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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 -
PloS One 2013Dopamine regulates the psychomotor stimulant activities of amphetamine-like substances in the brain. The effects of dopamine are mediated through five known dopamine...
Dopamine regulates the psychomotor stimulant activities of amphetamine-like substances in the brain. The effects of dopamine are mediated through five known dopamine receptor subtypes in mammals. The functional relevance of D5 dopamine receptors in the central nervous system is not well understood. To determine the functional relevance of D5 dopamine receptors, we created D5 dopamine receptor-deficient mice and then used these mice to assess the roles of D5 dopamine receptors in the behavioral response to methamphetamine. Interestingly, D5 dopamine receptor-deficient mice displayed increased ambulation in response to methamphetamine. Furthermore, dopamine transporter threonine phosphorylation levels, which regulate amphetamine-induced dopamine release, were elevated in D5 dopamine receptor-deficient mice. The increase in methamphetamine-induced locomotor activity was eliminated by pretreatment with the dopamine transporter blocker GBR12909. Taken together, these results suggest that dopamine transporter activity and threonine phosphorylation levels are regulated by D5 dopamine receptors.
Topics: Animals; Dopamine; Dopamine Plasma Membrane Transport Proteins; Locomotion; Methamphetamine; Mice; Mice, Inbred C57BL; Mice, Knockout; Phosphorylation; Receptors, Dopamine D5
PubMed: 24155877
DOI: 10.1371/journal.pone.0075975 -
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 -
ACS Chemical Neuroscience May 2022Fast-scan cyclic voltammetry (FSCV) is an effective tool for measuring dopamine release and clearance throughout the brain, especially the striatum where dopamine...
Fast-scan cyclic voltammetry (FSCV) is an effective tool for measuring dopamine release and clearance throughout the brain, especially the striatum where dopamine terminals are abundant and signals are heavily regulated by release machinery and the dopamine transporter (DAT). Peak height measurement is perhaps the most common method for measuring dopamine release, but it is influenced by changes in clearance. Michaelis-Menten-based modeling has been a standard in measuring dopamine clearance, but it is problematic in that it requires experimenter fitted modeling subject to experimenter bias. This study presents the use of the first derivative (velocity) of evoked dopamine signals as an alternative approach for measuring and distinguishing dopamine release from clearance. Maximal upward velocity predicts reductions in dopamine peak height due to D and GABA receptor stimulation and by alterations in calcium concentrations. The Michaelis-Menten maximal velocity () measure, an approximation for DAT levels, predicts maximal downward velocity in slices and in vivo. Dopamine peak height and upward velocity were similar between wild-type and DAT knock-out (DATKO) mice. In contrast, downward velocity was lower and exponential decay (tau) was higher in DATKO mice, supporting the use of both measures for extreme changes in DAT activity. In slices, the competitive DAT inhibitors cocaine, PTT, and WF23 increased peak height and upward velocity differentially across increasing concentrations, with PTT and cocaine reducing these measures at high concentrations. Downward velocity and tau values decreased and increased respectively across concentrations, with greater potency and efficacy observed with WF23 and PTT. In vivo recordings demonstrated similar effects of WF23, PTT, and cocaine on measures of release and clearance. Tau was a more sensitive measure at low concentrations, supporting its use as a surrogate for the Michaelis-Menten measure of apparent affinity (). Together, these results inform on the use of these various measures for dopamine release and clearance.
Topics: Animals; Cocaine; Corpus Striatum; Dopamine; Dopamine Plasma Membrane Transport Proteins; Dopamine Uptake Inhibitors; Mice; Rats; Rats, Sprague-Dawley
PubMed: 35482592
DOI: 10.1021/acschemneuro.2c00033 -
The Journal of Neuroscience : the... Feb 2017Midbrain dopamine neuron dysfunction contributes to various psychiatric and neurological diseases, including drug addiction and Parkinson's disease. Because of its well...
Midbrain dopamine neuron dysfunction contributes to various psychiatric and neurological diseases, including drug addiction and Parkinson's disease. Because of its well established dopaminotrophic effects, the therapeutic potential of glial cell line-derived neurotrophic factor (GDNF) has been studied extensively in various disorders with disturbed dopamine homeostasis. However, the outcomes from preclinical and clinical studies vary, highlighting a need for a better understanding of the physiological role of GDNF on striatal dopaminergic function. Nevertheless, the current lack of appropriate animal models has limited this understanding. Therefore, we have generated novel mouse models to study conditional deletion in the CNS during embryonic development and reduction of striatal GDNF levels in adult mice via AAV-Cre delivery. We found that both of these mice have reduced amphetamine-induced locomotor response and striatal dopamine efflux. Embryonic GDNF deletion in the CNS did not affect striatal dopamine levels or dopamine release, but dopamine reuptake was increased due to increased levels of both total and synaptic membrane-associated dopamine transporters. Collectively, these results suggest that endogenous GDNF plays an important role in regulating the function of dopamine transporters in the striatum. Delivery of ectopic glial cell line-derived neurotrophic factor (GDNF) promotes the function, plasticity, and survival of midbrain dopaminergic neurons, the dysfunction of which contributes to various neurological and psychiatric diseases. However, how the deletion or reduction of GDNF in the CNS affects the function of dopaminergic neurons has remained unknown. Using conditional knock-out mice, we found that endogenous GDNF affects striatal dopamine homeostasis and regulates amphetamine-induced behaviors by regulating the level and function of dopamine transporters. These data regarding the physiological role of GDNF are relevant in the context of neurological and neurodegenerative diseases that involve changes in dopamine transporter function.
Topics: Amphetamine; Animals; Brain; Dopamine Plasma Membrane Transport Proteins; Dopamine Uptake Inhibitors; Glial Cell Line-Derived Neurotrophic Factor; Male; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; Random Allocation
PubMed: 28096470
DOI: 10.1523/JNEUROSCI.1673-16.2016 -
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 -
Journal of Nuclear Medicine : Official... Aug 2013This article gives an update on nigrostriatal dopamine terminal imaging, with emphasis on SPECT performed with the presynaptic dopamine transporter (DAT) ligand... (Review)
Review
This article gives an update on nigrostriatal dopamine terminal imaging, with emphasis on SPECT performed with the presynaptic dopamine transporter (DAT) ligand (123)I-FP-CIT. The paper covers the rational use of this technique in the diagnostic work-up of patients with known or suspected parkinsonian syndromes. In detail, it addresses the impact of the method for the proof or exclusion of neurodegenerative parkinsonism, for its early and preclinical diagnosis, and for the evaluation of disease progression. The importance of normal DAT binding for differentiating symptomatic parkinsonism and relevant tremor syndromes from neurodegeneration is highlighted. Particularly emphasized is the role of DAT SPECT for diagnosing Lewy body dementia and its separation from Alzheimer dementia. Finally, some remarks deal with the economic aspects of the use of these imaging techniques in the clinical setting.
Topics: Animals; Brain; Dopamine; Dopamine Plasma Membrane Transport Proteins; Humans; Neurodegenerative Diseases; Tomography, Emission-Computed, Single-Photon
PubMed: 23864718
DOI: 10.2967/jnumed.112.105379 -
PloS One 2015Dopamine plays an important role in the development of alcohol dependence, cognitive dysfunction, and is regulated via dopamine transporter activity. Although dopamine...
Dopamine plays an important role in the development of alcohol dependence, cognitive dysfunction, and is regulated via dopamine transporter activity. Although dopamine transporter activity is critically involved in alcohol dependence, studies observing this relationship are limited. Thus the current study examined whether dopamine transporter availability is associated with developing of alcohol dependence and cognitive dysfunction. Brain imaging with 99mTc-TRODAT-1 as a ligand was used to measure dopamine transporter availability among 26 male patients with pure alcohol dependence and 22 age- and sex- matched healthy volunteers. The Wisconsin Card Sorting Test (WCST) and Tridimensional Personality Questionnaire (TPQ) were administered to assess neurocognitive functioning and personality traits, respectively. Compared to healthy controls, patients with alcohol dependence showed a significant reduction in dopamine transporter availability (p < 0.001), as well as diminished performance on the WCST (p < 0.001). Dopamine transporter availability was negatively correlated with both total and perseverative WCST errors among healthy controls, but only patients with alcohol dependence showed a positive correlation between dopamine transporter availability and a harm avoidance personality profile. Thus, reductions in dopamine transporter availability may play a pathophysiological role in the development of pure alcohol dependence, given its association with neurocognitive deficits. Moreover, personality may influence the development of pure alcohol dependence; however, additional clinical subgroups should be examined to confirm this possibility.
Topics: Alcoholism; Case-Control Studies; Cognition Disorders; Demography; Dopamine Plasma Membrane Transport Proteins; Humans; Male; Organotechnetium Compounds; Smoking; Statistics, Nonparametric; Tropanes
PubMed: 26120847
DOI: 10.1371/journal.pone.0131017 -
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