Dopaminergic Ric GTPase activity impacts amphetamine sensitivity and sleep quality in a dopamine transporter-dependent manner in Drosophila melanogaster.

Dopamine (DA) is required for movement, sleep, and reward, and DA signaling is tightly controlled by the presynaptic DA transporter (DAT). Therapeutic and addictive psychostimulants, including methylphenidate (Ritalin; MPH), cocaine, and amphetamine (AMPH), markedly elevate extracellular DA via their actions as competitive DAT inhibitors (MPH, cocaine) and substrates (AMPH). DAT silencing in mice and invertebrates results in hyperactivity, reduced sleep, and blunted psychostimulant responses, highlighting DAT's essential role in DA-dependent behaviors. DAT surface expression is not static; rather it is dynamically regulated by endocytic trafficking. PKC-stimulated DAT endocytosis requires the neuronal GTPase, Rit2, and Rit2 silencing in mouse DA neurons impacts psychostimulant sensitivity. However, it is unknown whether or not Rit2-mediated changes in psychostimulant sensitivity are DAT-dependent. Here, we leveraged Drosophila melanogaster to test whether the Drosophila Rit2 ortholog, Ric, impacts dDAT function, trafficking, and DA-dependent behaviors. Orthologous to hDAT and Rit2, dDAT and Ric directly interact, and the constitutively active Ric mutant Q117L increased dDAT surface levels and function in cell lines and ex vivo Drosophila brains. Moreover, DAergic RicQ117L expression caused sleep fragmentation in a DAT-dependent manner but had no effect on total sleep and daily locomotor activity. Importantly, we found that Rit2 is required for AMPH-stimulated DAT internalization in mouse striatum, and that DAergic RicQ117L expression significantly increased Drosophila AMPH sensitivity in a DAT-dependent manner, suggesting a conserved impact of Ric-dependent DAT trafficking on AMPH sensitivity. These studies support that the DAT/Rit2 interaction impacts both baseline behaviors and AMPH sensitivity, potentially by regulating DAT trafficking.

Dopamine transporter trafficking and Rit2 GTPase: Mechanism of action and in vivo impact.

Following its evoked release, dopamine (DA) signaling is rapidly terminated by presynaptic reuptake, mediated by the cocaine-sensitive DA transporter (DAT). DAT surface availability is dynamically regulated by endocytic trafficking, and direct protein kinase C (PKC) activation acutely diminishes DAT surface expression by accelerating DAT internalization. Previous cell line studies demonstrated that PKC-stimulated DAT endocytosis requires both Ack1 inactivation, which releases a DAT-specific endocytic brake, and the neuronal GTPase, Rit2, which binds DAT. However, it is unknown whether Rit2 is required for PKC-stimulated DAT endocytosis in DAergic terminals or whether there are region- and/or sex-dependent differences in PKC-stimulated DAT trafficking. Moreover, the mechanisms by which Rit2 controls PKC-stimulated DAT endocytosis are unknown. Here, we directly examined these important questions. Ex vivo studies revealed that PKC activation acutely decreased DAT surface expression selectively in ventral, but not dorsal, striatum. AAV-mediated, conditional Rit2 knockdown in DAergic neurons impacted baseline DAT surface:intracellular distribution in DAergic terminals from female ventral, but not dorsal, striatum. Further, Rit2 was required for PKC-stimulated DAT internalization in both male and female ventral striatum. FRET and surface pulldown studies in cell lines revealed that PKC activation drives DAT-Rit2 surface dissociation and that the DAT N terminus is required for both PKC-mediated DAT-Rit2 dissociation and DAT internalization. Finally, we found that Rit2 and Ack1 independently converge on DAT to facilitate PKC-stimulated DAT endocytosis. Together, our data provide greater insight into mechanisms that mediate PKC-regulated DAT internalization and reveal unexpected region-specific differences in PKC-stimulated DAT trafficking in bona fide DAergic terminals.

SLC6 transporter oligomerization.

Transporters of the solute carrier 6 (SLC6) family mediate the reuptake of neurotransmitters such as dopamine, norepinephrine, serotonin, GABA, and glycine. SLC6 family members are 12 transmembrane helix-spanning proteins that operate using the transmembrane sodium gradient for transport. These transporters assume various quaternary arrangements ranging from monomers to complex stoichiometries with multiple subunits. Dopamine and serotonin transporter oligomerization has been implicated in trafficking of newly formed proteins from the endoplasmic reticulum to the plasma membrane with a pre-fixed assembly. Once at the plasma membrane, oligomers are kept fixed in their quaternary assembly by interaction with phosphoinositides. While it remains unclear how oligomer formation precisely affects physiological transporter function, it has been shown that oligomerization supports the activity of release-type psychostimulants. Most recently, single molecule microscopy experiments unveiled that the stoichiometry differs between individual members of the SLC6 family. The present overview summarizes our understanding of the influence of plasma membrane constituents on transporter oligomerization, describes the known interfaces between protomers and discusses open questions.

Designer drugs: mechanism of action and adverse effects.

Psychoactive substances with chemical structures or pharmacological profiles that are similar to traditional drugs of abuse continue to emerge on the recreational drug market. Internet vendors may at least temporarily sell these so-called designer drugs without adhering to legal statutes or facing legal consequences. Overall, the mechanism of action and adverse effects of designer drugs are similar to traditional drugs of abuse. Stimulants, such as amphetamines and cathinones, primarily interact with monoamine transporters and mostly induce sympathomimetic adverse effects. Agonism at µ-opioid receptors and γ-aminobutyric acid-A (GABAA) or GABAB receptors mediates the pharmacological effects of sedatives, which may induce cardiorespiratory depression. Dissociative designer drugs primarily act as N-methyl-D-aspartate receptor antagonists and pose similar health risks as the medically approved dissociative anesthetic ketamine. The cannabinoid type 1 (CB1) receptor is thought to drive the psychoactive effects of synthetic cannabinoids, which are associated with a less desirable effect profile and more severe adverse effects compared with cannabis. Serotonergic 5-hydroxytryptamine-2A (5-HT2A) receptors mediate alterations of perception and cognition that are induced by serotonergic psychedelics. Because of their novelty, designer drugs may remain undetected by routine drug screening, thus hampering evaluations of adverse effects. Intoxication reports suggest that several designer drugs are used concurrently, posing a high risk for severe adverse effects and even death.

Molecular Toxicological Mechanisms of Synthetic Cathinones on C2C12 Myoblasts.

Synthetic cathinones are popular psychoactive substances that may cause skeletal muscle damage. In addition to indirect sympathomimetic myotoxicity, these substances could be directly myotoxic. Since studies in myocytes are currently lacking, the aim of the present study was to investigate potential toxicological effects by synthetic cathinones on C2C12 myoblasts (mouse skeletal muscle cell line). We exposed C2C12 myoblasts to 3-methylmethcathinone, 4-methylmethcathinone (mephedrone), 3,4-methylenedioxymethcathinone (methylone), 3,4-methylenedioxypyrovalerone (MDPV), alpha-pyrrolidinovalerophenone (α-PVP), and naphthylpyrovalerone (naphyrone) for 1 or 24 h before cell membrane integrity, ATP content, mitochondrial oxygen consumption, and mitochondrial superoxide production was measured. 3,4-Methylenedioxymethamphetamine (MDMA) was included as a reference compound. All investigated synthetic cathinones, as well as MDMA, impaired cell membrane integrity, depleted ATP levels, and increased mitochondrial superoxide concentrations in a concentration-dependent manner in the range of 50⁻2000 µM. The two pyrovalerone derivatives α-PVP and naphyrone, and MDMA, additionally impaired basal and maximal cellular respiration, suggesting mitochondrial dysfunction. Alpha-PVP inhibited complex I, naphyrone complex II, and MDMA complex I and III, whereas complex IV was not affected. We conclude that, in addition to sympathetic nervous system effects and strenuous muscle exercise, direct effects of some cathinones on skeletal muscle mitochondria may contribute to myotoxicity in susceptible synthetic cathinone drugs users.

Monoamine Transporter and Receptor Interaction Profiles in Vitro Predict Reported Human Doses of Novel Psychoactive Stimulants and Psychedelics.

Background: Pharmacological profiles of new psychoactive substances can be established rapidly in vitro and provide information on potential psychoactive effects in humans. The present study investigated whether specific in vitro monoamine transporter and receptor interactions can predict effective psychoactive doses in humans. Methods: We correlated previously assessed in vitro data of stimulants and psychedelics with human doses that are reported on the Internet and in books. Results: For stimulants, dopamine and norepinephrine transporter inhibition potency was positively correlated with human doses, whereas serotonin transporter inhibition potency was inversely correlated with human doses. Serotonin 5-hydroxytryptamine-2A (5-HT2A) and 5-HT2C receptor affinity was significantly correlated with psychedelic doses, but 5-HT1A receptor affinity and 5-HT2A and 5-HT2B receptor activation potency were not. Conclusions: The rapid assessment of in vitro pharmacological profiles of new psychoactive substances can help to predict psychoactive doses and effects in humans and facilitate the appropriate scheduling of new psychoactive substances.

Liquid chromatography-tandem mass spectrometric assay for the multikinase inhibitor regorafenib in plasma.

Regorafenib has recently been approved for the treatment of colorectal cancer. A bioanalytical liquid chromatography-tandem mass spectrometric assay for this multikinase inhibitor was developed and validated in plasma. The concentration range of the assay was 25-25,000 ng/mL. Protein precipitation with acetonitrile was used as sample pre-treatment with sorafenib as internal standard. The extract was diluted with methanol (25%, v/v) and then injected onto the sub-2 µm particle, bridged ethylsilicia hybrid trifunctional bonded C18 column. Isocratic elution using 0.02% (v/v) formic acid in a methanol-water mixture was used. Compounds were monitored by a triple quadrupole mass spectrometer in the selected reaction monitoring mode after positive electrospray ionization. Double logarithmic calibration was used; within-day precisions, between-day precisions, and accuracies were 3.2-9.2, 4.1-12.3 and 94.8-103.0%, respectively. High drug stability was observed under all relevant storage conditions. The assay was used to measure drug concentrations in a pharmacokinetic study in wild-type FVB mice.

Derivatization-free determination of chiral plasma pharmacokinetics of MDMA and its enantiomers.

3,4-Methylenedioxymethamphetamine (MDMA) is an entactogen with therapeutic potential. The two enantiomers of MDMA differ regarding their pharmacokinetics and pharmacodynamics but the chiral pharmacology of MDMA needs further study in clinical trials. Here, an achiral and an enantioselective high performance liquid chromatography-tandem mass spectrometry method for the quantification of MDMA and its psychoactive phase I metabolite 3,4-methylenedioxyamphetamine (MDA) in human plasma were developed and validated. The analytes were detected by positive electrospray ionization followed by multiple reaction monitoring. The calibration range was 0.5-500 ng/mL for the achiral analysis of both analytes, 0.5-1,000 ng/mL for chiral MDMA analysis, and 1-1,000 ng/mL for chiral MDA analysis. Accuracy, precision, selectivity, and sensitivity of both bioanalytical methods were in accordance with regulatory guidelines. Furthermore, accuracy and precision of the enantioselective method were maintained when racemic calibrations were used to measure quality control samples containing only one of the enantiomers. Likewise, enantiomeric calibrations could be used to reliably quantify enantiomers in racemic samples. The achiral and enantioselective methods were employed to assess pharmacokinetic parameters in clinical study participants treated with racemic MDMA or one of its enantiomers. The pharmacokinetic parameters assessed with both bioanalytical methods were comparable. In conclusion, the enantioselective method is useful for the simultaneous quantification of both enantiomers in subjects treated with racemic MDMA. However, as MDMA and MDA do not undergo chiral inversion, enantioselective separation is not necessary in subjects treated with only one of the enantiomers.

Development and validation of an LC-MS/MS method for quantifying diamorphine and its major metabolites 6-monoacetylmorphine, morphine, morphine-3-glucuronide, and morphine-6-glucuronide in human plasma.

Diamorphine, commonly known as heroin, is a semi-synthetic opioid analgesic. In the context of heroin-assisted treatment for opioid-dependent patients, diamorphine is mostly administered intravenously. However, recent attention has shifted towards intranasal administration as a better-tolerated alternative to the intravenous route. Here, we developed and validated a rapid bioanalytical method for the simultaneous quantification of diamorphine and its major metabolites 6-monoacetylmorphine, morphine, morphine-3-glucuronide, and morphine-6-glucuronide in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A straightforward protein precipitation extraction step was used for sample preparation. Chromatographic analyte separation was achieved using a Kinetex EVO C18 analytical column and a mobile phase gradient comprising an aqueous solution of ammonium hydrogen carbonate and methanol supplied with formic acid. Employing positive electrospray ionization and scheduled multiple reaction monitoring, we established a quantification range of 1-1,000 ng/mL for all analytes. Our validation results demonstrate a mean intra-assay accuracy of 91-106% and an intra-assay precision (CV) between 2 and 9% for all analytes and over three validation runs. The method exhibits a high extraction recovery (> 87%) and a negligible matrix effect (99-125%). Furthermore, no interferences with endogenous plasma compounds were detected. Lastly, we applied the method to assess the plasma concentrations of an opioid-dependent patient after the intranasal administration of diamorphine in a clinical study. In summary, we have successfully developed a rapid, highly reliable, and straightforward bioanalytical method for quantifying diamorphine and its metabolites in low amounts of clinical plasma samples.

Use of the head-twitch response to investigate the structure-activity relationships of 4-thio-substituted 2,5-dimethoxyphenylalkylamines.

RATIONALE: 4-Thio-substituted phenylalkylamines such as 2,5-dimethoxy-4-ethylthiophenethylamine (2C-T-2) and 2,5-dimethoxy-4-n-propylthiophenethylamine (2C-T-7) produce psychedelic effects in humans and have been distributed as recreational drugs. OBJECTIVES: The present studies were conducted to examine the structure-activity relationships (SAR) of a series of 4-thio-substituted phenylalkylamines using the head twitch response (HTR), a 5-HT2A receptor-mediated behavior induced by psychedelic drugs in mice. The HTR is commonly used as a behavioral proxy in rodents for human psychedelic effects and can be used to discriminate hallucinogenic and non-hallucinogenic 5-HT2A agonists. METHODS: HTR dose-response studies with twelve different 4-thio-substituted phenylalkylamines were conducted in male C57BL/6 J mice. To detect the HTR, head movement was recorded electronically using a magnetometer coil and then head twitches were identified in the recordings using a validated method based on artificial intelligence. RESULTS: 2C-T, the parent compound of this series, had relatively low potency in the HTR paradigm, but adding an α-methyl group increased potency fivefold. Potency was also increased when the 4-methylthio group was extended by one to three methylene units. Fluorination of the 4-position alkylthio chain, however, was detrimental for activity, as was the presence of a 4-allylthio substituent versus a propylthio group. 2C-T analogs containing a 4-benzylthio group showed little or no effect in the HTR paradigm, which is consistent with evidence that bulky 4-substituents can dampen agonist efficacy at the 5-HT2A receptor. Binding and functional studies confirmed that the compounds have nanomolar affinity for 5-HT2 receptor subtypes and act as partial agonists at 5-HT2A. CONCLUSIONS: In general, there were close parallels between the HTR data and the known SAR governing activity of phenylalkylamines at the 5-HT2A receptor. These findings further support the classification of 2C-T compounds as psychedelic drugs.

Acute effects of intravenous DMT in a randomized placebo-controlled study in healthy participants.

N,N-dimethyltryptamine (DMT) is distinct among classic serotonergic psychedelics because of its short-lasting effects when administered intravenously. Despite growing interest in the experimental and therapeutic use of intravenous DMT, data are lacking on its clinical pharmacology. We conducted a double-blind, randomized, placebo-controlled crossover trial in 27 healthy participants to test different intravenous DMT administration regimens: placebo, low infusion (0.6 mg/min), high infusion (1 mg/min), low bolus + low infusion (15 mg + 0.6 mg/min), and high bolus + high infusion (25 mg + 1 mg/min). Study sessions lasted for 5 h and were separated by at least 1 week. Participant's lifetime use of psychedelics was ≤20 times. Outcome measures included subjective, autonomic, and adverse effects, pharmacokinetics of DMT, and plasma levels of brain-derived neurotropic factor (BDNF) and oxytocin. Low (15 mg) and high (25 mg) DMT bolus doses rapidly induced very intense psychedelic effects that peaked within 2 min. DMT infusions (0.6 or 1 mg/min) without a bolus induced slowly increasing and dose-dependent psychedelic effects that reached plateaus after 30 min. Both bolus doses produced more negative subjective effects and anxiety than infusions. After stopping the infusion, all drug effects rapidly decreased and completely subsided within 15 min, consistent with a short early plasma elimination half-life (t1/2α) of 5.0-5.8 min, followed by longer late elimination (t1/2ß = 14-16 min) after 15-20 min. Subjective effects of DMT were stable from 30 to 90 min, despite further increasing plasma concentrations, thus indicating acute tolerance to continuous DMT administration. Intravenous DMT, particularly when administered as an infusion, is a promising tool for the controlled induction of a psychedelic state that can be tailored to the specific needs of patients and therapeutic sessions.Trial registration: ClinicalTrials.gov identifier: NCT04353024.

Comparative acute effects of mescaline, lysergic acid diethylamide, and psilocybin in a randomized, double-blind, placebo-controlled cross-over study in healthy participants.

Mescaline, lysergic acid diethylamide (LSD), and psilocybin are classic serotonergic psychedelics. A valid, direct comparison of the effects of these substances is lacking. The main goal of the present study was to investigate potential pharmacological, physiological and phenomenological differences at psychoactive-equivalent doses of mescaline, LSD, and psilocybin. The present study used a randomized, double-blind, placebo-controlled, cross-over design to compare the acute subjective effects, autonomic effects, and pharmacokinetics of typically used, moderate to high doses of mescaline (300 and 500 mg), LSD (100 µg), and psilocybin (20 mg) in 32 healthy participants. A mescaline dose of 300 mg was used in the first 16 participants and 500 mg was used in the subsequent 16 participants. Acute subjective effects of 500 mg mescaline, LSD, and psilocybin were comparable across various psychometric scales. Autonomic effects of 500 mg mescaline, LSD, and psilocybin were moderate, with psilocybin causing a higher increase in diastolic blood pressure compared with LSD, and LSD showing a trend toward an increase in heart rate compared with psilocybin. The tolerability of mescaline, LSD, and psilocybin was comparable, with mescaline at both doses inducing slightly more subacute adverse effects (12-24 h) than LSD and psilocybin. Clear distinctions were seen in the duration of action between the three substances. Mescaline had the longest effect duration (mean: 11.1 h), followed by LSD (mean: 8.2 h), and psilocybin (mean: 4.9 h). Plasma elimination half-lives of mescaline and LSD were similar (approximately 3.5 h). The longer effect duration of mescaline compared with LSD was due to the longer time to reach maximal plasma concentrations and related peak effects. Mescaline and LSD, but not psilocybin, enhanced circulating oxytocin. None of the substances altered plasma brain-derived neurotrophic factor concentrations. In conclusion, the present study found no evidence of qualitative differences in altered states of consciousness that were induced by equally strong doses of mescaline, LSD, and psilocybin. The results indicate that any differences in the pharmacological profiles of mescaline, LSD, and psilocybin do not translate into relevant differences in the subjective experience. ClinicalTrials.gov identifier: NCT04227756.

Pharmacological characterization of 3,4-methylenedioxyamphetamine (MDA) analogs and two amphetamine-based compounds: N,α-DEPEA and DPIA.

3,4-methylenedioxyamphetamine (MDA) is a psychoactive compound chemically related to the entactogen MDMA. MDA shares some of the entactogenic effects of MDMA but also exerts stimulant effects and psychedelic properties at higher doses. Here, we examined the pharmacological properties of MDA analogs and related amphetamine-based compounds detected in street drug samples or in sport supplements. We examined the key pharmacological mechanisms including monoamine uptake inhibition and release using human embryonic kidney 293 cells stably transfected with the respective human transporters. Additionally, we assessed monoamine transporter and receptor binding and activation properties. MDA, its fluorinated analogs, as well as the α-ethyl containing BDB and the dimeric amphetamine DPIA inhibited NET with the greatest potency and preferentially inhibited 5-HT vs. dopamine uptake. The ß­methoxy MDA analog 3C-BOH and the amphetamine-based N,α-DEPEA inhibited NET and preferentially inhibited dopamine vs. 5-HT uptake. The test drugs mediated efflux of at least one monoamine with the exception of DPIA. Most compounds bound to 5-HT2A and 5-HT2C receptors (Ki ≤ 10 µM) and several substances activated the 5-HT2A and 5-HT2B receptor as partial or full agonists. Furthermore, several compounds interacted with adrenergic receptors and the trace amine-associated receptor 1 (TAAR1) in the micromolar range. The pharmacological profiles of some fluorinated and nonfluorinated MDA analogs resemble the profile of MDMA. In contrast, 3C-BOH and N,α-DEPEA displayed more pronounced dopaminergic activity similar to amphetamine. Pharmacokinetics and pharmacodynamics studies are necessary to better establish the risks and therapeutic potential of the tested drugs.

Corrigendum: Cell-Based Radiotracer Binding and Uptake Inhibition Assays: A Comparison of In Vitro Methods to Assess the Potency of Drugs That Target Monoamine Transporters.

[This corrects the article DOI: 10.3389/fphar.2020.00673.].

Liquid chromatography-tandem mass spectrometry method for the bioanalysis of N,N-dimethyltryptamine (DMT) and its metabolites DMT-N-oxide and indole-3-acetic acid in human plasma.

The indole alkaloid N,N-dimethyltryptamine (DMT) induces psychedelic effects in humans. In addition to ceremonial and recreational use, DMT is subject to clinical investigations. Sensitive bioanalytical methods are required to assess the pharmacokinetics of DMT and its metabolites in human plasma. Here, a high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of DMT and its major metabolites indole-3-acetic acid (IAA) and DMT-N-oxide (DMT-NO) was developed and validated. As IAA is an endogenous component of human plasma, 13C6-IAA was used to determine IAA concentrations. After simple protein precipitation with methanol, analytes were separated on a pentafluorophenyl column. A gradient consisting of 0.1% (v/v) formic acid in a methanol-water mixture was applied for analyte separation. The analytes were detected by positive electrospray ionization followed by multiple reaction monitoring. The calibration range of the assay was 0.25-250 ng/mL for DMT, 0.1-100 ng/mL for DMT-NO, and 25-25,000 ng/mL for 13C6-IAA. The intra- and inter-assay accuracy was 93-113% for all analytes at all quality control levels, with coefficient of variation ≤ 11%. All analytes were stable under storage conditions relevant for the analysis of large batches of study samples. The validated method was capable of assessing pharmacokinetic (PK) parameters of DMT and its metabolites in study participants intravenously perfused with 1 mg/min DMT for 90 min. Overall, the developed method is easy-to-use, has a short run time, and qualifies for PK and metabolism studies of DMT in clinical settings.

(2-Aminopropyl)benzo[ß]thiophenes (APBTs) are novel monoamine transporter ligands that lack stimulant effects but display psychedelic-like activity in mice.

Derivatives of (2-aminopropyl)indole (API) and (2-aminopropyl)benzofuran (APB) are new psychoactive substances which produce stimulant effects in vivo. (2-Aminopropyl)benzo[ß]thiophene (APBT) is a novel sulfur-based analog of API and APB that has not been pharmacologically characterized. In the current study, we assessed the pharmacological effects of six APBT positional isomers in vitro, and three of these isomers (3-APBT, 5-APBT, and 6-APBT) were subjected to further investigations in vivo. Uptake inhibition and efflux assays in human transporter-transfected HEK293 cells and in rat brain synaptosomes revealed that APBTs inhibit monoamine reuptake and induce transporter-mediated substrate release. Despite being nonselective transporter releasers like MDMA, the APBT compounds failed to produce locomotor stimulation in C57BL/6J mice. Interestingly, 3-APBT, 5-APBT, and 6-APBT were full agonists at 5-HT2 receptor subtypes as determined by calcium mobilization assays and induced the head-twitch response in C57BL/6J mice, suggesting psychedelic-like activity. Compared to their APB counterparts, ABPT compounds demonstrated that replacing the oxygen atom with sulfur results in enhanced releasing potency at the serotonin transporter and more potent and efficacious activity at 5-HT2 receptors, which fundamentally changed the in vitro and in vivo profile of APBT isomers in the present studies. Overall, our data suggest that APBT isomers may exhibit psychedelic and/or entactogenic effects in humans, with minimal psychomotor stimulation. Whether this unique pharmacological profile of APBT isomers translates into potential therapeutic potential, for instance as candidates for drug-assisted psychotherapy, warrants further investigation.

Structural basis of organic cation transporter-3 inhibition.

Organic cation transporters (OCTs) facilitate the translocation of catecholamines, drugs and xenobiotics across the plasma membrane in various tissues throughout the human body. OCT3 plays a key role in low-affinity, high-capacity uptake of monoamines in most tissues including heart, brain and liver. Its deregulation plays a role in diseases. Despite its importance, the structural basis of OCT3 function and its inhibition has remained enigmatic. Here we describe the cryo-EM structure of human OCT3 at 3.2 Å resolution. Structures of OCT3 bound to two inhibitors, corticosterone and decynium-22, define the ligand binding pocket and reveal common features of major facilitator transporter inhibitors. In addition, we relate the functional characteristics of an extensive collection of previously uncharacterized human genetic variants to structural features, thereby providing a basis for understanding the impact of OCT3 polymorphisms.

Phosphatidylinositol 4,5-bisphosphate (PIP2) facilitates norepinephrine transporter dimerization and modulates substrate efflux.

The plasmalemmal norepinephrine transporter (NET) regulates cardiovascular sympathetic activity by clearing extracellular norepinephrine in the synaptic cleft. Here, we investigate the subunit stoichiometry and function of NET using single-molecule fluorescence microscopy and flux assays. In particular, we show the effect of phosphatidylinositol 4,5-bisphosphate (PIP2) on NET oligomerization and efflux. NET forms monomers (~60%) and dimers (~40%) at the plasma membrane. PIP2 depletion results in a decrease in the average oligomeric state and decreases NET-mediated substrate efflux while not affecting substrate uptake. Mutation of the putative PIP2 binding residues R121, K334, and R440 to alanines does not affect NET dimerization but results in decreased substrate efflux that is not altered upon PIP2 depletion; this indicates that PIP2 interactions with these residues affect NET-mediated efflux. A dysregulation of norepinephrine and PIP2 signaling have both been implicated in neuropsychiatric and cardiovascular diseases. This study provides evidence that PIP2 directly regulates NET organization and function.

Molecular and clinical aspects of potential neurotoxicity induced by new psychoactive stimulants and psychedelics.

New psychoactive stimulants and psychedelics continue to play an important role on the illicit new psychoactive substance (NPS) market. Designer stimulants and psychedelics both affect monoaminergic systems, although by different mechanisms. Stimulant NPS primarily interact with monoamine transporters, either as inhibitors or as substrates. Psychedelic NPS most potently interact with serotonergic receptors and mediate their mind-altering effects mainly through agonism at serotonin 5-hydroxytryptamine-2A (5-HT2A) receptors. Rarely, designer stimulants and psychedelics are associated with potentially severe adverse effects. However, due to the high number of emerging NPS, it is not possible to investigate the toxicity of each individual substance in detail. The brain is an organ particularly sensitive to substance-induced toxicity due to its high metabolic activity. In fact, stimulant and psychedelic NPS have been linked to neurological and cognitive impairments. Furthermore, studies using in vitro cell models or rodents indicate a variety of mechanisms that could potentially lead to neurotoxic damage in NPS users. Cytotoxicity, mitochondrial dysfunction, and oxidative stress may potentially contribute to neurotoxicity of stimulant NPS in addition to altered neurochemistry. Serotonin 5-HT2A receptor-mediated toxicity, oxidative stress, and activation of mitochondrial apoptosis pathways could contribute to neurotoxicity of some psychedelic NPS. However, it remains unclear how well the current preclinical data of NPS-induced neurotoxicity translate to humans.