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Neurochemical Research Jun 2020Dopamine (DA) is critical for motivation, reward, movement initiation, and learning. Mechanisms that control DA signaling have a profound impact on these important... (Review)
Review
Dopamine (DA) is critical for motivation, reward, movement initiation, and learning. Mechanisms that control DA signaling have a profound impact on these important behaviors, and additionally play a role in DA-related neuropathologies. The presynaptic SLC6 DA transporter (DAT) limits extracellular DA levels by clearing released DA, and is potently inhibited by addictive and therapeutic psychostimulants. Decades of evidence support that the DAT is subject to acute regulation by a number of signaling pathways, and that endocytic trafficking strongly regulates DAT availability and function. DAT trafficking studies have been performed in a variety of model systems, including both in vitro and ex vivo preparations. In this review, we focus on the breadth of DAT trafficking studies, with specific attention to, and comparison of, how context may influence DAT's response to different stimuli. In particular, this overview highlights that stimulated DAT trafficking not only differs between in vitro and ex vivo environments, but also is influenced by both sex and anatomical subregions.
Topics: Animals; Brain; Dopamine; Dopamine Plasma Membrane Transport Proteins; GABA Plasma Membrane Transport Proteins; Humans; Protein Transport; Signal Transduction
PubMed: 32146647
DOI: 10.1007/s11064-020-03001-6 -
The Journal of Pharmacology and... Aug 2023The alkylamine stimulant 1,3-dimethylamylamine (DMAA) is used nonmedically as an appetite suppressant and exercise performance enhancer despite adverse cardiovascular...
The alkylamine stimulant 1,3-dimethylamylamine (DMAA) is used nonmedically as an appetite suppressant and exercise performance enhancer despite adverse cardiovascular effects that have limited its legal status. There is scant research describing the mechanism of action of DMAA, making it difficult to gauge risks or therapeutic potential. An important molecular target of structurally related phenethylamines, such as amphetamine, for regulating mood, cognition, movement, and the development of substance use disorder is the dopamine transporter, which limits the range and magnitude of dopamine signaling via reuptake from the extracellular space. The present studies were therefore initiated to characterize the effects of DMAA on dopamine transporter function. Specifically, we tested the hypothesis that DMAA exhibits substrate-like effects on dopamine transporter function and trafficking. In transport assays in human embryonic kidney cells, DMAA inhibited dopamine uptake by the human dopamine transporter in a competitive manner. Docking analysis and molecular dynamics simulations supported these findings, revealing that DMAA binds to the S1 substrate binding site and induces a conformational change from outward-facing open to outward-facing closed states, similar to the known substrates. Further supporting substrate-like effects of DMAA, the drug stimulated dopamine transporter endocytosis in a heterologous expression system via cocaine- and protein kinase A-sensitive mechanisms, mirroring findings with amphetamine. Together, these data indicate that DMAA elicits neurologic effects by binding to and regulating function of the dopamine transporter. Furthermore, pharmacologic distinctions from amphetamine reveal structural determinants for regulating transporter conformation and add mechanistic insight for the regulation of dopamine transporter endocytosis. SIGNIFICANCE STATEMENT: The alkylamine stimulant 1,3-dimethylamylamine (DMAA) is used as an appetite suppressant and athletic performance enhancer and is structurally similar to amphetamine, but there is scant research describing its mechanism of action. Characterizing the effects of DMAA on dopamine transporter function supports evaluation of potential risks and therapeutic potential while also revealing mechanistic details of dynamic transporter-substrate interactions.
Topics: Humans; Dopamine Plasma Membrane Transport Proteins; Appetite Depressants; Dopamine; Cocaine; Amphetamine; Central Nervous System Agents
PubMed: 37348963
DOI: 10.1124/jpet.122.001573 -
Journal of Chemical Information and... Jul 2023Human dopamine transporter (hDAT) regulates the reuptake of extracellular dopamine (DA) and is an essential therapeutic target for central nervous system (CNS) diseases....
Human dopamine transporter (hDAT) regulates the reuptake of extracellular dopamine (DA) and is an essential therapeutic target for central nervous system (CNS) diseases. The allosteric modulation of hDAT has been identified for decades. However, the molecular mechanism underlying the transportation is still elusive, which hinders the rational design of allosteric modulators against hDAT. Here, a systematic structure-based method was performed to explore allosteric sites on hDAT in inward-open (IO) conformation and to screen compounds with allosteric affinity. First, the model of the hDAT structure was constructed based on the recently reported Cryo-EM structure of the human serotonin transporter (hSERT) and Gaussian-accelerated molecular dynamics (GaMD) simulation was further utilized for the identification of intermediate energetic stable states of the transporter. Then, with the potential druggable allosteric site on hDAT in IO conformation, virtual screening of seven enamine chemical libraries (∼440,000 compounds) was processed, resulting in 10 compounds being purchased for assay and with Z1078601926 discovered to allosterically inhibit hDAT (IC = 0.527 [0.284; 0.988] μM) when nomifensine was introduced as an orthosteric ligand. Finally, the synergistic effect underlying the allosteric inhibition of hDAT by Z1078601926 and nomifensine was explored using additional GaMD simulation and postbinding free energy analysis. The hit compound discovered in this work not only provides a good starting point for lead optimization but also demonstrates the usability of the method for the structure-based discovery of novel allosteric modulators of other therapeutic targets.
Topics: Humans; Dopamine Plasma Membrane Transport Proteins; Nomifensine; Molecular Dynamics Simulation; Allosteric Site; Ligands
PubMed: 37410882
DOI: 10.1021/acs.jcim.3c00477 -
Journal of Nuclear Medicine Technology Jun 2020Racial and ethnic disparities in the prevalence of neurodegenerative diseases exist. This study examined the agreement between gold standard diagnosis and visual... (Comparative Study)
Comparative Study
Racial and ethnic disparities in the prevalence of neurodegenerative diseases exist. This study examined the agreement between gold standard diagnosis and visual assessment of dopamine transporter (DaT) imaging in Hispanic and non-Hispanic patients being evaluated for Parkinsonian syndromes (PS). A retrospective review of DaT imaging and demographic data was performed with institutional review board approval. Documented interpretation by visual assessment was used to classify scans as normal or abnormal. The gold standard for the final diagnosis of PS was determined by a neurologist after 2 or more years of clinical follow-up. Data were analyzed with a -test for uncorrelated samples. In 30 Hispanic patients, DaT imaging was abnormal in 17, normal in 12, and nondiagnostic in 1. Of those with abnormal imaging, PS was confirmed in 16 of 17. Of those with normal imaging, no PS was confirmed in any patient. Sensitivity was 100%, and specificity was 92%. The single patient with nondiagnostic imaging was excluded. Of 77 non-Hispanic patients, visual assessment of DaT imaging was abnormal in 51. Of those with abnormal imaging, PS was confirmed in 48 of 51. Of those with normal imaging, no PS was confirmed in 22 of 26. Sensitivity was 92%, and specificity was 88%. There was no statistically significant difference ( = 0.34) in the rates of agreement between the gold standard and DaT imaging in Hispanic versus non-Hispanic patients. The study sample size afforded a power of 0.60. No significant difference was found in the accuracy of DaT imaging between Hispanic and non-Hispanic patients. Accuracy was high for both groups.
Topics: Adult; Aged; Aged, 80 and over; Diagnostic Imaging; Dopamine Plasma Membrane Transport Proteins; Female; Hispanic or Latino; Humans; Iodine Radioisotopes; Male; Middle Aged; Parkinsonian Disorders; Retrospective Studies
PubMed: 31604897
DOI: 10.2967/jnmt.119.231423 -
Psychiatry Research. Neuroimaging Nov 2020Drug dependence associated with increased dopamine neurotransmission and neuroplastic changes is influenced by Dopamine transporters (DAT) which are modulated by genetic...
Drug dependence associated with increased dopamine neurotransmission and neuroplastic changes is influenced by Dopamine transporters (DAT) which are modulated by genetic and epigenetic factors. This study assesses DAT availability in relation to the 40bp DAT1 VNTR (genetic) and DAT1 promoter methylation (epigenetic) changes in patients with alcohol dependence (AD) and opioid dependence (OD). A total of 60 subjects (n=20 each of AD, OD and controls) were recruited. SPECT/CT imaging using Tc-TRODAT-1 was performed for measuring striatal DAT availability and DNA screened to check DAT1promoter methylation and 40bp VNTR polymorphism. SPECT/CT imaging revealed significant decrease in DAT availability in the striatum and putamen and significant increase in DAT1 promoter methylation in AD compared to control and OD. The 40bp VNTR distribution was similar in all three groups with 10repeat and 9repeat alleles being the most common. The AD individuals with DAT1promoter methylation showed significantly lower TRODAT-1 uptake compared to the ones with no methylation. AD individuals homozygous for the 10repeat VNTR also showed reduced DAT availability. This is the first imaging study using Tc-TRODAT-1 from India documenting significantly reduced striatal DAT availability, increased DAT methylation and frequency of 10repeat individuals associated with decreased DAT availability in AD.
Topics: Alcoholism; Corpus Striatum; DNA Methylation; Dopamine Plasma Membrane Transport Proteins; Genetic Association Studies; Humans; India; Minisatellite Repeats; Opioid-Related Disorders; Organotechnetium Compounds; Polymorphism, Genetic; Promoter Regions, Genetic; Putamen; Tomography, Emission-Computed, Single-Photon; Tropanes
PubMed: 32947183
DOI: 10.1016/j.pscychresns.2020.111187 -
Gynecological Endocrinology : the... Jul 2022Worldwide, approximately 10-15% of couples is affected by infertility and infertility is associated with disturbances in female and/or male reproductive systems. The aim...
OBJECTIVE
Worldwide, approximately 10-15% of couples is affected by infertility and infertility is associated with disturbances in female and/or male reproductive systems. The aim of the current study is to investigate the relationship between DAT1 (SLC6A3) VNTR polymorphism with female infertility.
METHODS
Genomic DNA extractions were performed in 98 fertile and 90 infertile females, 3' untranslated region (3' UTR) variable number tandem repeat (VNTR) polymorphism of DAT1/SLC6A3 was determined by the use of Polymerase Chain Reaction (PCR) method.
RESULTS
It has been demonstrated that there was no statistically significant difference between female infertile and fertile groups in the terms of DAT1 genotypes ( > .05). Moreover, there was no significant difference regarding the frequencies of 9R and 10R alleles in infertile and fertile groups ( > .05).
CONCLUSION
This is the first study for investigating the relationship between DAT1/SLC6A3 gene polymorphism and infertility in females. Our study contributes to the growing awareness of the relationship between dopaminergic system and female infertility despite no significant differences were reported between infertile females and corresponding fertile subjects in DAT1/SLC6A3 gene.
Topics: 3' Untranslated Regions; Dopamine Plasma Membrane Transport Proteins; Female; Genotype; Humans; Infertility, Female; Male; Minisatellite Repeats; Polymorphism, Genetic; Turkey
PubMed: 35536100
DOI: 10.1080/09513590.2022.2073992 -
Neuroscience and Biobehavioral Reviews Jan 2016The gene coding for the dopamine transporter (DAT), SLC6A3, contains a 40-base pair variable number of tandem repeats (VNTR) polymorphism (rs28363170) in its 3'... (Meta-Analysis)
Meta-Analysis Review
The gene coding for the dopamine transporter (DAT), SLC6A3, contains a 40-base pair variable number of tandem repeats (VNTR) polymorphism (rs28363170) in its 3' untranslated region. This VNTR has been associated with attention deficit hyperactivity disorder (ADHD) and has been investigated in relation to cognition and brain function. Here, we report the results of a comprehensive meta-analysis with meta-regression examining the association of the VNTR with different domains of cognition in healthy adults. We extracted data from 28 independent studies and carried out meta-analyses for associations with working memory (k=10 samples, N=1193 subjects), inhibition (k=8 samples, N=829 subjects), executive functions including inhibition (k=10 samples, N=984 subjects), attention (k=6 samples, N=742 subjects) and declarative long-term memory (k=5 samples, N=251 subjects). None of the investigated dimensions showed significant associations with the VNTR (all p>0.26). Meta-regression including year of publication, gender, age, ethnicity and percentage of 10R-homozygotes similarly did not attain significance. We conclude that there is no evidence that rs28363170 may be a significant predictor of cognitive function in healthy adults.
Topics: Cognition; Dopamine Plasma Membrane Transport Proteins; Humans; Minisatellite Repeats; Polymorphism, Genetic
PubMed: 26593110
DOI: 10.1016/j.neubiorev.2015.09.021 -
ACS Chemical Neuroscience Jun 2019The dopamine transporter (DAT) is a plasma membrane protein that mediates the reuptake of extracellular dopamine (DA) and controls the spatiotemporal dynamics of...
The dopamine transporter (DAT) is a plasma membrane protein that mediates the reuptake of extracellular dopamine (DA) and controls the spatiotemporal dynamics of dopaminergic neurotransmission. The transporter is subject to fine control that tailors clearance of transmitter to physiological demands, and dysregulation of reuptake induced by psychostimulant drugs, transporter polymorphisms, and signaling defects may impact transmitter tone in disease states. We previously demonstrated that DAT undergoes complex regulation by palmitoylation, with acute inhibition of the modification leading to rapid reduction of transport activity and sustained inhibition of the modification leading to transporter degradation and reduced expression. Here, to examine mechanisms and outcomes related to increased modification, we coexpressed DAT with palmitoyl acyltransferases (PATs), also known as DHHC enzymes, which catalyze palmitate addition to proteins. Of 12 PATs tested, DAT palmitoylation was stimulated by DHHC2, DHHC3, DHHC8, DHHC15, and DHHC17, with others having no effect. Increased modification was localized to previously identified palmitoylation site Cys580 and resulted in upregulation of transport kinetics and elevated transporter expression mediated by reduced degradation. These findings confirm palmitoylation as a regulator of multiple DAT properties crucial for appropriate DA homeostasis and identify several potential PAT pathways linked to these effects. Defects in palmitoylation processes thus represent possible mechanisms of transport imbalances in DA disorders.
Topics: Acyltransferases; Animals; Dopamine; Dopamine Plasma Membrane Transport Proteins; Lipoylation; Protein Stability; Rats; Synaptic Transmission
PubMed: 30965003
DOI: 10.1021/acschemneuro.8b00558 -
Advances in Pharmacology (San Diego,... 2018Amphetamines (AMPHs) are potent psychostimulants that are widely used and abused, with profound medical and societal impact. Their actions at dopaminergic neurons are... (Review)
Review
Amphetamines (AMPHs) are potent psychostimulants that are widely used and abused, with profound medical and societal impact. Their actions at dopaminergic neurons are thought to mediate their therapeutic efficacy as well as their liability for abuse and dependence. AMPHs target the dopamine transporter (DAT), the plasmalemmal membrane protein that mediates the inactivation of released dopamine (DA) through its reuptake. AMPHs act as substrates for DAT and are known to cause mobilization of dopamine (DA) to the cell exterior via DAT-mediated reverse transport (efflux). It has become increasingly evident that the mechanisms that regulate AMPH-induced DA efflux are distinct from those that regulate DA uptake. Central to these mechanisms is the phosphorylation of the DAT amino (N)-terminus, which has been repeatedly demonstrated to facilitate DAT-mediated DA efflux, without impacting other aspects of DAT physiology. This review aims to summarize the current status of knowledge regarding DAT N-terminal phosphorylation and its regulation by protein modulators and the membrane microenvironment. A better understanding of these mechanisms may lead to the identification of novel therapeutic approaches that interfere selectively with the pharmacological effects of AMPHs without altering the physiological function of DAT.
Topics: Amphetamine; Animals; Cell Membrane; Cellular Microenvironment; Dopamine; Dopamine Plasma Membrane Transport Proteins; Humans; Phosphorylation
PubMed: 29413521
DOI: 10.1016/bs.apha.2017.09.002 -
Neuropharmacology Dec 2019Recent work demonstrated the propensity of dopamine transporters (DATs) to form trimers or higher oligomers, enhanced upon binding a furopyrimidine, AIM-100. AIM-100...
Recent work demonstrated the propensity of dopamine transporters (DATs) to form trimers or higher oligomers, enhanced upon binding a furopyrimidine, AIM-100. AIM-100 binding promotes DAT endocytosis and thereby moderates dopaminergic transmission. Despite the neurobiological significance of these events, the molecular mechanisms that underlie the stabilization of DAT trimer and the key interactions that modulate the trimerization of DAT, and not serotonin transporter SERT, remain unclear. In the present study, we determined three structural models, termed trimer-W238, -C306 and -Y303, for possible trimerization of DATs . To this aim, we used structural data resolved for DAT and its structural homologs that share the LeuT fold, advanced computational modeling and simulations, site-directed mutagenesis experiments and live-cell imaging assays. The models are in accord with the versatility of LeuT fold to stabilize dimeric or higher order constructs. Selected residues show a high propensity to occupy interfacial regions. Among them, D231-W238 in the extracellular loop EL2, including the intersubunit salt-bridge forming pair D231/D232-R237 (not present in SERT) (in trimer-W238), the loop EL3 (trimers-C306 and -Y303), and W497 on the intracellularly exposed IL5 loop (trimer-C306) and its spatial neighbors (e.g. K525) near the C-terminus are computationally predicted and experimentally confirmed to play important roles in enabling the correct folding and/or oligomerization of DATs in the presence of AIM-100. The study suggests the possibility of controlling the effective transport of dopamine by altering the oligomerization state of DAT upon small molecule binding, as a possible intervention strategy to modulate dopaminergic signaling. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.
Topics: Binding Sites; Cells, Cultured; Computer Simulation; Dopamine Plasma Membrane Transport Proteins; Furans; Humans; Models, Molecular; Molecular Docking Simulation; Molecular Dynamics Simulation; Mutagenesis, Site-Directed; Protein Binding; Protein Folding; Pyrimidines; Serotonin Plasma Membrane Transport Proteins
PubMed: 31228486
DOI: 10.1016/j.neuropharm.2019.107676