Felix P. Mayer

A quantitative model of amphetamine action on the 5‐HT transporter

Amphetamines bind to the plasmalemmal transporters for the monoamines dopamine (DAT), noradrenaline (NET) and 5‐HT (SERT); influx of amphetamine leads to efflux of substrates. Various models have been proposed to account for this amphetamine‐induced reverse transport in mechanistic terms. A most notable example is the molecular stent hypothesis, which posits a special amphetamine‐induced conformation that is not likely in alternative access models of transport. The current study was designed to evaluate the explanatory power of these models and the molecular stent hypothesis.

Phase I metabolites of mephedrone display biological activity as substrates at monoamine transporters

4‐Methyl‐N‐methylcathinone (mephedrone) is a synthetic stimulant that acts as a substrate‐type releaser at transporters for dopamine (DAT), noradrenaline (NET) and 5‐HT (SERT). Upon systemic administration, mephedrone is metabolized to several phase I compounds: the N‐demethylated metabolite, 4‐methylcathinone (nor‐mephedrone); the ring‐hydroxylated metabolite, 4‐hydroxytolylmephedrone (4‐OH‐mephedrone); and the reduced keto‐metabolite, dihydromephedrone.

Occupancy of the zinc-binding site by transition metals decreases the substrate affinity of the human dopamine transporter by an allosteric mechanism.

The human dopamine transporter (DAT) has a tetrahedral Zn2+-binding site. Zn2+-binding sites are also recognized by other first-row transition metals. Excessive accumulation of manganese or of copper can lead to parkinsonism because of dopamine deficiency. Accordingly, we examined the effect of Mn2+, Co2+, Ni2+, and Cu2+ on transport-associated currents through DAT and DAT-H193K, a mutant with a disrupted Zn2+-binding site. All transition metals except Mn2+ modulated the transport cycle of wild-type DAT with affinities in the low micromolar range. In this concentration range, they were devoid of any action on DAT-H193K. The active transition metals reduced the affinity of DAT for dopamine. The affinity shift was most pronounced for Cu2+, followed by Ni2+ and Zn2+ (= Co2+). The extent of the affinity shift and the reciprocal effect of substrate on metal affinity accounted for the different modes of action: Ni2+ and Cu2+ uniformly stimulated and inhibited, respectively, the substrate-induced steady-state currents through DAT. In contrast, Zn2+ elicited biphasic effects on transport, i.e. stimulation at 1 μm and inhibition at 10 μm. A kinetic model that posited preferential binding of transition metal ions to the outward-facing apo state of DAT and a reciprocal interaction of dopamine and transition metals recapitulated all experimental findings. Allosteric activation of DAT via the Zn2+-binding site may be of interest to restore transport in loss-of-function mutants.

An unsuspected role for organic cation transporter 3 in the actions of amphetamine

Amphetamine abuse is a major public health concern for which there is currently no effective treatment. To develop effective treatments, the mechanisms by which amphetamine produces its abuse-related effects need to be fully understood. It is well known that amphetamine exerts its actions by targeting high-affinity transporters for monoamines, in particular the cocaine-sensitive dopamine transporter. Organic cation transporter 3 (OCT3) has recently been found to play an important role in regulating monoamine signaling. However, whether OCT3 contributes to the actions of amphetamine is unclear. We found that OCT3 is expressed in dopamine neurons. Then, applying a combination of in vivo, ex vivo, and in vitro approaches, we revealed that a substantial component of amphetamine’s actions is OCT3-dependent and cocaine insensitive. Our findings support OCT3 as a new player in the actions of amphetamine and encourage investigation of this transporter as a potential new target for the treatment of psychostimulant abuse.

A kinetic account for amphetamine-induced monoamine release

The plasmalemmal monoamine transporters for dopamine, norepinephrine, and serotonin (SERT) are targets for amphetamines. In vivo, amphetamines elicit most, if not all, of their actions by triggering monoamine efflux. This is thought to be accomplished by an amphetamine-induced switch from the forward-transport to the substrate-exchange mode. The mechanism underlying this switch has remained elusive; available kinetic models posit that substrates and cosubstrate Na+ ions bind either in a random or in a sequential order. Neither can account for all reported experimental observations. We used electrophysiological recordings to interrogate crucial conformational transitions associated with the binding of five different substrates (serotonin, para-chloroamphetamine, and the high-affinity naphthyl-propan-amines PAL-287, PAL-1045, and PAL-1046) to human SERT expressed in HEK293 cells; specifically, we determined the relaxation kinetics of SERT from a substrate-loaded to a substrate-free state at various intracellular and extracellular Na+ concentrations. These rates and their dependence on intracellular and extracellular Na+ concentrations differed considerably between substrates. We also examined the effect of K+ on substrate affinity and found that K+ enhanced substrate dissociation. A kinetic model was developed, which allowed for random, but cooperative, binding of substrate and Na+ (or K+). The synthetic data generated by this model recapitulated the experimental observations. More importantly, the cooperative binding model accounted for the releasing action of amphetamines without any digression from alternating access. To the best of our knowledge, this model is the first to provide a mechanistic framework for amphetamine-induced monoamine release and to account for the findings that some substrates are less efficacious than others in promoting the substrate-exchange mode.

The psychostimulant (±)- cis -4,4′-dimethylaminorex (4,4′-DMAR) interacts with human plasmalemmal and vesicular monoamine transporters

ABSTRACT (±)‐cis‐4,4′‐Dimethylaminorex (4,4′‐DMAR) is a new psychoactive substance (NPS) that has been associated with 31 fatalities and other adverse events in Europe between June 2013 and February 2014. We used in vitro uptake inhibition and transporter release assays to determine the effects of 4,4′‐DMAR on human high‐affinity transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT). In addition, we assessed its binding affinities to monoamine receptors and transporters. Furthermore, we investigated the interaction of 4,4′‐DMAR with the vesicular monoamine transporter 2 (VMAT2) in rat phaeochromocytoma (PC12) cells and synaptic vesicles prepared from human striatum. 4,4′‐DMAR inhibited uptake mediated by human DAT, NET or SERT, respectively in the low micromolar range (IC50 values<2&mgr;M). Release assays identified 4,4′‐DMAR as a substrate type releaser, capable of inducing transporter‐mediated reverse transport via DAT, NET and SERT. Furthermore, 4,4′‐DMAR inhibited both the rat and human isoforms of VMAT2 at a potency similar to 3,4‐methylenedioxymethylamphetamine (MDMA). This study identified 4,4′‐DMAR as a potent non‐selective monoamine releasing agent. In contrast to the known effects of aminorex and 4‐methylaminorex, 4,4′‐DMAR exerts profound effects on human SERT. The latter finding is consistent with the idea that fatalities associated with its abuse may be linked to monoaminergic toxicity including serotonin syndrome. The activity at VMAT2 suggests that chronic abuse of 4,4′‐DMAR may result in long‐term neurotoxicity. HIGHLIGHTS4,4′‐Dimethylaminorex targets plasmalemmal and vesicular monoamine transporters.4,4′‐DMAR is a non‐selective monoamine transporter releasing agent.4,4′‐DMARs profile of action is similar to MDMA but it is a more potent releaser.4,4′‐DMAR related deaths can be related to serotonin and norepinephrine toxicity.4,4′‐DMAR inhibits VMAT2, suggestive of its long‐term neurotoxic effects.

Comparative analysis of novel decynium-22 analogs to inhibit transport by the low-affinity, high-capacity monoamine transporters, organic cation transporters 2 and 3, and plasma membrane monoamine transporter

Abstract Growing evidence supports involvement of low‐affinity/high‐capacity organic cation transporters (OCTs) and plasma membrane monoamine transporter (PMAT) in regulating clearance of monoamines. Currently decynium‐22 (D22) is the best pharmacological tool to study these transporters, however it does not readily discriminate among them, underscoring a need to develop compounds with greater selectivity for each of these transporters. We developed seven D22 analogs, and previously reported that some have lower affinity for &agr;1‐adrenoceptors than D22 and showed antidepressant‐like activity in mice. Here, we extend these findings to determine the affinity of these analogs for OCT2, OCT3 and PMAT, as well as serotonin, norepinephrine and dopamine transporters (SERT, NET and DAT) using a combination of uptake competition with [3H]methyl‐4‐phenylpyridinium acetate in overexpressed HEK cells and [3H]citalopram, [3H]nisoxetine and [3H]WIN 35428 displacement binding in mouse hippocampal and striatal preparations. Like D22, all analogs showed greater binding affinities for OCT3 than OCT2 and PMAT. However, unlike D22, some analogs also showed modest affinity for SERT and DAT. Dual OCT3/SERT and/or OCT3/DAT actions of certain analogs may help explain their ability to produce antidepressant‐like effects in mice and help account for our previous findings that D22 lacks antidepressant‐like effects unless SERT function is either genetically or pharmacologically compromised. Though these analogs are not superior than D22 in discriminating among OCTs/PMAT, our findings point to development of compounds with combined ability to inhibit both low‐affinity/high‐capacity transporters, such as OCT3, and high‐affinity/low‐capacity transporters, such as SERT, as therapeutics with potentially improved efficacy for treatment of psychiatric disorders.

DARK Classics in Chemical Neuroscience: Aminorex Analogues

Aminorex (5-phenyl-4,5-dihydro-1,3-oxazol-2-amine) and 4-methylaminorex (4-methyl-5phenyl-4,5-dihydro-1,3-oxazol-2-amine) are psychostimulants that have long been listed in Schedules IV and I of the UN Convention on Psychotropic Substances of 1971. However, a range of psychoactive analogs exist that are not internationally controlled and therefore often classified as new psychoactive substances (NPS). Aminorex analogs encompass failed pharmaceuticals that reemerged as drugs of abuse, and newly synthesized substances that were solely designed for recreational use by clandestine chemists. NPS, sometimes also referred to as “designer drugs” in alignment with a phenomenon arising in the early 1980s, serve as alternatives to controlled drugs. Aminorex and its derivatives interact with monoaminergic neurotransmission by interfering with the function of monoamine transporters. Hence, these compounds share pharmacological and neurochemical similarities with amphetamines and cocaine. The consumption of aminorex, 4-methylaminorex and 4,4’dimethylaminorex (4-methyl-5-(4-methylphenyl)-4,5-dihydro-1,3-oxazol-2-amine) has been associated with adverse events including death, bestowing an inglorious fame on aminorexderived drugs. In this review, a historical background is presented, as well as an account of the pharmacodynamic and pharmacokinetic properties of aminorex and various analogs. Light is shed on their misuse as drug adulterants of well-established drugs on the market. This review not only provides a detailed overview of an abused substance-class, but also emphasizes the darkest aspect of the NPS market, i.e. deleterious side effects that arise from the ingestion of certain NPS, as knowledge of the pharmacology, the potency or the identity of the active ingredients remains obscure to NPS users.

Real-time uptake of fluorescent ASP+ via the organic cation transporter 3

Background The organic cation transporter 3 (OCT3) shows a broad expression pattern in the nervous system and has been detected in neurons and glial cells. Here, OCT3 serves as an additional re-uptake system for neurotransmitters, such as dopamine, serotonin and norepinephrine and therefore OCT3 mediated uptake has been termed “uptake 2”. Methods We measured OCT3 mediated uptake of the fluorescent OCT3 substrate 4-Di-1-ASP (4-(-4(dimethylamino(styryl)-N-methylpyridinium (ASP) in real-time in HEK293 cells stably expressing the human and the mouse isoforms of OCT3. Data obtained on OCT3 mediated uptake of fluorescent ASP were compared to uptake of tritiated 1-methyl-4-phenylpyridinium (MPP). Results We show that ASP is selectively taken up via OCT3 in real time and this allows for sensitive assessment of transport via OCT3. Hence, we analyzed the mode of action of several OCT3 substrates and transport inhibitors, such as the stress hormone corticosterone and decynium-22. All results obtained from ASP uptake studies are comparable with data obtained from uptake studies with tritiated MPP and in line with previously published reports. Finally, we tested if OCT-mediated uptake is sensitive to phosphorylation and found that the protein kinase C inhibitor GF109203X inhibited uptake. Discussion The use of ASP as substrate for OCT3 provides the opportunity to analyze OCT3 mediated transport with a high temporal resolution and is comparable to OCT3 mediated uptake of tritiated MPP. Uptake inhibition by corticosterone was comparable using either ASP or MPP and similar to inhibition of protein kinase C.