Reverse Transport Of Dopamine Research Articles

Amphetamine-induced reverse transport of dopamine does not require cytosolic Ca2+

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 Ca2+-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 Ca2+ levels, we employ the fluorescent Ca2+ sensor jRGECO1a expressed in cultured dopaminergic neurons together with the fluorescent DA sensor GRABDA1H 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 Ca2+ and was unaffected by blockade of voltage-gated calcium channels or chelation of cytosolic Ca2+. The independence of cytosolic Ca2+ was further supported by activation of N-methyl-D-aspartate-type ionotropic glutamate receptors leading to a marked increase in cytosolic Ca2+ without affecting AMPH-induced DA efflux. Curiously, AMPH elicited spontaneous Ca2+ spikes upon blockade of the D2 receptor, suggesting that AMPH can regulate intracellular Ca2+ in an autoreceptor-dependent manner regardless of the apparent independence of Ca2+ for AMPH-induced efflux. We conclude that AMPH-induced DA efflux in dopaminergic neurons does not require cytosolic Ca2+ but is strictly dependent on the concerted action of AMPH on both vesicular monoamine transporter-2 and DAT.

Sigma receptor ligands haloperidol and ifenprodil attenuate hypoxia induced dopamine release in rat striatum

ABSTRACT Objective We aimed to investigate the hypothesis that sigma receptor ligands, haloperidol and ifenprodil, attenuate hypoxia-induced striatal dopamine release in vitro and determine the possible mechanisms. Methods Extracellular concentrations of dopamine were measured using acute brain slices method under hypoxic, aglycemic and ischemic conditions. Sigma receptor ligands haloperidol and ifenprodil attenuate striatal dopamine release induced by hypoxia in contrast to aglycemia and ischemia. To determine the possible contribution of glutamatergic system on this effect, we compared the effect of NMDA receptor antagonist MK-801 and haloperidol in hypoxia induced by Na-K-ATPaz enzyme inhibitor ouabain. Also, we compared the effect of dopamine uptake blocker nomifensine and haloperidol to determine the role of dopamine transporter on this effect. Results Haloperidol and nomifensine almost completely abolish ouabain-induced dopamine release unlike MK-801. Different effects of sigma ligands and glutamate receptor antagonists on the hypoxia and ouabain induced dopamine release show that glutamate receptor blockade is partial involved in inhibitory effect of sigma ligand on dopamine release under hypoxic conditions. Similar effect of dopamine uptake blocker nomifensine and sigma receptor ligand haloperidol on ouabain induced dopamine release supports the possibility that inhibition of reverse dopamine transport by sigma ligands might be involved in their protective effect. Conclusions Data in this study suggest that sigma ligands may be a new therapeutic intervention for the management of hypoxic conditions.

Functional characterization of the biogenic amine transporters on human macrophages.

Monocyte-derived macrophages (MDMs) are key players in tissue homeostasis and diseases regulated by a variety of signaling molecules. Recent literature has highlighted the ability for biogenic amines to regulate macrophage functions, but the mechanisms governing biogenic amine signaling in and around immune cells remain nebulous. In the CNS, biogenic amine transporters are regarded as the master regulators of neurotransmitter signaling. While we and others have shown that macrophages express these transporters, relatively little is known of their function in these cells. To address these knowledge gaps, we investigated the function of norepinephrine transporter (NET) and dopamine transporter (DAT) on human MDMs. We found that both NET and DAT are present and can uptake substrate from the extracellular space at baseline. Not only was DAT expressed in cultured MDMs, but it was also detected in a subset of intestinal macrophages in situ. Surprisingly, we discovered a NET-independent, DAT-mediated immunomodulatory mechanism in response to LPS. LPS induced reverse transport of dopamine through DAT, engaging an autocrine/paracrine signaling loop that regulated the macrophage response. Removing this signaling loop enhanced the proinflammatory response to LPS. Our data introduce a potential role for DAT in the regulation of innate immunity.

The Role of the Dopamine Transporter in the Effects of Amphetamine on Sleep and Sleep Architecture in Drosophila.

The dopamine transporter (DAT) mediates the inactivation of released dopamine (DA) through its reuptake, and thereby plays an important homeostatic role in dopaminergic neurotransmission. Amphetamines exert their stimulant effects by targeting DAT and inducing the reverse transport of DA, leading to a dramatic increase of extracellular DA. Animal models have proven critical to investigating the molecular and cellular mechanisms underlying transporter function and its modulation by psychostimulants such as amphetamine. Here we establish a behavioral model for amphetamine action using adult Drosophila melanogaster. We use it to characterize the effects of amphetamine on sleep and sleep architecture. Our data show that amphetamine induces hyperactivity and disrupts sleep in a DA-dependent manner. Flies that do not express a functional DAT (dDAT null mutants) have been shown to be hyperactive and to exhibit significantly reduced sleep at baseline. Our data show that, in contrast to its action in control flies, amphetamine decreases the locomotoractivity of dDAT null mutants and restores their sleep by modulating distinct aspects of sleep structure. To begin to explore the circuitry involved in the actions of amphetamine on sleep, we also describe the localization of dDAT throughout the fly brain, particularly in neuropils known to regulate sleep. Together, our data establish Drosophila as a robust model for studying the regulatory mechanisms that govern DAT function and psychostimulant action.

Autism-linked dopamine transporter mutation alters striatal dopamine neurotransmission and dopamine-dependent behaviors.

The precise regulation of synaptic dopamine (DA) content by the dopamine transporter (DAT) ensures the phasic nature of the DA signal, which underlies the ability of DA to encode reward prediction error, thereby driving motivation, attention, and behavioral learning. Disruptions to the DA system are implicated in a number of neuropsychiatric disorders, including attention deficit hyperactivity disorder (ADHD) and, more recently, Autism Spectrum Disorder (ASD). An ASD-associated de novo mutation in the SLC6A3 gene resulting in a threonine to methionine substitution at site 356 (DAT T356M) was recently identified and has been shown to drive persistent reverse transport of DA (i.e. anomalous DA efflux) in transfected cells and to drive hyperlocomotion in Drosophila melanogaster. A corresponding mutation in the leucine transporter, a DAT-homologous transporter, promotes an outward-facing transporter conformation upon substrate binding, a conformation possibly underlying anomalous dopamine efflux. Here we investigated in vivo the impact of this ASD-associated mutation on DA signaling and ASD-associated behaviors. We found that mice homozygous for this mutation display impaired striatal DA neurotransmission and altered DA-dependent behaviors that correspond with some of the behavioral phenotypes observed in ASD.

Phosphatidylinositol (4, 5)‐bisphosphate coordinates functional interactions in the dopamine transporter to promote amphetamine behaviors

The psychostimulant amphetamine (AMPH) mainly mediates its pharmacological and behavioral effects by increasing extracellular dopamine (DA) availability. DA homeostasis is maintained by the dopamine transporter (DAT), a presynaptic membrane protein that mediates the high‐affinity reuptake of released DA from the synaptic cleft. We have previously demonstrated that AMPH induces N‐term phosphorylation of the DAT, which leads to transport‐mediated efflux of DA. Furthermore, we have shown that phosphatidylinositol (4, 5)‐bisphosphate (PIP2) directly interacts with the DAT and facilitates AMPH‐induced DA efflux, but is not required for DA uptake. Specially, PIP2 binds DAT through electrostatic interactions with positively charged DAT N‐terminal residues. Disrupting the interactions between DAT and PIP2 or depleting PIP2 diminishes reverse transport (efflux) of DA. Previous studies on the human serotonin transporter show that non‐N‐terminal PIP2 binding sites (within the fourth intracellular loop) are essential for AMPH‐induced reverse transport of serotonin, but not substrate uptake. Together, these data led us to hypothesize that PIP2 coordinates functional interactions between the N‐terminus and the fourth intracellular loop to promote reverse transport of DA. Here, we demonstrate that depletion of PIP2 after DAT N‐terminus phosphorylation does not affect AMPH‐induced DA efflux, suggesting that N‐terminus phosphorylation is the limiting step of reverse transport of DA. Notably, we show that residue R443, which resides in the fourth intracellular loop, also modulates AMPH‐induced DA efflux. Thus, we focus on the role of the interaction between the DAT N‐terminus and R443 in mediating the reverse transport of DA. Furthermore, we translate our molecular discoveries in vivo by using a coordinated genetic and pharmacological approach to study AMPH‐induced behaviors in Drosophila melanogasterSupport or Funding Information1F31MH114316 (JIA), AHA Predoctoral Award (AS), DA035263(AG)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Neurotoxic Dopamine Transporter Substrates Differentially Affect DAT Regulation, Uptake, and Reverse Transport

Synaptic action of the neurotransmitter dopamine (DA) is primarily terminated by reuptake into presynaptic dopaminergic neurons via dopamine transporters (DATs). DATs are integral plasmalemmal proteins whose reuptake function is tightly coordinated by interplay between the post translational modifications phosphorylation and palmitoylation. The psychostimulant amphetamine (AMPH) is a DAT substrate known to empty sequestered DA from vesicles and induce protein kinase C (PKC)‐dependent DAT phosphorylation, resulting in reverse transport of DA (efflux) greatly increased DA signaling. Elevated DA concentrations similar to those produced by AMPH create neurotoxic conditions as DA oxidation produces free radicals and hinders mitochondrial respiration, thus strict spatial and temporal control of cytosolic and synaptic DA is essential for neuronal health. DA induced oxidative stress is an important pathogenic mediator in Parkinson's disease (PD), and animal models of PD are generated by the neurotoxins 1‐methyl‐4‐phenylpyridinium ion (MPP+) and 6‐hydroxydopamine (6OHDA) which selectively target DAT‐expressing neurons, are translocated to the cytosol by DAT, and perturb the oxidative balance within the cell, producing nigrostriatal DA neuron death observed in PD. It is therefore intriguing to investigate DAT‐involved mechanisms in terms of PD etiology as atypical DAT function could contribute to DA oxidative stress and is hypothesized to play a role in other neurological disorders including attention deficit/hyperactivity disorder (ADHD) and addiction. DAT expression occurs around synaptic terminals, along axons, and on dendritic spines, and efflux via DATs located on dendrites in the substantia nigra has been shown to diminish dopaminergic signaling. It is possible, therefore, for aberrant regulation and localization of DA clearance and DAT‐mediated DA efflux to profoundly influence the excitability of neurons and play a significant role in neurodegeneration in the progression of PD. This is even more interesting in the light of recent studies revealing DAT polymorphisms in patients diagnosed with a psychiatric disorder or early onset PD that display an elevated basal DA efflux phenotype. In this study, we are investigating the effect of DAT substrates and PD‐inducing neurotoxins on DAT phosphorylation, palmitoylation, down‐regulation, and reverse transport to better understand aberrant DAT mechanisms which may contribute the onset of PD. Our findings thus far indicate MPP+ and 6OHDA differentially alter DAT function. 6OHDA inhibits basal and attenuates PMA‐induced DAT phosphorylation, does not significantly reduce DAT function, and decreases DA efflux. MPP+ does not change basal or PMA stimulated DAT phosphorylation yet it induces significant DA efflux which may be an additional means by which MPP+ produces neurodegeneration. This MPP+ induced efflux was not blunted by pretreatment with the PKC inhibitor bisindolylmaleimide (BIM). Although AMPH‐induced efflux was blunted after BIM treatment, significant efflux remained suggesting mechanisms independent of PKC mediated DAT phosphorylation for the stimulation of DA efflux.Support or Funding InformationSupported by the National Institute on Drug Abuse Grant DA 031991 JDF, P20 RR017699 (to U.N.D.) from the COBRE program of the National Center for Research Resources, and P20 RR016741 (to U.N.D.) from the INBRE program of the National Center for Research Resources.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In Vitro Assays for the Functional Characterization of the Dopamine Transporter (DAT).

Detailed in this unit are protocols for studying the in vitro uptake of dopamine (DA) as a means for defining the functional characteristics of dopamine transporters. All assays are performed using commercially available cell lines that transiently express the transporter under investigation. The three main assays provided are: a kinetic assay to calculate the affinity (KM ) and maximal velocity (Vmax ) of radiolabeled DA uptake into cells; concentration-response assays to measure the potencies (IC50 /Ki values) of test compounds as transport inhibitors; and an efflux assay to assess the ability and potency (EC50 ) of a ligand to elicit reverse transport of DA accumulated in the cell. Although the methods are described using DAT and its ligands, the same procedure can be employed for studying serotonin and norepinephrine transporters as well. © 2017 by John Wiley & Sons, Inc.

Atypical dopamine efflux caused by 3,4-methylenedioxypyrovalerone (MDPV) via the human dopamine transporter

Synthetic cathinones are similar in chemical structure to amphetamines, and their behavioral effects are associated with enhanced dopaminergic signaling. The past ten years of research on the common constituent of bath salts, MDPV (the synthetic cathinone 3,4-methylenedioxypyrovalerone), has aided the understanding of how synthetic cathinones act at the dopamine (DA) transporter (DAT). Several groups have described the ability of MDPV to block the DAT with high-affinity. In this study, we demonstrate for the first time a new mode of action of MDPV, namely its ability to promote DAT-mediated DA efflux. Using single cell amperometric assays, we determined that low concentrations of MDPV (1nM) can cause reverse transport of DA via DAT. Notably, administration of MDPV leads to hyperlocomotion in Drosophila melanogaster. These data describe further how MDPV acts at the DAT, possibly paving the way for novel treatment strategies for individuals who abuse bath salts.

Combined in Vitro and in Silico Approaches to the Assessment of Stimulant Properties of Novel Psychoactive Substances

Novel psychoactive substances (NPS) are increasingly prevalent world-wide although their pharmacological characteristics are largely unknown; those with stimulant properties, due to interactions with the dopamine transporter (DAT), have addictive potential which their users may not realise. We evaluated the binding of 1-(1-benzofuran-5-yl)-N-methylpropan-2-amine (5-MAPB) to rat striatal DAT by means of quantitative autoradiography with [125I]RTI-121, and the effects of 5-MAPB on electrically-evoked dopamine efflux by fast-cyclic voltammetry in rat brain slices. 5-MAPB displaced [125I]RTI-121 in a concentration-dependent manner, with significant effects at 10 and 30 µM. The voltammetry data suggest that 5-MAPB reduces the rate of dopamine reuptake; while the peak dopamine efflux was not increased, the area under the curve was augmented. 5-MAPB can also cause reverse dopamine transport consistent with stimulant properties, more similar to amphetamine than cocaine. Molecular modelling and docking studies compared the binding site of DAT in complex with 5-MAPB to dopamine, amphetamine, 5-APB, MDMA, cocaine and RTI-121. This structural comparison reveals a binding mode for 5-MAPB found in the primary binding (S1) site, central to transmembrane domains 1, 3, 6 and 8, which overlaps with the binding modes of dopamine, cocaine and its analogues. Atomistic molecular dynamics simulations further show that, when in complex with 5-MAPB, DAT can exhibit conformational transitions that spontaneously isomerize the transporter into inward-facing state, similarly to that observed in dopamine-bound DAT. These novel insights, offered by the combination of computational methods of biophysics with neurobiological procedures, provide structural context for NPS at DAT and relate them with their functional properties at DAT as the molecular target of stimulants.

Combined in vitro and in silico approaches to the assessment of stimulant properties of novel psychoactive substances – The case of the benzofuran 5-MAPB

Novel psychoactive substances (NPS) are increasingly prevalent world-wide although their pharmacological characteristics are largely unknown; those with stimulant properties, due to interactions with the dopamine transporter (DAT), have addictive potential which their users may not realise.We evaluated the binding of 1-(1-benzofuran-5-yl)-N-methylpropan-2-amine (5-MAPB) to rat striatal DAT by means of quantitative autoradiography with [125I]RTI-121, and the effects of 5-MAPB on electrically-evoked dopamine efflux by fast-cyclic voltammetry in rat brain slices. 5-MAPB displaced [125I]RTI-121 in a concentration-dependent manner, with significant effects at 10 and 30μM. The voltammetry data suggest that 5-MAPB reduces the rate of dopamine reuptake; while the peak dopamine efflux was not increased, the area under the curve was augmented. 5-MAPB can also cause reverse dopamine transport consistent with stimulant properties, more similar to amphetamine than cocaine. Molecular modelling and docking studies compared the binding site of DAT in complex with 5-MAPB to dopamine, amphetamine, 5-APB, MDMA, cocaine and RTI-121. This structural comparison reveals a binding mode for 5-MAPB found in the primary binding (S1) site, central to transmembrane domains 1, 3, 6 and 8, which overlaps with the binding modes of dopamine, cocaine and its analogues. Atomistic molecular dynamics simulations further show that, when in complex with 5-MAPB, DAT can exhibit conformational transitions that spontaneously isomerize the transporter into inward-facing state, similarly to that observed in dopamine-bound DAT.These novel insights, offered by the combination of computational methods of biophysics with neurobiological procedures, provide structural context for NPS at DAT and relate them with their functional properties at DAT as the molecular target of stimulants.

Methamphetamine Regulation of Firing Activity of Dopamine Neurons.

Methamphetamine (METH) competes with dopamine uptake, increases dopamine efflux via the dopamine transporter, and affects the excitability of dopamine neurons. Here, we identified an unexpected property of METH on dopamine neuron firing activity. METH transiently increased the spontaneous spike activity of dopamine neurons followed by a progressive reduction of the spontaneous spike activity. METH broadened the action potentials, increased coefficients of variation of the interspike interval, and decreased the amplitude of afterhyperpolarization, which are consistent with changes in the activity of Ca2+-activated potassium (BK) channels. We found that METH decreased the activity of BK channels by stimulating BK-α subunit trafficking. Thus, METH modulation of dopamine neurotransmission and resulting behavioral responses is, in part, due to METH regulation of BK channel activity.

PKCβ Inhibitors Attenuate Amphetamine-Stimulated Dopamine Efflux.

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.

Does reverse transport of dopamine play a role in autism?

genetic variants affecting the reverse transport of DA might influence the risk of common, sporadic forms of autism remains open. It is now widely accepted that rare genetic variants could play an important role in susceptibility to common diseases. In light of Cartier et al.'s results, major resequencing efforts of the SLC6A3 and STX1A genes should be undertaken in large cohorts of individuals with autism, with significant insights into disease biology likely from these results. Another interesting implication of the work by Cartier and colleagues lies in unraveling the therapeutic potential of DA reverse transport in autism. Starting from the observation that rare autism-causing variants are associated with inhibition of reverse transport of DA, it could be hypothesized that psychostimulants may serve as a potential therapeutic aid in autism. Notably, not only have psychostimulants been suggested to be effective for ADHD-like symptoms in autism spectrumdisorderindividuals,buttheprevalenceratesoftheiruseinautism

Rare Autism-Associated Variants Implicate Syntaxin 1 (STX1 R26Q) Phosphorylation and the Dopamine Transporter (hDAT R51W) in Dopamine Neurotransmission and Behaviors

BackgroundSyntaxin 1 (STX1) is a presynaptic plasma membrane protein that coordinates synaptic vesicle fusion. STX1 also regulates the function of neurotransmitter transporters, including the dopamine (DA) transporter (DAT). The DAT is a membrane protein that controls DA homeostasis through the high-affinity re-uptake of synaptically released DA. MethodsWe adopt newly developed animal models and state-of-the-art biophysical techniques to determine the contribution of the identified gene variants to impairments in DA neurotransmission observed in autism spectrum disorder (ASD). OutcomesHere, we characterize two independent autism-associated variants in the genes that encode STX1 and the DAT. We demonstrate that each variant dramatically alters DAT function. We identify molecular mechanisms that converge to inhibit reverse transport of DA and DA-associated behaviors. These mechanisms involve decreased phosphorylation of STX1 at Ser14 mediated by casein kinase 2 as well as a reduction in STX1/DAT interaction. These findings point to STX1/DAT interactions and STX1 phosphorylation as key regulators of DA homeostasis. InterpretationWe determine the molecular identity and the impact of these variants with the intent of defining DA dysfunction and associated behaviors as possible complications of ASD.

Adaptation and Dysadaptation of Abuse-Independent Physiological Responses to Amphetamine

Although amphetamine (AMPH) is an addictive drug of abuse which exhibits toxicity to dopaminergic neurons in long-term abusers, AMPH shows some abuse-independent effects. AMPH increases dopamine (DA) release from striatum by enhancing cytosolic DA ＂via＂ the redistribution of neurotransmitters from synaptic vesicles to neuronal cytoplasm and the reverse transport of DA from cytoplasm to synapse ＂via＂ the dopamine transporter (DAT). AMPH competitively binds DAT by facilitating the exchange of cytosolic DA, and inhibits the monoamine oxidase to decreasing the metabolism of postsynaptic DA. AMPH locally applied in ventral tegmental area (VTA) enhances DA release in nucleus accumbens and medial prefrontal cortex by activating noradrenergic neurotransmission in VTA. DA levels in striatum can be enhanced by estradiol ＂via＂ an increase of DAT expression following the administration of AMPH. In male rat tests, AMPH inhibits the basal and human chorionic gonadotropin (hCG)-stimulated secretion of testosterone by increasing cyclic AMP production and decreasing the activities of calcium channel and steroidogenic enzymes. AMPH also inhibits gastric emptying and intestinal transit ＂via＂ a mechanism associated with the hypersecretion of endogenous cholecystokinin (CCK). The offspring of pregnant rats administrated with AMPH shows ventricular hypertrophy, systolic dysfunction, and apoptosis in cardiac muscles. The above findings indicate that AMPH demonstrates multiple effects on cardiac, endocrine and gastrointestinal systems. Both adaptation and dysadaptation of abuse-independent physiological responses to AMPH are highly suggested.

A short review on the relation between the dopamine transporter 10/10-repeat allele and ADHD: implications for HIV infection

The dopamine transporter (DAT) is a functional element of the dopaminergic synapse in the brain. Its primary role is the regulation of dopamine (DA) availability by forward and reverse transport of DA from and to the synaptic cleft by which extracellular DA concentrations are being regulated. The DAT gene and especially the DAT 10/10 genotype have been intensively discussed as a candidate for several neuropsychiatric disorders including attention-deficit-hyperactivity disorder (ADHD). We found recently that the DAT 10/10 genotype is associated with increased levels of CSF DA and is present more frequently in HIV-infected individuals than in uninfected subjects, suggesting that personality traits related to this polymorphism may increase the risk of acquisition of HIV. In this article, we review studies on the DAT 10/10 genotype and the association with ADHD and its endophenotypes, express concerns on the reported DA neurochemistry in ADHD and discuss consequences of the DAT 10/10 genotype on the epidemiology of HIV infection.

The effects of benzofury (5-APB) on the dopamine transporter and 5-HT2-dependent vasoconstriction in the rat

5-APB, commonly marketed as 'benzofury' is a new psychoactive substance and erstwhile 'legal high' which has been implicated in 10 recent drug-related deaths in the UK. This drug was available on the internet and in 'head shops' and was one of the most commonly sold legal highs up until its recent UK temporary ban (UK Home Office). Despite its prominence, very little is known about its pharmacology. This study was undertaken to examine the pharmacology of 5-APB in vitro. We hypothesised that 5-APB would activate the dopamine and 5-HT systems which may underlie its putative stimulant and hallucinogenic effects. Autoradiographic studies showed that 5-APB displaced both [(125)I] RTI-121 and [(3)H] ketanserin from rat brain tissue suggesting affinity at the dopamine transporter and 5-HT2 receptor sites respectively. Voltammetric studies in rat accumbens brain slices revealed that 5-APB slowed dopamine reuptake, and at high concentrations caused reverse transport of dopamine. 5-APB also caused vasoconstriction of rat aorta, an effect antagonised by the 5-HT2A receptor antagonist ketanserin, and caused contraction of rat stomach fundus, which was reversed by the 5-HT2B receptor antagonist RS-127445. These data show that 5-APB interacts with the dopamine transporter and is an agonist at the 5-HT2A and 5-HT2B receptors in the rat. Thus 5-APB's pharmacology is consistent with it having both stimulant and hallucinogenic properties. In addition, 5-APB's activity at the 5-HT2B receptor may cause cardiotoxicity.

De novo mutation in the dopamine transporter gene associates dopamine dysfunction with autism spectrum disorder

De novo genetic variation is an important class of risk factors for autism spectrum disorder (ASD). Recently, whole-exome sequencing of ASD families has identified a novel de novo missense mutation in the human dopamine (DA) transporter (hDAT) gene, which results in a Thr to Met substitution at site 356 (hDAT T356M). The dopamine transporter (DAT) is a presynaptic membrane protein that regulates dopaminergic tone in the central nervous system by mediating the high-affinity reuptake of synaptically released DA, making it a crucial regulator of DA homeostasis. Here, we report the first functional, structural and behavioral characterization of an ASD-associated de novo mutation in the hDAT. We demonstrate that the hDAT T356M displays anomalous function, characterized as a persistent reverse transport of DA (substrate efflux). Importantly, in the bacterial homolog leucine transporter, substitution of A289 (the homologous site to T356) with a Met promotes an outward-facing conformation upon substrate binding. In the substrate-bound state, an outward-facing transporter conformation is required for substrate efflux. In Drosophila melanogaster, the expression of hDAT T356M in DA neurons-lacking Drosophila DAT leads to hyperlocomotion, a trait associated with DA dysfunction and ASD. Taken together, our findings demonstrate that alterations in DA homeostasis, mediated by aberrant DAT function, may confer risk for ASD and related neuropsychiatric conditions.

Amphetamine Activates an Amine-gated Chloride Channel to Generate Behavioral Effects in Caenorhabditis elegans

Amphetamine is a highly addictive psychostimulant, which is thought to generate its effects by promoting release of dopamine through reverse activation of dopamine transporters. However, some amphetamine-mediated behaviors persist in dopamine transporter knock-out animals, suggesting the existence of alternative amphetamine targets. Here we demonstrate the identification of a novel amphetamine target by showing that in Caenorhabditis elegans, a large fraction of the behavioral effects of amphetamine is mediated through activation of the amine-gated chloride channel, LGC-55. These findings bring to light alternative pathways engaged by amphetamine, and urge rethinking of the molecular mechanisms underlying the effects of this highly-addictive psychostimulant.