine from the synaptic clefts. The psychostimulatory actions of cocaine and amphetamine are primarily the result of a direct interaction of these compounds with DAT leading to attenuated dopamine clearance and for amphetamine even increased dopamine release. In the last decade, intensive efforts have been directed towards understanding the molecular and cellular mechanisms governing the activity and availability of DAT in the plasma membrane of the pre-synaptic neurons. This has led to the identification of a plethora of different kinases, receptors and scaffolding proteins that interact with DAT and hereby either modulate the catalytic activity of the transporter or regulate its trafficking and degradation. Several new tools for studying DAT regulation in live cells have also recently become available such as fluorescently tagged cocaine analogues and fluorescent substrates. Here we review the current knowledge about the role of protein-protein interactions in DAT regulation as well as we describe the most recent methodological developments that have been established to overcome the challenges associated with the study of DAT in endogenous systems.
Topics: Animals; Brain; Dopamine; Dopamine Agents; Dopamine Plasma Membrane Transport Proteins; Electric Conductivity; Fluorescent Antibody Technique; Fluorescent Dyes; Ion Transport; Models, Biological; Neurons; Protein Conformation; Protein Processing, Post-Translational; Protein Transport; Receptors, G-Protein-Coupled; Ubiquitination; alpha-Synuclein
PubMed: 20085610
DOI: 10.1111/j.1471-4159.2010.06599.x -
The Journal of Pharmacology and... Jul 2013The dopamine transporter (DAT) is a sodium-coupled symporter protein responsible for modulating the concentration of extraneuronal dopamine in the brain. The DAT is a... (Review)
Review
The dopamine transporter (DAT) is a sodium-coupled symporter protein responsible for modulating the concentration of extraneuronal dopamine in the brain. The DAT is a principle target of various psychostimulant, nootropic, and antidepressant drugs, as well as certain drugs used recreationally, including the notoriously addictive stimulant cocaine. DAT ligands have traditionally been divided into two categories: cocaine-like inhibitors and amphetamine-like substrates. Whereas inhibitors block monoamine uptake by the DAT but are not translocated across the membrane, substrates are actively translocated and trigger DAT-mediated release of dopamine by reversal of the translocation cycle. Because both inhibitors and substrates increase extraneuronal dopamine levels, it is often assumed that all DAT ligands possess an addictive liability equivalent to that of cocaine. However, certain recently developed ligands, such as atypical benztropine-like DAT inhibitors with reduced or even a complete lack of cocaine-like rewarding effects, suggest that addictiveness is not a constant property of DAT-affecting compounds. These atypical ligands do not conform to the classic preconception that all DAT inhibitors (or substrates) are functionally and mechanistically alike. Instead, they suggest the possibility that the DAT exhibits some of the ligand-specific pleiotropic functional qualities inherent to G-protein-coupled receptors. That is, ligands with different chemical structures induce specific conformational changes in the transporter protein that can be differentially transduced by the cell, ultimately eliciting unique behavioral and psychological effects. The present overview discusses compounds with conformation-specific activity, useful not only as tools for studying the mechanics of dopamine transport, but also as leads for medication development in addictive disorders.
Topics: Allosteric Regulation; Animals; Biological Transport; Brain; Dopamine; Dopamine Plasma Membrane Transport Proteins; Humans; Ligands; Membrane Transport Modulators; Nerve Tissue Proteins; Neurons; Protein Conformation; Protein Isoforms
PubMed: 23568856
DOI: 10.1124/jpet.111.191056 -
The Journal of Biological Chemistry Jan 2017Extracellular dopamine and serotonin concentrations are determined by the presynaptic dopamine (DAT) and serotonin (SERT) transporters, respectively. Numerous studies...
Extracellular dopamine and serotonin concentrations are determined by the presynaptic dopamine (DAT) and serotonin (SERT) transporters, respectively. Numerous studies have investigated the DAT and SERT structural elements contributing to inhibitor and substrate binding. To date, crystallographic studies have focused on conserved transmembrane domains, where multiple substrate binding and translocation features are conserved. However, it is unknown what, if any, role the highly divergent intracellular N and C termini contribute to these processes. Here, we used chimeric proteins to test whether DAT and SERT N and C termini contribute to transporter substrate and inhibitor affinities. Replacing the DAT N terminus with that of SERT had no effect on DA transport V but significantly decreased DAT substrate affinities for DA and amphetamine. Similar losses in uptake inhibition were observed for small DAT inhibitors, whereas substituting the DAT C terminus with that of SERT affected neither substrate nor inhibitor affinities. In contrast, the N-terminal substitution was completely tolerated by the larger DAT inhibitors, which exhibited no loss in apparent affinity. Remarkably, all affinity losses were rescued in DAT chimeras encoding both SERT N and C termini. The sensitivity to amino-terminal substitution was specific for DAT, because replacing the SERT N and/or C termini affected neither substrate nor inhibitor affinities. Taken together, these findings provide compelling experimental evidence that DAT N and C termini synergistically contribute to substrate and inhibitor affinities.
Topics: Amino Acid Substitution; Biological Transport, Active; Cell Line; Dopamine Plasma Membrane Transport Proteins; Humans; Mutation, Missense; Protein Domains; Serotonin Plasma Membrane Transport Proteins
PubMed: 27986813
DOI: 10.1074/jbc.M116.762872 -
Biomolecules Jun 2022Dopamine (DA), the most abundant human brain catecholaminergic neurotransmitter, modulates key behavioral and neurological processes in young and senescent brains,...
Dopamine (DA), the most abundant human brain catecholaminergic neurotransmitter, modulates key behavioral and neurological processes in young and senescent brains, including motricity, sleep, attention, emotion, learning and memory, and social and reward-seeking behaviors. The DA transporter (DAT) regulates transsynaptic DA levels, influencing all these processes. Compounds targeting DAT (e.g., cocaine and amphetamines) were historically used to shape mood and cognition, but these substances typically lead to severe negative side effects (tolerance, abuse, addiction, and dependence). DA/DAT signaling dysfunctions are associated with neuropsychiatric and progressive brain disorders, including Parkinson's and Alzheimer diseases, drug addiction and dementia, resulting in devastating personal and familial concerns and high socioeconomic costs worldwide. The development of low-side-effect, new/selective medicaments with reduced abuse-liability and which ameliorate DA/DAT-related dysfunctions is therefore crucial in the fields of medicine and healthcare. Using the rat as experimental animal model, the present work describes the synthesis and pharmacological profile of ()-MK-26, a new modafinil analogue with markedly improved potency and selectivity for DAT over parent drug. Ex vivo electrophysiology revealed significantly augmented hippocampal long-term synaptic potentiation upon acute, intraperitoneally delivered ()-MK-26 treatment, whereas in vivo experiments in the hole-board test showed only lesser effects on reference memory performance in aged rats. However, in effort-related FR5/chow and PROG/chow feeding choice experiments, ()-MK-26 treatment reversed the depression-like behavior induced by the dopamine-depleting drug tetrabenazine (TBZ) and increased the selection of high-effort alternatives. Moreover, in in vivo microdialysis experiments, ()-MK-26 significantly increased extracellular DA levels in the prefrontal cortex and in nucleus accumbens core and shell. These studies highlight ()-MK-26 as a potent enhancer of transsynaptic DA and promoter of synaptic plasticity, with predominant beneficial effects on effort-related behaviors, thus proposing therapeutic potentials for ()-MK-26 in the treatment of low-effort exertion and motivational dysfunctions characteristic of depression and aging-related disorders.
Topics: Animals; Dopamine; Dopamine Plasma Membrane Transport Proteins; Hippocampus; Humans; Motivation; Neuronal Plasticity; Rats
PubMed: 35883437
DOI: 10.3390/biom12070881 -
The Journal of Biological Chemistry 2021The dopamine transporter (DAT) is essential for the reuptake of the released neurotransmitter dopamine (DA) in the brain. Psychostimulants, methamphetamine and cocaine,...
The dopamine transporter (DAT) is essential for the reuptake of the released neurotransmitter dopamine (DA) in the brain. Psychostimulants, methamphetamine and cocaine, have been reported to induce the formation of DAT multimeric complexes in vivo and in vitro. The interpretation of DAT multimer function has been primarily in the context of compounds that induce structural and functional modifications of the DAT, complicating the understanding of the significance of DAT multimers. To examine multimerization in the absence of DAT ligands as well as in their presence, we developed a novel, optogenetic fusion chimera of cryptochrome 2 and DAT with an mCherry fluorescent reporter (Cry2-DAT). Using blue light to induce Cry2-DAT multimeric protein complex formation, we were able to simultaneously test the functional contributions of DAT multimerization in the absence or presence of substrates or inhibitors with high spatiotemporal precision. We found that blue light-stimulated Cry2-DAT multimers significantly increased IDT307 uptake and MFZ 9-18 binding in the absence of ligands as well as after methamphetamine and nomifensine treatment. Blue light-induced Cry2-DAT multimerization increased colocalization with recycling endosomal marker Rab11 and had decreased presence in Rab5-positive early endosomes and Rab7-positive late endosomes. Our data suggest that the increased uptake and binding results from induced and rapid trafficking of DAT multimers to the plasma membrane. Our data suggest that DAT multimers may function to help maintain DA homeostasis.
Topics: Animals; Biological Transport; Cell Membrane; Dopamine; Dopamine Plasma Membrane Transport Proteins; Gene Expression; HEK293 Cells; Humans; Neurons; Optogenetics; Protein Multimerization
PubMed: 34015332
DOI: 10.1016/j.jbc.2021.100787 -
Neuropharmacology Dec 2021Clandestine chemists are currently exploiting the pyrrolidinophenone scaffold to develop new designer drugs that carry the risk of abuse and overdose. These drugs...
Clandestine chemists are currently exploiting the pyrrolidinophenone scaffold to develop new designer drugs that carry the risk of abuse and overdose. These drugs promote addiction through the rewarding effects of increased dopaminergic neurotransmission. 3,4-Methylenedioxypyrovalerone (MDPV) and its analogs are illicit psychostimulants of this class that are ∼50-fold more potent than cocaine at inhibiting the human dopamine transporter (hDAT). In contrast, MDPV is a weak inhibitor at both the human serotonin transporter (hSERT) and, as it is shown here, the Drosophila melanogaster DAT (dDAT). We studied three conserved residues between hSERT and dDAT that are unique in hDAT (A117, F318, and P323 in dDAT), and one residue that is different in all three transporters (D121 in dDAT). hDAT residues were replaced in the dDAT sequence at these positions using site-directed mutagenesis and stable cell lines were generated expressing these mutant transporters. The potencies of MDPV and two of its analogs were determined using a Ca-mobilization assay. In this assay, voltage-gated Ca channels are expressed to sense the membrane electrical depolarization evoked when dopamine is transported through DAT. Each individual mutant slightly improved MDPV's potency, but the combination of all four increased its potency ∼100-fold (2 log units) in inhibiting dDAT activity. Molecular modeling and docking studies were conducted to explore the possible mode of interaction between MDPV and DAT in silico. Two of the studied residues (F318 and P323) are at the entrance of the S1 binding site, whereas the other two (A117 and D121) face the aryl moiety of MDPV when bound to this site. Therefore, these four non-conserved residues can influence MDPV selectivity not only by stabilizing binding, but also by controlling access to its binding site at DAT.
Topics: Animals; Benzodioxoles; Biological Transport; Calcium Channels; Cell Line; Designer Drugs; Dopamine Plasma Membrane Transport Proteins; Dopamine Uptake Inhibitors; Drosophila melanogaster; Molecular Docking Simulation; Pyrrolidines; Serotonin Plasma Membrane Transport Proteins; Synthetic Cathinone
PubMed: 34619165
DOI: 10.1016/j.neuropharm.2021.108820