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Nature Communications Jun 2019The dopamine transporter is a member of the neurotransmitter:sodium symporters (NSSs), which are responsible for termination of neurotransmission through Na-driven...
The dopamine transporter is a member of the neurotransmitter:sodium symporters (NSSs), which are responsible for termination of neurotransmission through Na-driven reuptake of neurotransmitter from the extracellular space. Experimental evidence elucidating the coordinated conformational rearrangements related to the transport mechanism has so far been limited. Here we probe the global Na- and dopamine-induced conformational dynamics of the wild-type Drosophila melanogaster dopamine transporter using hydrogen-deuterium exchange mass spectrometry. We identify Na- and dopamine-induced changes in specific regions of the transporter, suggesting their involvement in protein conformational transitions. Furthermore, we detect ligand-dependent slow cooperative fluctuations of helical stretches in several domains of the transporter, which could be a molecular mechanism that assists in the transporter function. Our results provide a framework for understanding the molecular mechanism underlying the function of NSSs by revealing detailed insight into the state-dependent conformational changes associated with the alternating access model of the dopamine transporter.
Topics: Dopamine; Dopamine Plasma Membrane Transport Proteins; Drosophila Proteins; HEK293 Cells; Humans; Ligands; Mass Spectrometry; Molecular Dynamics Simulation; Protein Conformation, alpha-Helical; Protein Domains; Recombinant Proteins; Sodium
PubMed: 31221956
DOI: 10.1038/s41467-019-10449-w -
The Journal of Biological Chemistry Nov 2015The dopamine transporter is a neuronal protein that drives the presynaptic reuptake of dopamine (DA) and is the major determinant of transmitter availability in the...
The dopamine transporter is a neuronal protein that drives the presynaptic reuptake of dopamine (DA) and is the major determinant of transmitter availability in the brain. Dopamine transporter function is regulated by protein kinase C (PKC) and other signaling pathways through mechanisms that are complex and poorly understood. Here we investigate the role of Ser-7 phosphorylation and Cys-580 palmitoylation in mediating steady-state transport kinetics and PKC-stimulated transport down-regulation. Using both mutational and pharmacological approaches, we demonstrate that these post-translational modifications are reciprocally regulated, leading to transporter populations that display high phosphorylation-low palmitoylation or low phosphorylation-high palmitoylation. The balance between the modifications dictates transport capacity, as conditions that promote high phosphorylation or low palmitoylation reduce transport Vmax and enhance PKC-stimulated down-regulation, whereas conditions that promote low phosphorylation or high palmitoylation increase transport Vmax and suppress PKC-stimulated down-regulation. Transitions between these functional states occur when endocytosis is blocked or undetectable, indicating that the modifications kinetically regulate the velocity of surface transporters. These findings reveal a novel mechanism for control of DA reuptake that may represent a point of dysregulation in DA imbalance disorders.
Topics: Cell Line; Dopamine; Dopamine Plasma Membrane Transport Proteins; Down-Regulation; Endocytosis; Humans; Kinetics; Lipoylation; Protein Kinase C
PubMed: 26424792
DOI: 10.1074/jbc.M115.667055 -
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 -
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 -
No link between striatal dopaminergic axons and dopamine transporter imaging in Parkinson's disease.Movement Disorders : Official Journal... Oct 2019Brain dopamine transporter binding has been considered a possible biomarker for nigrostriatal degeneration in PD.
BACKGROUND
Brain dopamine transporter binding has been considered a possible biomarker for nigrostriatal degeneration in PD.
OBJECTIVE
To investigate whether dopamine transporter binding is associated with the number of dopaminergic neurites in the putamen.
METHODS
Tyrosine hydroxylase-positive nerve fibers were counted from postmortem putamen sections taken from 14 parkinsonism patients who had been scanned with dopamine transporter single-photon emission computed tomography antemortem. Fiber counts were correlated with putamen dopamine transporter binding and SN neuron counts.
RESULTS
The putamen dopamine transporter specific binding ratio did not correlate with the putamen tyrosine hydroxylase-positive axon counts (r = 0.00; P = 1.0; PD patients: r = 0.07; P = 0.86). The nigra neuron counts had a positive correlation with the putamen tyrosine hydroxylase-positive axon counts.
CONCLUSIONS
Striatal dopamine transporter imaging does not associate with axonal nor somal loss of the nigrostriatal neurons in PD. It may reflect dopaminergic activity rather than number of surviving neurons or their striatal projection axons. © 2019 International Parkinson and Movement Disorder Society.
Topics: Aged; Aged, 80 and over; Axons; Corpus Striatum; Dopamine; Dopamine Plasma Membrane Transport Proteins; Female; Humans; Male; Middle Aged; Neurons; Parkinson Disease; Putamen; Substantia Nigra; Tomography, Emission-Computed, Single-Photon
PubMed: 31234224
DOI: 10.1002/mds.27777 -
Neuroscience Jan 2021Recent work indicates a role for RING finger protein 11 (RNF11) in Parkinson disease (PD) pathology, which involves the loss of dopaminergic neurons. However, the role...
Recent work indicates a role for RING finger protein 11 (RNF11) in Parkinson disease (PD) pathology, which involves the loss of dopaminergic neurons. However, the role of RNF11 in regulating dopamine neurotransmission has not been studied. In this work, we tested the effect of RNF11 RNAi knockdown or overexpression on stimulated dopamine release in the larval Drosophila central nervous system. Dopamine release was stimulated using optogenetics and monitored in real-time using fast-scan cyclic voltammetry at an electrode implanted in an isolated ventral nerve cord. RNF11 knockdown doubled dopamine release, but there was no decrease in dopamine from RNF11 overexpression. RNF11 knockdown did not significantly increase stimulated serotonin or octopamine release, indicating the effect is dopamine specific. Dopamine clearance was also changed, as RNF11 RNAi flies had a higher V and RNF11 overexpressing flies had a lower V than control flies. RNF11 RNAi flies had increased mRNA levels of dopamine transporter (DAT) in RNF11, confirming changes in DAT. In RNF11 RNAi flies, release was maintained better for stimulations repeated at short intervals, indicating increases in the recycled releasable pool of dopamine. Nisoxetine, a DAT inhibitor, and flupenthixol, a D2 antagonist, did not affect RNF11 RNAi or overexpressing flies differently than control. Thus, RNF11 knockdown causes early changes in dopamine neurotransmission, and this is the first work to demonstrate that RNF11 affects both dopamine release and uptake. RNF11 expression decreases in human dopaminergic neurons during PD, and that decrease may be protective by increasing dopamine neurotransmission in the surviving dopaminergic neurons.
Topics: Animals; Central Nervous System; DNA-Binding Proteins; Dopamine; Dopamine Plasma Membrane Transport Proteins; Dopaminergic Neurons; Drosophila; Drosophila Proteins; Humans; Serotonin
PubMed: 33176188
DOI: 10.1016/j.neuroscience.2020.10.021 -
Handbook of Experimental Pharmacology 2018Synthetic cathinones are derivatives of the naturally occurring compound cathinone, the main psychoactive ingredient in the khat plant Catha edulis. Cathinone is the...
Synthetic cathinones are derivatives of the naturally occurring compound cathinone, the main psychoactive ingredient in the khat plant Catha edulis. Cathinone is the β-keto analog of amphetamine, and all synthetic cathinones display a β-keto moiety in their structure. Several synthetic cathinones are widely prescribed medications (e.g., bupropion, Wellbutrin), while others are problematic drugs of abuse (e.g., 4-methylmethcathinone, mephedrone). Similar to amphetamines, synthetic cathinones are psychomotor stimulants that exert their effects by impairing the normal function of plasma membrane transporters for dopamine (DAT), norepinephrine (NET), and 5-HT (SERT). Ring-substituted cathinones like mephedrone are transporter substrates that evoke neurotransmitter release by reversing the normal direction of transporter flux (i.e., releasers), whereas pyrrolidine-containing cathinones like 3,4-methylenedioxypyrovalerone (MDPV) are potent transporter inhibitors that block neurotransmitter uptake (i.e., blockers). Regardless of molecular mechanism, all synthetic cathinones increase extracellular monoamine concentrations in the brain, thereby enhancing cell-to-cell monoamine signaling. Here, we briefly review the mechanisms of action, structure-activity relationships, and in vivo pharmacology of synthetic cathinones. Overall, the findings show that certain synthetic cathinones are powerful drugs of abuse that could pose significant risk to users.
Topics: Alkaloids; Central Nervous System Stimulants; Dopamine Plasma Membrane Transport Proteins; Humans; Neuropharmacology; Norepinephrine Plasma Membrane Transport Proteins; Serotonin Antagonists; Structure-Activity Relationship
PubMed: 30406443
DOI: 10.1007/164_2018_178 -
ACS Chemical Neuroscience Jun 2016Amphetamine abuse afflicts over 13 million people, and there is currently no universally accepted treatment for amphetamine addiction. Amphetamine serves as a substrate...
Amphetamine abuse afflicts over 13 million people, and there is currently no universally accepted treatment for amphetamine addiction. Amphetamine serves as a substrate for the dopamine transporter and reverses the transporter to cause an increase in extracellular dopamine. Activation of the beta subunit of protein kinase C (PKCβ) enhances extracellular dopamine in the presence of amphetamine by facilitating the reverse transport of dopamine and internalizing the D2 autoreceptor. We previously demonstrated that PKCβ inhibitors block amphetamine-stimulated dopamine efflux in synaptosomes from rat striatum in vitro. In this study, we utilized in vivo microdialysis in live, behaving rats to assess the effect of the PKCβ inhibitors, enzastaurin and ruboxistaurin, on amphetamine-stimulated locomotion and increases in monoamines and their metabolites. A 30 min perfusion of the nucleus accumbens core with 1 μM enzastaurin or 1 μM ruboxistaurin reduced efflux of dopamine and its metabolite 3-methoxytyramine induced by amphetamine by approximately 50%. The inhibitors also significantly reduced amphetamine-stimulated extracellular levels of norepinephrine. The stimulation of locomotor behavior by amphetamine, measured simultaneously with the analytes, was comparably reduced by the PKCβ inhibitors. Using a stable isotope label retrodialysis procedure, we determined that ruboxistaurin had no effect on basal levels of dopamine, norepinephrine, glutamate, or GABA. In addition, normal uptake function through the dopamine transporter was unaltered by the PKCβ inhibitors, as measured in rat synaptosomes. Our results support the utility of using PKCβ inhibitors to reduce the effects of amphetamine.
Topics: Amphetamine; Animals; Autoreceptors; Corpus Striatum; Dopamine; Dopamine Plasma Membrane Transport Proteins; Dopamine Uptake Inhibitors; Male; Microdialysis; Neostriatum; Protein Kinase C beta; Protein Kinase Inhibitors; Rats, Sprague-Dawley
PubMed: 26996926
DOI: 10.1021/acschemneuro.6b00028 -
JCI Insight Sep 2021Dysfunctional dopaminergic neurotransmission is central to movement disorders and mental diseases. The dopamine transporter (DAT) regulates extracellular dopamine...
Dysfunctional dopaminergic neurotransmission is central to movement disorders and mental diseases. The dopamine transporter (DAT) regulates extracellular dopamine levels, but the genetic and mechanistic link between DAT function and dopamine-related pathologies is not clear. Particularly, the pathophysiological significance of monoallelic missense mutations in DAT is unknown. Here, we use clinical information, neuroimaging, and large-scale exome-sequencing data to uncover the occurrence and phenotypic spectrum of a DAT coding variant, DAT-K619N, which localizes to the critical C-terminal PSD-95/Discs-large/ZO-1 homology-binding motif of human DAT (hDAT). We identified the rare but recurrent hDAT-K619N variant in exome-sequenced samples of patients with neuropsychiatric diseases and a patient with early-onset neurodegenerative parkinsonism and comorbid neuropsychiatric disease. In cell cultures, hDAT-K619N displayed reduced uptake capacity, decreased surface expression, and accelerated turnover. Unilateral expression in mouse nigrostriatal neurons revealed differential effects of hDAT-K619N and hDAT-WT on dopamine-directed behaviors, and hDAT-K619N expression in Drosophila led to impairments in dopamine transmission with accompanying hyperlocomotion and age-dependent disturbances of the negative geotactic response. Moreover, cellular studies and viral expression of hDAT-K619N in mice demonstrated a dominant-negative effect of the hDAT-K619N mutant. Summarized, our results suggest that hDAT-K619N can effectuate dopamine dysfunction of pathological relevance in a dominant-negative manner.
Topics: Adult; Animals; Behavior, Animal; Biological Transport; Cells, Cultured; Databases, Genetic; Dopamine; Dopamine Plasma Membrane Transport Proteins; Drosophila; Exome; Female; Humans; Hypokinesia; Male; Mental Disorders; Mesencephalon; Mice; Middle Aged; Motor Activity; Mutation; Neurons; Parkinsonian Disorders; Phenotype; Synaptic Transmission; Tomography, Emission-Computed, Single-Photon; Transfection
PubMed: 34375312
DOI: 10.1172/jci.insight.151496 -
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