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Biophysical Journal Nov 2007By performing homology modeling, molecular docking, and molecular dynamics simulations, we have developed three-dimensional (3D) structural models of both dopamine...
By performing homology modeling, molecular docking, and molecular dynamics simulations, we have developed three-dimensional (3D) structural models of both dopamine transporter and dopamine transporter-dopamine complex in the environment of lipid bilayer and solvent water. According to the simulated structure of dopamine transporter-dopamine complex, dopamine was orientated in a hydrophobic pocket at the midpoint of the membrane. The modeled 3D structures provide some detailed structural and mechanistic insights concerning how dopamine transporter (DAT) interacts with dopamine at atomic level, extending our mechanistic understanding of the dopamine reuptake with the help of Na(+) ions. The general features of the modeled 3D structures are consistent with available experimental data. Based on the modeled structures, our calculated binding free energy (DeltaG(bind) = -6.4 kcal/mol) for dopamine binding with DAT is also reasonably close to the experimentally derived DeltaG(bind) value of -7.4 kcal/mol. Finally, a possible dopamine-entry pathway, which involves formation and breaking of the salt bridge between side chains of Arg(85) and Asp(476), is proposed based on the results obtained from the modeling and molecular dynamics simulation. The new structural and mechanistic insights obtained from this computational study are expected to stimulate future, further biochemical and pharmacological studies on the detailed structures and mechanisms of DAT and other homologous transporters.
Topics: Amino Acid Sequence; Computer Simulation; Databases, Protein; Dopamine; Dopamine Plasma Membrane Transport Proteins; Humans; Ions; Models, Molecular; Molecular Conformation; Molecular Sequence Data; Protein Binding; Protein Conformation; Sequence Homology, Amino Acid; Sodium; Software; Thermodynamics; Time Factors
PubMed: 17704152
DOI: 10.1529/biophysj.107.110924 -
PloS One 2018The human dopamine transporter (hDAT) plays a major role in dopamine homeostasis and regulation of neurotransmission by clearing dopamine from the extracellular space...
The human dopamine transporter (hDAT) plays a major role in dopamine homeostasis and regulation of neurotransmission by clearing dopamine from the extracellular space using secondary active transport. Dopamine is an essential monoamine chemical messenger that regulates reward seeking behavior, motor control, hormonal release, and emotional response in humans. Psychostimulants such as cocaine primarily target the central binding site of hDAT and lock the transporter in an outward-facing conformation, thereby inhibiting dopamine reuptake. The inhibition of dopamine reuptake leads to accumulation of dopamine in the synapse causing heightened signaling. In addition, hDAT is implicated in various neurological disorders and disease-associated neurodegeneration. Despite its significance, the structural studies of hDAT have proven difficult. Instability of hDAT in detergent micelles has been a limiting factor in its successful biochemical, biophysical, and structural characterization. To overcome this hurdle, we identified ligands that stabilize hDAT in detergent micelles. We then screened ~200 single residue mutants of hDAT using a high-throughput scintillation proximity assay and identified a thermostable variant (I248Y). Here we report a robust strategy to overexpress and successfully purify a thermostable variant of hDAT in an inhibitor and allosteric ligand bound conformation.
Topics: Allosteric Regulation; Cell Membrane; Dopamine; Dopamine Plasma Membrane Transport Proteins; HEK293 Cells; High-Throughput Screening Assays; Humans; Ligands; Micelles; Mutagenesis, Site-Directed; Mutation; Protein Conformation; Protein Stability; Temperature
PubMed: 29965988
DOI: 10.1371/journal.pone.0200085 -
Journal of Neurochemistry Apr 2017As one of the primary mechanisms by which dopamine signaling is regulated, the dopamine transporter (DAT) is an attractive pharmacological target for the treatment of...
As one of the primary mechanisms by which dopamine signaling is regulated, the dopamine transporter (DAT) is an attractive pharmacological target for the treatment of diseases based in dopaminergic dysfunction. In this work we demonstrate for the first time that the commonly prescribed breast cancer therapeutic tamoxifen and its major metabolites, 4-hydroxytamoxifen and endoxifen, inhibit DAT function. Tamoxifen inhibits [ H]dopamine uptake into human DAT (hDAT)-N2A cells via an uncompetitive or mixed mechanism. Endoxifen, an active metabolite of tamoxifen, asymmetrically inhibits DAT function in hDAT-N2A cells, showing a preference for the inhibition of amphetamine-stimulated dopamine efflux as compared to dopamine uptake. Importantly, we demonstrate that the effects of tamoxifen and its metabolites on the DAT occur independently of its activity as selective estrogen receptor modulators. This work suggests that tamoxifen is inhibiting DAT function through a previously unidentified mechanism.
Topics: Animals; Cell Line, Tumor; Dopamine Plasma Membrane Transport Proteins; Dose-Response Relationship, Drug; Estrogen Antagonists; Humans; Mice; Receptors, Estrogen; Selective Estrogen Receptor Modulators; Tamoxifen
PubMed: 28075498
DOI: 10.1111/jnc.13955 -
Trends in Neurosciences Apr 2014Phasic increases in brain dopamine are required for cue-directed reward seeking. Although compelling within the framework of appetitive behavior, the view that illicit... (Review)
Review
Phasic increases in brain dopamine are required for cue-directed reward seeking. Although compelling within the framework of appetitive behavior, the view that illicit drugs hijack reward circuits by hyperactivating these dopamine transients is inconsistent with established psychostimulant pharmacology. However, recent work reclassifying amphetamine (AMPH), cocaine, and other addictive dopamine-transporter inhibitors (DAT-Is) supports transient hyperactivation as a unifying hypothesis of abused drugs. We argue here that reclassification also identifies generating burst firing by dopamine neurons as a keystone action. Unlike natural rewards, which are processed by sensory systems, drugs act directly on the brain. Consequently, to mimic natural rewards and exploit reward circuits, dopamine transients must be elicited de novo. Of available drug targets, only burst firing achieves this essential outcome.
Topics: Animals; Brain; Dopamine; Dopamine Plasma Membrane Transport Proteins; Dopaminergic Neurons; Humans; Illicit Drugs; Models, Neurological; Substance-Related Disorders
PubMed: 24656971
DOI: 10.1016/j.tins.2014.02.002 -
Movement Disorders : Official Journal... Oct 2019Recent work has shown loss of phosphodiesterase 10A levels in middle-stage and advanced treated patients with PD, which was associated with motor symptom severity. (Comparative Study)
Comparative Study
BACKGROUND
Recent work has shown loss of phosphodiesterase 10A levels in middle-stage and advanced treated patients with PD, which was associated with motor symptom severity.
OBJECTIVES
To assess phosphodiesterase 10A levels in early PD and compare with loss of dopamine transporter as markers of disease burden.
METHODS
Seventy-eight subjects were included in this study (17 early de novo, 15 early l-dopa-treated, 24 moderate-advanced l-dopa-treated patients with PD, and 22 healthy controls). All participants underwent [ C]IMA107 PET, [ C]PE2I PET, and 3-Tesla MRI scan.
RESULTS
Early de novo PD patients showed loss of [ C]IMA107 and of [ C]PE2I binding in caudate and putamen (P < 0.001); early l-dopa-treated PD patients showed additional loss of [ C]IMA107 in the caudate (P < 0.001; annual decline 3.6%) and putamen (P < 0.001; annual decline 2.8%), but loss of [ C]PE2I only in the putamen (P < 0.001; annual decline 6.8%). Lower [ C]IMA107 correlated with lower [ C]PE2I in the caudate (rho = 0.51; P < 0.01) and putamen (rho = 0.53; P < 0.01). Longer disease duration correlated with lower [ C]IMA107 in the caudate (rho = -0.72; P < 0.001) and putamen (rho = -0.48; P < 0.01), and with lower [ C]PE2I only in the putamen (rho = -0.65; P < 0.001). Higher burden of motor symptoms correlated with lower [ C]IMA107 in the caudate (rho = -0.42; P < 0.05) and putamen (rho = -0.41; P < 0.05), and with lower [ C]PE2I only in the putamen (rho = -0.69; P < 0.001).
CONCLUSION
Our findings demonstrate loss of phosphodiesterase 10A levels very early in the course of PD and is associated with the gradual and progressive increase of motor symptoms. Phosphodiesterase 10A imaging shows similar potential with dopamine transporter imaging to follow disease progression. © 2019 International Parkinson and Movement Disorder Society.
Topics: Disease Progression; Dopamine Plasma Membrane Transport Proteins; Heterocyclic Compounds, 2-Ring; Humans; Magnetic Resonance Imaging; Nortropanes; Parkinson Disease; Positron-Emission Tomography; Putamen; Quinoxalines
PubMed: 31158314
DOI: 10.1002/mds.27733 -
The Journal of Neuroscience : the... Apr 2009Uptake through the dopamine transporter (DAT) represents the primary mechanism used to terminate dopaminergic transmission in brain. Although it is well known that... (Comparative Study)
Comparative Study
Uptake through the dopamine transporter (DAT) represents the primary mechanism used to terminate dopaminergic transmission in brain. Although it is well known that dopamine (DA) taken up by the transporter is used to replenish synaptic vesicle stores for subsequent release, the molecular details of this mechanism are not completely understood. Here, we identified the synaptic vesicle protein synaptogyrin-3 as a DAT interacting protein using the split ubiquitin system. This interaction was confirmed through coimmunoprecipitation experiments using heterologous cell lines and mouse brain. DAT and synaptogyrin-3 colocalized at presynaptic terminals from mouse striatum. Using fluorescence resonance energy transfer microscopy, we show that both proteins interact in live neurons. Pull-down assays with GST (glutathione S-transferase) proteins revealed that the cytoplasmic N termini of both DAT and synaptogyrin-3 are sufficient for this interaction. Furthermore, the N terminus of DAT is capable of binding purified synaptic vesicles from brain tissue. Functional assays revealed that synaptogyrin-3 expression correlated with DAT activity in PC12 and MN9D cells, but not in the non-neuronal HEK-293 cells. These changes were not attributed to changes in transporter cell surface levels or to direct effect of the protein-protein interaction. Instead, the synaptogyrin-3 effect on DAT activity was abolished in the presence of the vesicular monoamine transporter-2 (VMAT2) inhibitor reserpine, suggesting a dependence on the vesicular DA storage system. Finally, we provide evidence for a biochemical complex involving DAT, synaptogyrin-3, and VMAT2. Collectively, our data identify a novel interaction between DAT and synaptogyrin-3 and suggest a physical and functional link between DAT and the vesicular DA system.
Topics: Animals; Dopamine Plasma Membrane Transport Proteins; Humans; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Nerve Tissue Proteins; PC12 Cells; Rats; Synaptic Vesicles; Synaptogyrins; Transfection
PubMed: 19357284
DOI: 10.1523/JNEUROSCI.4559-08.2009 -
The Journal of Biological Chemistry Feb 2011Palmitoylation is a lipid modification that confers diverse functions to target proteins and is a contributing factor for many neuronal diseases. In this study, we...
Palmitoylation is a lipid modification that confers diverse functions to target proteins and is a contributing factor for many neuronal diseases. In this study, we demonstrate using [(3)H]palmitic acid labeling and acyl-biotinyl exchange that native and expressed dopamine transporters (DATs) are palmitoylated, and using the palmitoyl acyltransferase inhibitor 2-bromopalmitate (2BP), we identify several associated functions. Treatment of rat striatal synaptosomes with 2BP using lower doses or shorter times caused robust inhibition of transport V(max) that occurred with no losses of DAT protein or changes in DAT surface levels, indicating that acute loss of palmitoylation leads to reduction of transport kinetics. Treatment of synaptosomes or cells with 2BP using higher doses or longer times resulted in DAT protein losses and production of transporter fragments, implicating palmitoylation in regulation of transporter degradation. Site-directed mutagenesis indicated that palmitoylation of rat DAT occurs at Cys-580 at the intracellular end of transmembrane domain 12 and at one or more additional unidentified site(s). Cys-580 mutation also led to production of transporter degradation fragments and to increased phorbol ester-induced down-regulation, further supporting palmitoylation in opposing DAT turnover and in opposing protein kinase C-mediated regulation. These results identify S-palmitoylation as a major regulator of DAT properties that could significantly impact acute and long term dopamine transport capacity.
Topics: Animals; Biological Transport; Cell Line; Dopamine; Dopamine Plasma Membrane Transport Proteins; Enzyme Inhibitors; Hypoglycemic Agents; Lipoylation; Male; Mutagenesis, Site-Directed; Mutation, Missense; Palmitates; Protein Kinase C; Rats; Rats, Sprague-Dawley; Swine; Synaptosomes
PubMed: 21118819
DOI: 10.1074/jbc.M110.187872 -
Neuroscience Bulletin Dec 2016The dopamine transporter (DAT) is involved in the regulation of extracellular dopamine levels. A 40-bp variable-number tandem repeat (VNTR) polymorphism in the...
The dopamine transporter (DAT) is involved in the regulation of extracellular dopamine levels. A 40-bp variable-number tandem repeat (VNTR) polymorphism in the 3'-untranslated region (3'UTR) of the DAT has been reported to be associated with various phenotypes that are involved in the aberrant regulation of dopaminergic neurotransmission. In the present study, we found that miR-137 and miR-491 caused a marked reduction of DAT expression, thereby influencing neuronal dopamine transport. Moreover, the regulation of miR-137 and miR-491 on this transport disappeared after the DAT was silenced. The miR-491 seed region that is located on the VNTR sequence in the 3'UTR of the DAT and the regulatory effect of miR-491 on the DAT depended on the VNTR copy-number. These data indicate that miR-137 and miR-491 regulate DAT expression and dopamine transport at the post-transcriptional level, suggesting that microRNA may be targeted for the treatment of diseases associated with DAT dysfunction.
Topics: Cell Line, Tumor; Computational Biology; Dopamine; Dopamine Plasma Membrane Transport Proteins; Gene Expression Regulation, Neoplastic; Green Fluorescent Proteins; Humans; MicroRNAs; Minisatellite Repeats; Neuroblastoma; RNA, Messenger; RNA, Small Interfering; Time Factors; Transfection
PubMed: 27628529
DOI: 10.1007/s12264-016-0061-6 -
BMC Genetics May 2013The Belgian Malinois dog breed (MAL) is frequently used in law enforcement and military environments. Owners have reported seizures and unpredictable behavioral changes...
BACKGROUND
The Belgian Malinois dog breed (MAL) is frequently used in law enforcement and military environments. Owners have reported seizures and unpredictable behavioral changes including dogs' eyes "glazing over," dogs' lack of response to environmental stimuli, and loss of behavioral inhibition including owner-directed biting behavior. Dogs with severe behavioral changes may be euthanized as they can represent a danger to humans and other dogs. In the dog, the dopamine transporter gene (DAT) contains a 38-base pair variable number tandem repeat (DAT-VNTR); alleles have either one or two copies of the 38-base pair sequence. The objective of this study was to assess frequency of DAT-VNTR alleles, and characterize the association between DAT-VNTR alleles and behavior in MAL and other breeds.
RESULTS
In an American sample of 280 dogs comprising 26 breeds, most breeds are predominantly homozygous for the DAT-VNTR two-tandem-repeat allele (2/2). The one-tandem-repeat allele is over-represented in American MAL (AM-MAL) (n = 144), both as heterozygotes (1/2) and homozygotes (1/1). All AM-MAL with reported seizures (n = 5) were 1/1 genotype. For AM-MAL with at least one "1" allele (1/1 or 1/2 genotype, n = 121), owners reported higher levels of attention, increased frequency of episodic aggression, and increased frequency of loss of responsiveness to environmental stimuli. In behavior observations, Belgian Military Working Dogs (MWD) with 1/1 or 1/2 genotypes displayed fewer distracted behaviors and more stress-related behaviors such as lower posture and increased yawning. Handlers' treatment of MWD varied with DAT-VNTR genotype as did dogs' responses to handlers' behavior. For 1/1 or 1/2 genotype MWD, 1) lower posture after the first aversive stimulus given by handlers was associated with poorer obedience performance; 2) increased aversive stimuli during protection exercises were associated with decreased performance; 3) more aversive stimuli during obedience were associated with more aversive stimuli during protection; and 4) handlers used more aversive stimuli in protection compared with obedience exercises.
CONCLUSIONS
The single copy allele of DAT-VNTR is associated with owner-reported seizures, loss of responsiveness to environmental stimuli, episodic aggression, and hyper-vigilance in MAL. Behavioral changes are associated with differential treatment by handlers. Findings should be considered preliminary until replicated in a larger sample.
Topics: Animals; Behavior, Animal; Dogs; Dopamine Plasma Membrane Transport Proteins; Minisatellite Repeats; Polymorphism, Genetic
PubMed: 23718893
DOI: 10.1186/1471-2156-14-45 -
Journal of Neurochemistry Jul 2012Midbrain dopaminergic neurons are implicated in various neurological and psychiatric diseases as well as drug addiction. Thus, the study of their generation and...
Midbrain dopaminergic neurons are implicated in various neurological and psychiatric diseases as well as drug addiction. Thus, the study of their generation and maintenance is pivotal to further our understanding of these disease-underlying mechanisms and development of novel therapeutics. Here, using an embryonic stem cell in vitro differentiation system and mutant dreher mouse, we showed that Lmx1a, an early regulator of midbrain dopamine neural progenitor phenotype specification, is also involved in the regulation of midbrain dopaminergic maturation by regulating gene expression of the dopamine transporter. Forced expression of Lmx1a induced dopamine transporter expression precociously in immature dopaminergic neurons, accompanied by significant increase in specific dopamine uptake. Lmx1a binds to well-conserved sequences in the dopamine transporter promoter region, and this binding sequence directs Lmx1a-dependent activation of reporter gene expression. Furthermore, during mouse embryonic development, dopamine transporter was more severely affected by Lmx1a mutation compared to other dopamine markers such as tyrosine hydroxylase and dopa decarboxylase, again supporting the role of Lmx1a in midbrain dopaminergic maturation in vivo. Thus, this study demonstrates that dopamine transporter is a direct target of Lmx1a and emphasizes a novel role of Lmx1a as one of regulators of mature midbrain dopaminergic neurotransmitter phenotypes.
Topics: Animals; Blastocyst; Cell Differentiation; Chromatin Immunoprecipitation; DNA Primers; Dopamine; Dopamine Plasma Membrane Transport Proteins; Embryonic Development; Embryonic Stem Cells; Female; Gene Expression Regulation; Genes, Reporter; Immunohistochemistry; LIM-Homeodomain Proteins; Luciferases; Mice; Mice, Inbred C57BL; Mutation; Polymerase Chain Reaction; Pregnancy; Real-Time Polymerase Chain Reaction; Transcription Factors
PubMed: 22564125
DOI: 10.1111/j.1471-4159.2012.07779.x