Amphetamines, new psychoactive drugs and the monoamine transporter cycle
Authors: Harald H. Sitte, Michael Freissmuth
Journal: Trends in Pharmacological Sciences (2014)

•Amphetamines comprise both plasma-membrane transporter substrates and blockers.•Amphetamines elicit psychostimulant effects which can lead to recreational abuse.•New psychoactive substances bypass drug bans via structural variations.•Reverse operation of substrate transport is asymmetric for the reuptake of substrates. In monoaminergic neurons, the vesicular transporters and the plasma membrane transporters operate in a relay. Amphetamine and its congeners target this relay to elicit their actions: most amphetamines are substrates, which pervert the relay to elicit efflux of monoamines into the synaptic cleft. However, some amphetamines act as transporter inhibitors. Both compound classes elicit profound psychostimulant effects, which render them liable to recreational abuse. Currently, a surge of new psychoactive substances occurs on a global scale. Chemists bypass drug bans by ingenuous structural variations, resulting in a rich pharmacology. A credible transport model must account for their distinct mode of action and link this to subtle differences in activity and undesired, potentially deleterious effects. In monoaminergic neurons, the vesicular transporters and the plasma membrane transporters operate in a relay. Amphetamine and its congeners target this relay to elicit their actions: most amphetamines are substrates, which pervert the relay to elicit efflux of monoamines into the synaptic cleft. However, some amphetamines act as transporter inhibitors. Both compound classes elicit profound psychostimulant effects, which render them liable to recreational abuse. Currently, a surge of new psychoactive substances occurs on a global scale. Chemists bypass drug bans by ingenuous structural variations, resulting in a rich pharmacology. A credible transport model must account for their distinct mode of action and link this to subtle differences in activity and undesired, potentially deleterious effects. The amphetamines are a diverse class of chemical compounds. They comprise synthetic compounds and naturally occurring alkaloids such as ephedrine and cathinone (Box 1), which are synthesized in the plant species Ephedra and Catha, respectively. Extracts from these plants have been used for their remedial and psychostimulant effects for millenia (for an excellent historical overview see [1Sulzer D. et al.Mechanisms of neurotransmitter release by amphetamines: a review.Prog. Neurobiol. 2005; 75: 406-433Crossref PubMed Scopus (929) Google Scholar]). Lazăr Edeleanu synthesized the eponymous compound amphetamine in Berlin in 1887 (Box 1); many derivatives were produced within the next two decades [1Sulzer D. et al.Mechanisms of neurotransmitter release by amphetamines: a review.Prog. Neurobiol. 2005; 75: 406-433Crossref PubMed Scopus (929) Google Scholar]. The availability of pure compounds allowed their pharmacological characterization, which was initiated by Sir Henry Dale [2Barger G. Dale H.H. Chemical structure and sympathomimetic action of amines.J. Physiol. 1910; 41: 19-59Crossref PubMed Scopus (248) Google Scholar]. Biel and Bopp [3Biel J.H. Bopp B.A. Amphetamines: structure–activity relationships.in: Iversen L.L. Handbook of Psychopharmacology: Stimulants. Plenum, 1978: 1-40Google Scholar] chemically defined an amphetamine as containing: (i) an unsubstituted phenyl ring, (ii) a two-carbon side chain between the phenyl ring and nitrogen, (iii) an α-methyl group, and (iv) a primary amino group (Box 1). Many compounds meet these criteria. In addition, the targets (i.e., the monoamine transporters) apparently interpret the rules in a flexible manner: for instance, methamphetamine, ephedrine, and methylphenidate violate the primary-amine rule (Box 1). Hence, this large group of compounds is difficult to define on chemical grounds. An operational definition based on the pharmacology ('amphetamine-like action') falls short of accounting for the complex mode of action: in fact, there is a continuum ranging from amphetamine-triggered release to methylphenidate-induced blockage of uptake. A clear-cut definition would facilitate imposing legal restrictions on marketing activities. The difficulties arising from a chemistry-based definition are also exemplified by cathinones: the naturally occurring cathinone is a ketone. It serves as a scaffold for numerous substitutions: methylation produces meth(yl)cathinone, which allows regulations restricting the sales of cathinone to be bypassed. However, cathinone derivatives are not only used illicitly: they include the antidepressant drug bupropion and the anorectic agent diethylpropion. Synthetic analogs of methcathinone have recently become popular as 'designer drugs' or 'legal highs' on the illicit drug market [4Rosenbaum C.D. et al.Here today, gone tomorrow.. and back again? A review of herbal marijuana alternatives (K2, Spice), synthetic cathinones (bath salts), kratom, Salvia divinorum, methoxetamine, and piperazines.J. Med. Toxicol. 2012; 8: 15-32Crossref PubMed Scopus (289) Google Scholar]. Often, they are referred to as 'bath salts', 'plant food', or 'research chemicals' to facilitate their distribution and to obviate prosecution. Popular methcathinone derivatives are 4-methylmethcathinone (mephedrone, Box 1) and 3,4-methylenedioxymethcathinone (methylone). More recently, 3,4-methylenedioxypyrrovalerone (MDPV, Box 1) has reached the market; it is an example of a very potent, psychoactive synthetic cathinone and illustrates the problem of a laissez-faire approach to recreational drug use: MDPV exerts stimulant-like effects at low doses, but life-threatening side effects are seen at high doses or upon chronic use [5Ross E.A. et al.'Bath salts' intoxication.N. Engl. J. Med. 2011; 365: 967-968Crossref PubMed Scopus (96) Google Scholar]. Accordingly, the authorities have banned amphetamines including the first-generation synthetic cathinones such as mephedrone, methylone, and MDPV in the USA and the EU.Box 1Structural differences between amphetaminesThe chemical definition by Biel and Bopp [3Biel J.H. Bopp B.A. Amphetamines: structure–activity relationships.in: Iversen L.L. Handbook of Psychopharmacology: Stimulants. Plenum, 1978: 1-40Google Scholar] posits the presence of (i) an unsubstituted phenyl ring, (ii) a two-carbon side chain between the phenyl ring and a nitrogen, (iii) an α-methyl group, and (iv) a primary amino group in a compound to qualify as an amphetamine (Figure I). Panel (A) illustrates this rule. Panel (B) shows the trace amine phenethylamine which is produced in higher organisms, while panels (C,D) show the structures of the plant alkaloids ephedrine and cathinone. Panels (E–I) show the structural diversity in amphetamines, which either conform to (E) or violate the chemical definition of Biel and Bopp rule (F–I). Accordingly, amphetamines are a diverse group of compounds that target the transporters for monoamine neurotransmitters, but do not engage their cognate receptors [75Sulzer D. How addictive drugs disrupt presynaptic dopamine neurotransmission.Neuron. 2011; 69: 628-649Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar]. Moreover, the individual structural differences between the different amphetamines highlight the specificity between monoamine transporters, for example the SERT over DAT selectivity of pCA and fenfluramine [1Sulzer D. et al.Mechanisms of neurotransmitter release by amphetamines: a review.Prog. Neurobiol. 2005; 75: 406-433Crossref PubMed Scopus (929) Google Scholar, 76Baumann M.H. et al.Evidence for a role of transporter-mediated currents in the depletion of brain serotonin induced by serotonin transporter substrates.Neuropsychopharmacology. 2014; 39: 1355-1365Crossref PubMed Scopus (28) Google Scholar], or the DAT-preference of D-amphetamine [75Sulzer D. How addictive drugs disrupt presynaptic dopamine neurotransmission.Neuron. 2011; 69: 628-649Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar]. However, amphetamines also bind to non-monoamine transporter targets such as adrenergic receptors [1Sulzer D. et al.Mechanisms of neurotransmitter release by amphetamines: a review.Prog. Neurobiol. 2005; 75: 406-433Crossref PubMed Scopus (929) Google Scholar] or trace amine receptors [77Bunzow J.R. et al.Amphetamine, 3,4-methylenedioxymethamphetamine, lysergic acid diethylamide, and metabolites of the catecholamine neurotransmitters are agonists of a rat trace amine receptor.Mol. Pharmacol. 2001; 60: 1181-1188Crossref PubMed Scopus (484) Google Scholar]. These receptors form complexes with transporters [78Xie Z. Miller G.M. Trace amine-associated receptor 1 is a modulator of the dopamine transporter.J. Pharmacol. Exp. Ther. 2007; 321: 128-136Crossref PubMed Scopus (97) Google Scholar]; trace amine-associated receptor (TAR) agonists (including amphetamines) inhibit uptake [79Revel F.G. et al.Brain-specific overexpression of trace amine-associated receptor 1 alters monoaminergic neurotransmission and decreases sensitivity to amphetamine.Neuropsychopharmacology. 2012; 37: 2580-2592Crossref PubMed Scopus (81) Google Scholar, 80Wolinsky T.D. et al.The Trace Amine 1 receptor knockout mouse: an animal model with relevance to schizophrenia.Genes Brain Behav. 2007; 6: 628-639Crossref PubMed Scopus (174) Google Scholar]. These observations point to a possible role of TARs in the treatment of amphetamine addiction. The compound structures are reproduced from the PubChem database. The chemical definition by Biel and Bopp [3Biel J.H. Bopp B.A. Amphetamines: structure–activity relationships.in: Iversen L.L. Handbook of Psychopharmacology: Stimulants. Plenum, 1978: 1-40Google Scholar] posits the presence of (i) an unsubstituted phenyl ring, (ii) a two-carbon side chain between the phenyl ring and a nitrogen, (iii) an α-methyl group, and (iv) a primary amino group in a compound to qualify as an amphetamine (Figure I). Panel (A) illustrates this rule. Panel (B) shows the trace amine phenethylamine which is produced in higher organisms, while panels (C,D) show the structures of the plant alkaloids ephedrine and cathinone. Panels (E–I) show the structural diversity in amphetamines, which either conform to (E) or violate the chemical definition of Biel and Bopp rule (F–I). Accordingly, amphetamines are a diverse group of compounds that target the transporters for monoamine neurotransmitters, but do not engage their cognate receptors [75Sulzer D. How addictive drugs disrupt presynaptic dopamine neurotransmission.Neuron. 2011; 69: 628-649Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar]. Moreover, the individual structural differences between the different amphetamines highlight the specificity between monoamine transporters, for example the SERT over DAT selectivity of pCA and fenfluramine [1Sulzer D. et al.Mechanisms of neurotransmitter release by amphetamines: a review.Prog. Neurobiol. 2005; 75: 406-433Crossref PubMed Scopus (929) Google Scholar, 76Baumann M.H. et al.Evidence for a role of transporter-mediated currents in the depletion of brain serotonin induced by serotonin transporter substrates.Neuropsychopharmacology. 2014; 39: 1355-1365Crossref PubMed Scopus (28) Google Scholar], or the DAT-preference of D-amphetamine [75Sulzer D. How addictive drugs disrupt presynaptic dopamine neurotransmission.Neuron. 2011; 69: 628-649Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar]. However, amphetamines also bind to non-monoamine transporter targets such as adrenergic receptors [1Sulzer D. et al.Mechanisms of neurotransmitter release by amphetamines: a review.Prog. Neurobiol. 2005; 75: 406-433Crossref PubMed Scopus (929) Google Scholar] or trace amine receptors [77Bunzow J.R. et al.Amphetamine, 3,4-methylenedioxymethamphetamine, lysergic acid diethylamide, and metabolites of the catecholamine neurotransmitters are agonists of a rat trace amine receptor.Mol. Pharmacol. 2001; 60: 1181-1188Crossref PubMed Scopus (484) Google Scholar]. These receptors form complexes with transporters [78Xie Z. Miller G.M. Trace amine-associated receptor 1 is a modulator of the dopamine transporter.J. Pharmacol. Exp. Ther. 2007; 321: 128-136Crossref PubMed Scopus (97) Google Scholar]; trace amine-associated receptor (TAR) agonists (including amphetamines) inhibit uptake [79Revel F.G. et al.Brain-specific overexpression of trace amine-associated receptor 1 alters monoaminergic neurotransmission and decreases sensitivity to amphetamine.Neuropsychopharmacology. 2012; 37: 2580-2592Crossref PubMed Scopus (81) Google Scholar, 80Wolinsky T.D. et al.The Trace Amine 1 receptor knockout mouse: an animal model with relevance to schizophrenia.Genes Brain Behav. 2007; 6: 628-639Crossref PubMed Scopus (174) Google Scholar]. These observations point to a possible role of TARs in the treatment of amphetamine addiction. The compound structures are reproduced from the PubChem database. Ignorance is blatant with respect to new psychoactive substances (NPS) which flood the drug market at present (Box 2). Chemical substitutions may render a given drug compatible with current legislation, but they may also introduce dramatic changes in the activity of the compound and in its mode of action. Widespread consumption of NPS may be associated with health risks such as neurodegeneration. These are linked – at least in part – to the mode of action. Accordingly, it is of relevance to elucidate the different modes of action of amphetamine, its congeners, and in particular of stimulant NPS. The main targets of amphetamines (and presumably of many stimulant NPS) are the neurotransmitter:sodium symporters (NSS) for the monoamines dopamine (DAT/solute carrier protein SLC6A3), serotonin (SERT/SLC6A4), and noradrenaline/norepinephrine (NET/SLC6A2). Amphetamines are exogenous substrates of and generate a current through these transporters [6Robertson S.D. et al.A closer look at amphetamine-induced reverse transport and trafficking of the dopamine and norepinephrine transporters.Mol. Neurobiol. 2009; 39: 73-80Crossref PubMed Scopus (139) Google Scholar]. The action of amphetamine has been studied for more than 100 years [2Barger G. Dale H.H. Chemical structure and sympathomimetic action of amines.J. Physiol. 1910; 41: 19-59Crossref PubMed Scopus (248) Google Scholar]. Nevertheless, there are still many open questions: (i) how do these compounds differ from physiological substrates to act as efficient releasers? (ii) What is the role of protein kinases in triggering efflux? (iii) Does the oligomeric arrangement of monoamine transporters affect the action of amphetamine? (iv) Why does amphetamine elicit a blocking (paradoxical) action on disease-associated mutants of, for example, the dopamine transporter/SLC6A3? (v) Does the lipid composition of the plasma membrane affect the action of amphetamine? In this review we address these issues and argue that their resolution is important to understanding the differences in the actions of individual compounds.Box 2New psychoactive substances: scrutinizing their mode of actionThe term 'new psychoactive substances' (NPS) refers to a wide variety of compounds which elicit psychotomimetic effects. These drugs include stimulants that are congeners of amphetamine or synthetic cathinones. Their street market names 'designer drugs', 'bath salts', 'plant food', or 'research chemicals' imply an innocuous recreational consumption. However, NPS is not restricted to compounds that act like amphetamine: also cannabimimetics, sedative-hypnotics, and also hallucinogens such as 2-CB (2,5-dimethoxy-4-bromophenethylamine), a partial agonist at 5HT2A-receptors, and related receptor ligands are also classified as NPS [81Baumann M.H. et al.Psychoactive 'bath salts': not so soothing.Eur. J. Pharmacol. 2013; 698: 1-5Crossref PubMed Scopus (153) Google Scholar]. NPS are not harmless: 4-methyl-N-methylcathinone (mephedrone) and 3,4-methylenedioxypyrrovalerone (MDPV) do not only exert psychostimulant effects – they cause deleterious side effects at higher doses [82Dargan P.I. et al.The pharmacology and toxicology of the synthetic cathinone mephedrone (4-methylmethcathinone).Drug Test. Anal. 2011; 3: 454-463Crossref PubMed Scopus (114) Google Scholar]. Therefore, the marketing and the consumption of these drugs have been prohibited in many countries. However, this ban predictably drives an evolutionary arms race: ingenuous chemists create novel compounds and thus circumvent the legislation. Currently, there is a surge of NPS on the drug market, including some 'second-generation' cathinones and congeners. As with all novel active compounds – whether developed for the legal or the illicit drug market – it is worthwhile to elucidate the principal mechanism of action. This claim is supported by the following line of arguments. In the case of NPS, the candidate targets are monoamine transporters. It is necessary to explore whether the new substance acts as a releasing or an inhibiting agent. Both types of drugs increase the synaptic concentrations of monoamines in central nervous tissue, but they differ significantly in their mode of action: substrates induce transporter-mediated sodium currents (i.e., depolarization [1Sulzer D. et al.Mechanisms of neurotransmitter release by amphetamines: a review.Prog. Neurobiol. 2005; 75: 406-433Crossref PubMed Scopus (929) Google Scholar]) and initiate transporter-mediated monoamine efflux (i.e., reverse transport or release), whereas blockers do not. Importantly, amphetamine-associated depolarization puts neurons at risk: in fact, the use of fenfluramines has been associated not only with neuronal depletion of 5-HT [76Baumann M.H. et al.Evidence for a role of transporter-mediated currents in the depletion of brain serotonin induced by serotonin transporter substrates.Neuropsychopharmacology. 2014; 39: 1355-1365Crossref PubMed Scopus (28) Google Scholar], but also with pulmonary hypertension and valvular heart disease [83Rothman R.B. Baumann M.H. Therapeutic and adverse actions of serotonin transporter substrates.Pharmacol. Ther. 2002; 95: 73-88Crossref PubMed Scopus (152) Google Scholar]. The latter arises from the long-term effect of serotonin on the pulmonary vasculature.Two simple diagnostic assays can be employed to assess the nature of the compound under scrutiny: (i) electrophysiological recording of transport-associated currents and/or (ii) examination of the initiation of reverse transport comparing control conditions and intracellular high-sodium conditions. The first experiment employs either cells or Xenopus laevis oocytes, which heterologously express monoamine transporters, and compares the concentration–response curve of the NPS to that of reference compounds. The second experiment relies on the elevation of intracellular sodium concentrations by blockage of the Na+/K+-ATPase, or by applying the Na+/H+-ionophore monensin which selectively exchanges Na+ for H+ [14Scholze P. et al.Transporter-mediated release: a superfusion study on human embryonic kidney cells stably expressing the human serotonin transporter.J. Pharmacol. Exp. Ther. 2000; 293: 870-878PubMed Google Scholar]: under these conditions a bona fide releaser potentiates release, while a blocker is inactive (for an example see the characterization of MDPV in [84Baumann M.H. et al.Powerful cocaine-like actions of 3,4-methylenedioxypyrovalerone (MDPV), a principal constituent of psychoactive 'bath salts' products.Neuropsychopharmacology. 2013; 38: 552-562Crossref PubMed Scopus (336) Google Scholar].The illicit drug market provides a powerful incentive to circumvent the legal ban via chemical modifications. NPS are actually predicted to increase in numbers [85Leffler A.M. et al.The analytical investigation of synthetic street drugs containing cathinone analogs.Forensic Sci. Int. 2014; 234: 50-56Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar]. Furthermore, street drugs are increasingly adulterated with a plethora of different compounds, some of which may lead to most severe side effects [86Hofmaier T. et al.Aminorex, a metabolite of the cocaine adulterant levamisole, exerts amphetamine like actions at monoamine transporters.Neurochem. Int. 2014; 73: 32-41Crossref PubMed Scopus (78) Google Scholar]. This justifies the effort to not only analyze the composition of street drugs but also the mode of action of these combinations [86Hofmaier T. et al.Aminorex, a metabolite of the cocaine adulterant levamisole, exerts amphetamine like actions at monoamine transporters.Neurochem. Int. 2014; 73: 32-41Crossref PubMed Scopus (78) Google Scholar, 87Rosenauer R. et al.A combined approach using transporter-flux assays and mass spectrometry to examine psychostimulant street drugs of unknown content.ACS Chem. Neurosci. 2013; 4: 182-190Crossref PubMed Scopus (18) Google Scholar]. The term 'new psychoactive substances' (NPS) refers to a wide variety of compounds which elicit psychotomimetic effects. These drugs include stimulants that are congeners of amphetamine or synthetic cathinones. Their street market names 'designer drugs', 'bath salts', 'plant food', or 'research chemicals' imply an innocuous recreational consumption. However, NPS is not restricted to compounds that act like amphetamine: also cannabimimetics, sedative-hypnotics, and also hallucinogens such as 2-CB (2,5-dimethoxy-4-bromophenethylamine), a partial agonist at 5HT2A-receptors, and related receptor ligands are also classified as NPS [81Baumann M.H. et al.Psychoactive 'bath salts': not so soothing.Eur. J. Pharmacol. 2013; 698: 1-5Crossref PubMed Scopus (153) Google Scholar]. NPS are not harmless: 4-methyl-N-methylcathinone (mephedrone) and 3,4-methylenedioxypyrrovalerone (MDPV) do not only exert psychostimulant effects – they cause deleterious side effects at higher doses [82Dargan P.I. et al.The pharmacology and toxicology of the synthetic cathinone mephedrone (4-methylmethcathinone).Drug Test. Anal. 2011; 3: 454-463Crossref PubMed Scopus (114) Google Scholar]. Therefore, the marketing and the consumption of these drugs have been prohibited in many countries. However, this ban predictably drives an evolutionary arms race: ingenuous chemists create novel compounds and thus circumvent the legislation. Currently, there is a surge of NPS on the drug market, including some 'second-generation' cathinones and congeners. As with all novel active compounds – whether developed for the legal or the illicit drug market – it is worthwhile to elucidate the principal mechanism of action. This claim is supported by the following line of arguments. In the case of NPS, the candidate targets are monoamine transporters. It is necessary to explore whether the new substance acts as a releasing or an inhibiting agent. Both types of drugs increase the synaptic concentrations of monoamines in central nervous tissue, but they differ significantly in their mode of action: substrates induce transporter-mediated sodium currents (i.e., depolarization [1Sulzer D. et al.Mechanisms of neurotransmitter release by amphetamines: a review.Prog. Neurobiol. 2005; 75: 406-433Crossref PubMed Scopus (929) Google Scholar]) and initiate transporter-mediated monoamine efflux (i.e., reverse transport or release), whereas blockers do not. Importantly, amphetamine-associated depolarization puts neurons at risk: in fact, the use of fenfluramines has been associated not only with neuronal depletion of 5-HT [76Baumann M.H. et al.Evidence for a role of transporter-mediated currents in the depletion of brain serotonin induced by serotonin transporter substrates.Neuropsychopharmacology. 2014; 39: 1355-1365Crossref PubMed Scopus (28) Google Scholar], but also with pulmonary hypertension and valvular heart disease [83Rothman R.B. Baumann M.H. Therapeutic and adverse actions of serotonin transporter substrates.Pharmacol. Ther. 2002; 95: 73-88Crossref PubMed Scopus (152) Google Scholar]. The latter arises from the long-term effect of serotonin on the pulmonary vasculature. Two simple diagnostic assays can be employed to assess the nature of the compound under scrutiny: (i) electrophysiological recording of transport-associated currents and/or (ii) examination of the initiation of reverse transport comparing control conditions and intracellular high-sodium conditions. The first experiment employs either cells or Xenopus laevis oocytes, which heterologously express monoamine transporters, and compares the concentration–response curve of the NPS to that of reference compounds. The second experiment relies on the elevation of intracellular sodium concentrations by blockage of the Na+/K+-ATPase, or by applying the Na+/H+-ionophore monensin which selectively exchanges Na+ for H+ [14Scholze P. et al.Transporter-mediated release: a superfusion study on human embryonic kidney cells stably expressing the human serotonin transporter.J. Pharmacol. Exp. Ther. 2000; 293: 870-878PubMed Google Scholar]: under these conditions a bona fide releaser potentiates release, while a blocker is inactive (for an example see the characterization of MDPV in [84Baumann M.H. et al.Powerful cocaine-like actions of 3,4-methylenedioxypyrovalerone (MDPV), a principal constituent of psychoactive 'bath salts' products.Neuropsychopharmacology. 2013; 38: 552-562Crossref PubMed Scopus (336) Google Scholar]. The illicit drug market provides a powerful incentive to circumvent the legal ban via chemical modifications. NPS are actually predicted to increase in numbers [85Leffler A.M. et al.The analytical investigation of synthetic street drugs containing cathinone analogs.Forensic Sci. Int. 2014; 234: 50-56Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar]. Furthermore, street drugs are increasingly adulterated with a plethora of different compounds, some of which may lead to most severe side effects [86Hofmaier T. et al.Aminorex, a metabolite of the cocaine adulterant levamisole, exerts amphetamine like actions at monoamine transporters.Neurochem. Int. 2014; 73: 32-41Crossref PubMed Scopus (78) Google Scholar]. This justifies the effort to not only analyze the composition of street drugs but also the mode of action of these combinations [86Hofmaier T. et al.Aminorex, a metabolite of the cocaine adulterant levamisole, exerts amphetamine like actions at monoamine transporters.Neurochem. Int. 2014; 73: 32-41Crossref PubMed Scopus (78) Google Scholar, 87Rosenauer R. et al.A combined approach using transporter-flux assays and mass spectrometry to examine psychostimulant street drugs of unknown content.ACS Chem. Neurosci. 2013; 4: 182-190Crossref PubMed Scopus (18) Google Scholar]. In monoaminergic neurons, retrieval of neurotransmitters into the synaptic vesicles is accomplished by a relay that is made up of several components (Figure 1): (i) vesicles are tethered to the plasmalemmal monoamine transporter (DAT, SERT, or NET). The resulting spatial proximity promotes efficient (re)filling of the docked vesicle at the reuptake site [7Egana L.A. et al.Physical and functional interaction between the dopamine transporter and the synaptic vesicle protein synaptogyrin-3.J. Neurosci. 2009; 29: 4592-4604Crossref PubMed Scopus (104) Google Scholar]. (ii) The turnover number of the reserpine-sensitive, proton-driven vesicular monoamine transporter VMAT2 (400 min−1) [8Peter D. et al.The chromaffin granule and synaptic vesicle amine transporters differ in substrate recognition and sensitivity to inhibitors.J. Biol. Chem. 1994; 269: 7231-7237Abstract Full Text PDF PubMed Google Scholar] is higher than that of the plasmalemmal transporters (120–180 min−1) [9Erreger K. et al.Currents in response to rapid concentration jumps of amphetamine uncover novel aspects of human dopamine transporter function.J. Neurosci. 2008; 28: 976-989Crossref PubMed Scopus (54) Google Scholar, 10Sucic S. et al.The N terminus of monoamine transporters is a lever required for the action of amphetamines.J. Biol. Chem. 2010; 285: 10924-10938Crossref PubMed Scopus (107) Google Scholar]. This creates a sink and limits diffusion of monoamines within the cytosol of the presynaptic bouton. (iii) This is further enhanced by the action of mitochondrial monoamine oxidases (MAO-A and B) and catechol O-methyltransferase (COMT) [11Seiden L.S. et al.Amphetamine: effects on catecholamine systems and behavior.Annu. Rev. Pharmacol. Toxicol. 1993; 33: 639-677Crossref PubMed Google Scholar] which degrade cytosolic monoamines. Amphetamines interact with several targets within this relay; in other words, they are exogenous substrates of the plasmalemmal transporters (DAT, SERT, NET), and of the vesicular transporters VMAT1 and VMAT2, and inhibit MAO. The concerted action of amphetamines on these three targets is the core tenet of the 'weak base hypothesis' [12Sulzer D. et al.Amphetamine redistributes dopamine from synaptic vesicles to the cytosol and promotes reverse transport.J. Neurosci. 1995; 15: 4102-4108Crossref PubMed Google Scholar, 13Sulzer D. et al.Amphetamine and other weak bases act to promote reverse transport of dopamine in ventral midbrain neurons.J. Neurochem. 1993; 60: 527-535Crossref PubMed Scopus (253) Google Scholar]: amphetamines enter monoaminergic terminals via DAT, SERT, or NET and subsequently accumulate in the synaptic vesicles by the action of VMATs. Therein, by their physicochemical nature, they dissipate the proton gradient and preclude inward transport of monoamines. The resulting elevation in cytosolic monoamine neurotransmitters is further aggravated by MAO inhibition. Thus, they rise to concentrations that allow occupation of the internal substrate binding site [14Scholze P. et al.Transporter-mediated release: a superfusion study on human embryonic kidney cells stably expressing the human serotonin transporter.J. Pharmacol. Exp. Ther. 2000; 293: 870-878PubMed Google Scholar] and their subsequent outward transport by plasmalemmal transporters. Amphetamines als