Allosteric Modulation of Neurotransmitter Transporters as a Therapeutic Strategy
Authors: Marco Niello, Ralph Gradisch, Claus Juul Loland, Thomas Stockner, Harald H. Sitte
Journal: Trends in Pharmacological Sciences (2020)

Current research is showing an increasing heterogeneity in the binding sites available in NTTs. These binding sites are often located distant from the central binding sites, suggesting a higher level of complexity in the regulation of NTT function.Allosteric sites increase specificity, reducing off-target effects.Small molecules can be developed to target NTT allosteric sites, with the possibility to either increase or decrease their functions.NTT allosteric modulators are in advanced clinical trials, emphasizing their potential for the treatment of neuropsychiatric disorders. Neurotransmitter transporters (NTTs) are involved in the fine-tuning of brain neurotransmitter homeostasis. As such, they are implicated in a plethora of complex behaviors, including reward, movement, and cognition. During recent decades, compounds that modulate NTT functions have been developed. Some of them are in clinical use for the management of different neuropsychiatric conditions. The majority of these compounds have been found to selectively interact with the orthosteric site of NTTs. Recently, diverse allosteric sites have been described in a number of NTTs, modulating their function. A more complex NTT pharmacology may be useful in the development of novel therapeutics. Here, we summarize current knowledge on such modulatory allosteric sites, with specific focus on their pharmacological and therapeutic potential. Neurotransmitter transporters (NTTs) are involved in the fine-tuning of brain neurotransmitter homeostasis. As such, they are implicated in a plethora of complex behaviors, including reward, movement, and cognition. During recent decades, compounds that modulate NTT functions have been developed. Some of them are in clinical use for the management of different neuropsychiatric conditions. The majority of these compounds have been found to selectively interact with the orthosteric site of NTTs. Recently, diverse allosteric sites have been described in a number of NTTs, modulating their function. A more complex NTT pharmacology may be useful in the development of novel therapeutics. Here, we summarize current knowledge on such modulatory allosteric sites, with specific focus on their pharmacological and therapeutic potential. Synaptic transmission is a fundamental feature of the communication between neurons [1.Pereda A.E. Electrical synapses and their functional interactions with chemical synapses.Nat. Rev. Neurosci. 2014; 15: 250-263Crossref PubMed Scopus (175) Google Scholar]. After the quantal presynaptic release of neurotransmitter into the synaptic cleft, binding to pre- and postsynaptic receptors follows. Subsequently, the signal is quickly terminated by efficient reuptake of the neurotransmitter back into the presynaptic neuron (Figure 1A ). Reuptake is tightly regulated by neurotransmitter transporters (NTTs), a class of complex molecules, each holding specificity for only one or a few neurotransmitters. For instance, the dopamine and norepinephrine transporter (DAT and NET, respectively) can efficiently uptake both dopamine and norepinephrine with similar kinetics [2.Gu H. et al.Stable expression of biogenic amine transporters reveals differences in inhibitor sensitivity, kinetics, and ion dependence.J. Biol. Chem. 1994; 269: 7124-7130Abstract Full Text PDF PubMed Google Scholar,3.Morón J.A. et al.Dopamine uptake through the norepinephrine transporter in brain regions with low levels of the dopamine transporter: evidence from knock-out mouse lines.J. Neurosci. 2002; 22: 389-395Crossref PubMed Google Scholar], while the serotonin transporter (SERT) can efficiently transport serotonin but only binds dopamine with very low affinity [4.Larsen M.B. et al.Dopamine transport by the serotonin transporter: a mechanistically distinct mode of substrate translocation.J. Neurosci. 2011; 31: 6605-6615Crossref PubMed Scopus (0) Google Scholar]. Once inside the presynaptic neuron, the neurotransmitters are, in general, rapidly confined into vesicles by specific vesicular transporters (Figure 1A, grey transporter), in order to prevent toxicity [5.Blakely R.D. Edwards R.H. Vesicular and plasma membrane transporters for neurotransmitters.Cold Spring Harb. Perspect. Biol. 2012; 4: a005595Crossref PubMed Scopus (69) Google Scholar]. The rapid removal of neurotransmitters from extracellular fluids avoids an exaggerated signal and confines the neurotransmitters within the intended area, preventing chemical crosstalk [6.Focke P.J. et al.Neurotransmitter transporters: structure meets function.Structure. 2013; 21: 694-705Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar] (Figure 1). Because of their important role in synaptic transmission, dysfunctional or improper regulation of the NTTs contribute to neurological and neuropsychiatric disorders [7.Gether U. et al.Neurotransmitter transporters: molecular function of important drug targets.Trends Pharmacol. Sci. 2006; 27: 375-383Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar]. Therefore, NTTs are also clinically relevant pharmacological targets for the treatment of major neuropsychiatric diseases, such as depression, attention-deficit hyperactivity disorder, epilepsy, and narcolepsy [8.Kristensen A.S. et al.SLC6 neurotransmitter transporters: structure, function, and regulation.Pharmacol. Rev. 2011; 63: 585-640Crossref PubMed Scopus (420) Google Scholar]. All NTTs require the concentrating power of specific ions in order for the substrate to accumulate on its respective side of the membranes. The type and the stoichiometry of different ions vary among NTTs. In general, Na+ and Cl– are required in the case of DAT, NET, and SERT, and Na+ and K+ in the case of the excitatory amino acid transporters (EAATs) 1–5. Ionic concentration underlies the ability of the NTTs to accumulate substrate on one side of the cellular membrane [8.Kristensen A.S. et al.SLC6 neurotransmitter transporters: structure, function, and regulation.Pharmacol. Rev. 2011; 63: 585-640Crossref PubMed Scopus (420) Google Scholar,9.Amara S.G. Kuhar M.J. Neurotransmitter transporters: recent progress.Annu. Rev. Neurosci. 1993; 16: 73-93Crossref PubMed Google Scholar]. NTT structure can vary quite a lot between different classes of transporter, for instance, DAT, NET, and SERT are organized in 12 transmembrane helixes (TM), while other NTTs such as EAATs are instead organized in eight TMs. These TMs are connected by extracellular and intracellular loops, which work as regulatory sites through different post-translational modifications such as phosphorylation and glycosylation [10.Vaughan R.A. Foster J.D. Mechanisms of dopamine transporter regulation in normal and disease states.Trends Pharmacol. Sci. 2013; 34: 489-496Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar,11.Bermingham D.P. Blakely R.D. Kinase-dependent regulation of monoamine neurotransmitter transporters.Pharmacol. Rev. 2016; 68: 888-953Crossref PubMed Scopus (32) Google Scholar]. As far as NTTs are concerned, the 3D organization of the different TMs and loops determine a complex structure composed of a vestibule leading to the central binding site confined between an extracellular gate and an intracellular gate (Figure 1B) [8.Kristensen A.S. et al.SLC6 neurotransmitter transporters: structure, function, and regulation.Pharmacol. Rev. 2011; 63: 585-640Crossref PubMed Scopus (420) Google Scholar,12.Grewer C. et al.SLC1 glutamate transporters.Pflugers Arch. 2014; 466: 3-24Crossref PubMed Scopus (0) Google Scholar]. The dynamics of this structure follow the so-called 'alternate access model': upon binding of substrate and ions, an extracellular gate seals off the binding site and the protein alternates conformation between outward- and inward-facing modes, resulting in the transport of the substrate across the membrane [13.Jardetzky O. Simple allosteric model for membrane pumps.Nature. 1966; 211: 969-970Crossref PubMed Scopus (719) Google Scholar] (Figure 1B, bottom). This alternating process has been suggested to pass through different intermediate configurations, which are regulated in concert by the substrate and ions and is known as 'the transport cycle' [14.Hasenhuetl P.S. et al.Functional selectivity and partial efficacy at the monoamine transporters: a unified model of allosteric modulation and amphetamine-induced substrate release.Mol. Pharmacol. 2019; 95: 303-312Crossref PubMed Scopus (2) Google Scholar,15.Burtscher V. et al.Kinetic models of secondary active transporters.Int. J. Mol. Sci. 2019; 20E5365Crossref PubMed Scopus (0) Google Scholar]. NTTs are secondary active transporters and therefore they couple substrate transport to the electrochemical gradient of Na+. Most of them cotransport Cl– and some of them counter-transport K+ or H+ [8.Kristensen A.S. et al.SLC6 neurotransmitter transporters: structure, function, and regulation.Pharmacol. Rev. 2011; 63: 585-640Crossref PubMed Scopus (420) Google Scholar]. In the simplified version of the transport cycle (see Figure IC in Box 1), the transporter in the outward-facing conformation (To) needs to first bind Na+ (ToNa) and then the substrate (ToNaS) before it can transition to the inward-facing conformation (TiNaS). At this point, since the internal concentration of Na+ and S is very low, they can dissociate from the transporter (TiS and Ti) and the transporter in the inward-facing conformation can now return to the outward-facing conformation (To). In some cases, other ions (e.g., K+ and/or H+ in the case of SERT [16.Hasenhuetl P.S. et al.Electrogenic binding of intracellular cations defines a kinetic decision point in the transport cycle of the human serotonin transporter.J. Biol. Chem. 2016; 291: 25864-25876Crossref PubMed Scopus (15) Google Scholar]) can modulate the dissociation of the substrate (S) internally and therefore regulate the efficiency of the transport process.Box 1Methods for Studying Allosteric Modulation of TransportersHill-Langmuir EquationThe Hill-Langmuir equation describes the occupancy of a receptor by its ligand.θ=LηKd+Lη=11+KALηwhere θ is the fraction of the receptor–ligand complex, [L] is the concentration of unbound ligand, Kd is the dissociation constant, Ka is the ligand concentration producing half occupation, and η is the Hill coefficient assuming any positive value [53.Hill A.V. The combinations of haemoglobin with oxygen and with carbon monoxide. I.Biochem. J. 1913; 7: 471-480Crossref PubMed Google Scholar,131.Hill A.V. The possible effects of the aggregation of the molecules of haemoglobin on its oxygen dissociation curve.J. Physiol. 1910; 40: iv-viiGoogle Scholar]; η describes the degree of cooperativity between multiple binding sites for the same ligand. With η = 1 there is no cooperativity, with η > 1 the cooperativity is positive, and with η < 1 it is negative [53.Hill A.V. The combinations of haemoglobin with oxygen and with carbon monoxide. I.Biochem. J. 1913; 7: 471-480Crossref PubMed Google Scholar] (Figure IA).Uptake (Saturation) ExperimentsClassical uptake-saturation experiments are conducted in the presence of different concentrations of a putative allosteric ligand. The Michaelis-Menten parameters (Km and Vmax) are derived from the dose response curves using a linearized form of the Lineweaver-Burk plot or the Eadie-Hofstee diagram. Direct nonlinear regression analysis of the dose response data allows for alleviating the limitations of the linearized equations. Extrapolation of the Lineweaver-Burk plot to the abscissa for two measurements in the presence or absence of a second ligand allows for a simple detection of competitive or noncompetitive interaction (Figure IB).Alternatively, coupling between substrate and ions during the transport process generates transporter-mediated currents (measured electrophysiologically), which can be used as a surrogate of NTT activity (Figure IC). The whole-cell patch clamp approach provides access to the internal side of the cell. By changing the ionic compositions inside and outside of the cell, it is possible to isolate different components of the current (i.e., the steady-state, representing an average of all the transport partial reactions, and the peak, representing the outward-open, ToSNa, to inward-open transition, TiSNa) and to study partial reactions of the transport cycle [14.Hasenhuetl P.S. et al.Functional selectivity and partial efficacy at the monoamine transporters: a unified model of allosteric modulation and amphetamine-induced substrate release.Mol. Pharmacol. 2019; 95: 303-312Crossref PubMed Scopus (2) Google Scholar,23.Burtscher V. et al.A label-free approach to detect ligand binding to cell surface proteins in real time.Elife. 2018; 7e34944Crossref PubMed Scopus (6) Google Scholar] (Figure IC).Dissociation ExperimentsAllosteric ligands can change the dissociation of a prebound inhibitor. In brief, membrane preparations containing the protein of interest are incubated with a high affinity [3H]inhibitor. After the equilibrium is reached, the ligand of interest is added to the preparation and the reaction is stopped at different time-points. If the ligand binds to an allosteric site that increases the affinity in the central binding site, the dissociation rate is reduced [66.Plenge P. et al.Steric hindrance mutagenesis in the conserved extracellular vestibule impedes allosteric binding of antidepressants to the serotonin transporter.J. Biol. Chem. 2012; 287: 39316-39326Crossref PubMed Scopus (60) Google Scholar] (Figure ID).Atomic Force Microscopy (AFM)AFM can be used to study interactions of ligands and proteins at a single molecule level in native tissues. In brief, the tip of a highly sensitive cantilever is functionalized with the putative allosteric ligand and incubated with the cells expressing the desired transporter. Once at equilibrium, the AFM tip is lifted up and the bending of the cantilever allows the quantification of distribution of forces. A binomial distribution is indicative of a secondary binding site [69.Zhu R. et al.Nanopharmacological force sensing to reveal allosteric coupling in transporter binding sites.Angew. Chem. Int. Ed. 2016; 55: 1719-1722Crossref PubMed Scopus (15) Google Scholar,132.Puntheeranurak T. et al.Single-molecule recognition force spectroscopy of transmembrane transporters on living cells.Nat. Protoc. 2011; 6: 1443-1452Crossref PubMed Scopus (34) Google Scholar] (Figure IE). Hill-Langmuir Equation The Hill-Langmuir equation describes the occupancy of a receptor by its ligand.θ=LηKd+Lη=11+KALη where θ is the fraction of the receptor–ligand complex, [L] is the concentration of unbound ligand, Kd is the dissociation constant, Ka is the ligand concentration producing half occupation, and η is the Hill coefficient assuming any positive value [53.Hill A.V. The combinations of haemoglobin with oxygen and with carbon monoxide. I.Biochem. J. 1913; 7: 471-480Crossref PubMed Google Scholar,131.Hill A.V. The possible effects of the aggregation of the molecules of haemoglobin on its oxygen dissociation curve.J. Physiol. 1910; 40: iv-viiGoogle Scholar]; η describes the degree of cooperativity between multiple binding sites for the same ligand. With η = 1 there is no cooperativity, with η > 1 the cooperativity is positive, and with η < 1 it is negative [53.Hill A.V. The combinations of haemoglobin with oxygen and with carbon monoxide. I.Biochem. J. 1913; 7: 471-480Crossref PubMed Google Scholar] (Figure IA). Uptake (Saturation) Experiments Classical uptake-saturation experiments are conducted in the presence of different concentrations of a putative allosteric ligand. The Michaelis-Menten parameters (Km and Vmax) are derived from the dose response curves using a linearized form of the Lineweaver-Burk plot or the Eadie-Hofstee diagram. Direct nonlinear regression analysis of the dose response data allows for alleviating the limitations of the linearized equations. Extrapolation of the Lineweaver-Burk plot to the abscissa for two measurements in the presence or absence of a second ligand allows for a simple detection of competitive or noncompetitive interaction (Figure IB). Alternatively, coupling between substrate and ions during the transport process generates transporter-mediated currents (measured electrophysiologically), which can be used as a surrogate of NTT activity (Figure IC). The whole-cell patch clamp approach provides access to the internal side of the cell. By changing the ionic compositions inside and outside of the cell, it is possible to isolate different components of the current (i.e., the steady-state, representing an average of all the transport partial reactions, and the peak, representing the outward-open, ToSNa, to inward-open transition, TiSNa) and to study partial reactions of the transport cycle [14.Hasenhuetl P.S. et al.Functional selectivity and partial efficacy at the monoamine transporters: a unified model of allosteric modulation and amphetamine-induced substrate release.Mol. Pharmacol. 2019; 95: 303-312Crossref PubMed Scopus (2) Google Scholar,23.Burtscher V. et al.A label-free approach to detect ligand binding to cell surface proteins in real time.Elife. 2018; 7e34944Crossref PubMed Scopus (6) Google Scholar] (Figure IC). Dissociation Experiments Allosteric ligands can change the dissociation of a prebound inhibitor. In brief, membrane preparations containing the protein of interest are incubated with a high affinity [3H]inhibitor. After the equilibrium is reached, the ligand of interest is added to the preparation and the reaction is stopped at different time-points. If the ligand binds to an allosteric site that increases the affinity in the central binding site, the dissociation rate is reduced [66.Plenge P. et al.Steric hindrance mutagenesis in the conserved extracellular vestibule impedes allosteric binding of antidepressants to the serotonin transporter.J. Biol. Chem. 2012; 287: 39316-39326Crossref PubMed Scopus (60) Google Scholar] (Figure ID). Atomic Force Microscopy (AFM) AFM can be used to study interactions of ligands and proteins at a single molecule level in native tissues. In brief, the tip of a highly sensitive cantilever is functionalized with the putative allosteric ligand and incubated with the cells expressing the desired transporter. Once at equilibrium, the AFM tip is lifted up and the bending of the cantilever allows the quantification of distribution of forces. A binomial distribution is indicative of a secondary binding site [69.Zhu R. et al.Nanopharmacological force sensing to reveal allosteric coupling in transporter binding sites.Angew. Chem. Int. Ed. 2016; 55: 1719-1722Crossref PubMed Scopus (15) Google Scholar,132.Puntheeranurak T. et al.Single-molecule recognition force spectroscopy of transmembrane transporters on living cells.Nat. Protoc. 2011; 6: 1443-1452Crossref PubMed Scopus (34) Google Scholar] (Figure IE). The coupling between substrate and ions during the transport process results in the so-called 'transporter-mediated currents' that can be monitored by means of a whole-cell patch clamp (for a detailed review, consider [17.Gerstbrein K. Sitte H.H. Currents in neurotransmitter transporters.Handb. Exp. Pharmacol. 2006; 175: 95-111Crossref Google Scholar]). The typical transporter-mediated current is an inwardly-directed current. These are voltage-dependent and can be elicited by applying the substrate with a perfusion device able to switch between different solutions within 50–100 ms [16.Hasenhuetl P.S. et al.Electrogenic binding of intracellular cations defines a kinetic decision point in the transport cycle of the human serotonin transporter.J. Biol. Chem. 2016; 291: 25864-25876Crossref PubMed Scopus (15) Google Scholar,18.Bhat S. et al.Conformational state interactions provide clues to the pharmacochaperone potential of serotonin transporter partial substrates.J. Biol. Chem. 2017; 292: 16773-16786Crossref PubMed Scopus (14) Google Scholar,19.Li Y. et al.Dual action of Zn2+ on the transport cycle of the dopamine transporter.J. Biol. Chem. 2015; 290: 31069-31076Crossref PubMed Scopus (16) Google Scholar]. When cells are clamped at negative potential and under physiological conditions, they show a so-called 'steady-state current', which is present until the substrate is continuously applied to the cell, then decays back to the baseline as soon as the substrate is no longer present (see Figure IC in Box 1). This has been shown to be representative of the transporter completing the transport cycle and is therefore an average of all the charge movements happening during the transport cycle (see Figure IC in Box 1, bottom, blue and red). In some transporters, a peak current can also be present (see Figure IC in Box 1, top, red). If present, the peak current contains information about the outward-open to the inward-open transition and it can be elicited either by substrate application or by binding of inhibitors [20.Mager S. et al.Conducting states of a mammalian serotonin transporter.Neuron. 1994; 12: 845-859Abstract Full Text PDF PubMed Scopus (201) Google Scholar, 21.Sonders M.S. et al.Multiple ionic conductances of the human dopamine transporter: the actions of dopamine and psychostimulants.J. Neurosci. 1997; 17: 960-974Crossref PubMed Google Scholar, 22.Galli A. et al.Sodium-dependent norepinephrine-induced currents in norepinephrine-transporter-transfected HEK-293 cells blocked by cocaine and antidepressants.J. Exp. Biol. 1995; 198: 2197-2212Crossref PubMed Google Scholar, 23.Burtscher V. et al.A label-free approach to detect ligand binding to cell surface proteins in real time.Elife. 2018; 7e34944Crossref PubMed Scopus (6) Google Scholar]. The whole-cell patch clamp gives access to both the intracellular and the extracellular component. By substituting different ions or introducing compounds into the patch pipette, it is possible to isolate different states of the transport cycle and extract rate constants of all these partial reactions. These constants can then be used for developing kinetic models of the transport cycle [15.Burtscher V. et al.Kinetic models of secondary active transporters.Int. J. Mol. Sci. 2019; 20E5365Crossref PubMed Scopus (0) Google Scholar]. Detailed kinetic models exist already for GABA transporter-1 (GAT-1) [24.Hilgemann D.W. Lu C.C. GAT1 (GABA:Na+:Cl-) cotransport function. Database reconstruction with an alternating access model.J. Gen. Physiol. 1999; 114: 459-475Crossref PubMed Scopus (0) Google Scholar], DAT [25.Erreger 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 (0) Google Scholar], SERT [16.Hasenhuetl P.S. et al.Electrogenic binding of intracellular cations defines a kinetic decision point in the transport cycle of the human serotonin transporter.J. Biol. Chem. 2016; 291: 25864-25876Crossref PubMed Scopus (15) Google Scholar,26.Schicker K. et al.Unifying concept of serotonin transporter-associated currents.J. Biol. Chem. 2012; 287: 438-445Crossref PubMed Scopus (0) Google Scholar,27.Hasenhuetl P.S. et al.A kinetic account for amphetamine-induced monoamine release.J. Gen. Physiol. 2018; 150: 431-451Crossref PubMed Scopus (6) Google Scholar], and glycine transporters GlyT1 [28.Erdem F.A. et al.A comparison of the transport kinetics of glycine transporter 1 and glycine transporter 2.J. Gen. Physiol. 2019; 151: 1035-1050Crossref PubMed Google Scholar] and GlyT2 [28.Erdem F.A. et al.A comparison of the transport kinetics of glycine transporter 1 and glycine transporter 2.J. Gen. Physiol. 2019; 151: 1035-1050Crossref PubMed Google Scholar]. Keeping in mind the information present in such kinetic models during drug development may help in understanding differences between apparently similar drugs and transporters and provide information about allosteric effects [14.Hasenhuetl P.S. et al.Functional selectivity and partial efficacy at the monoamine transporters: a unified model of allosteric modulation and amphetamine-induced substrate release.Mol. Pharmacol. 2019; 95: 303-312Crossref PubMed Scopus (2) Google Scholar,19.Li Y. et al.Dual action of Zn2+ on the transport cycle of the dopamine transporter.J. Biol. Chem. 2015; 290: 31069-31076Crossref PubMed Scopus (16) Google Scholar,29.Niello M. et al.para-Trifluoromethyl-methcathinone is an allosteric modulator of the serotonin transporter.Neuropharmacology. 2019; 161107615Crossref PubMed Scopus (2) Google Scholar,30.Li Y. et al.Occupancy of the zinc-binding site by transition metals decreases the substrate affinity of the human dopamine transporter by an allosteric mechanism.J. Biol. Chem. 2017; 292: 4235-4243Crossref PubMed Scopus (10) Google Scholar]. Not only do transporters participate in the reuptake of neurotransmitters, shaping the input signaling (i.e., receptor activity), but they can also reverse their transport direction and release neurotransmitters in an exocytosis-independent manner and therefore participate in output signals. This process, also known as transporter-mediated 'efflux', has been mainly described for pharmacological agents like amphetamine and congeners targeting monoamine transporters [31.Sitte H.H. Freissmuth M. Amphetamines, new psychoactive drugs and the monoamine transporter cycle.Trends Pharmacol. Sci. 2015; 36: 41-50Abstract Full Text Full Text PDF PubMed Google Scholar]. As substrates of these transporters, they interact with the proton-driven vesicular monoamine transporters (VMATs) and are translocated into vesicles, dissipating their proton gradient and redistributing the vesicle content [32.Freyberg Z. et al.Mechanisms of amphetamine action illuminated through optical monitoring of dopamine synaptic vesicles in Drosophila brain.Nat. Commun. 2016; 7: 10652Crossref PubMed Scopus (37) Google Scholar] (Figure 1A). Concomitantly, amphetamines inhibit the activity of monoamine oxidases (MAO) [33.Mantle T.J. et al.Inhibition of monoamine oxidase by amphetamine and related compounds.Biochem. Pharmacol. 1976; 25: 2073-2077Crossref PubMed Scopus (0) Google Scholar], catechol-O-methyl-transferase (COMT), and activate different kinases (e.g., alpha-CamKII or protein kinase C [34.Reith M.E.A. Gnegy M.E. Molecular Mechanisms of Amphetamines.in: Handbook of Experimental Pharmacology. Springer, Berlin, Heidelberg2019Google Scholar]). The combined actions on the VMAT, MAO, and COMT determine the cytosolic increase in the endogenous neurotransmitter (e.g., dopamine). When the concentration of neurotransmitter is high enough in the presence of Na+, it can bind to the intracellular side of the NTT and translocate to the extracellular space [31.Sitte H.H. Freissmuth M. Amphetamines, new psychoactive drugs and the monoamine transporter cycle.Trends Pharmacol. Sci. 2015; 36: 41-50Abstract Full Text Full Text PDF PubMed Google Scholar]. In the case of SERT, this pharmacologically induced efflux has been recently suggested as a required mechanism for the prosocial effect of 3,4-methylenedioxy-N-methylamphetamine (MDMA), also known as 'ecstasy' [35.Heifets B.D. et al.Distinct neural mechanisms for the prosocial and rewarding properties of MDMA.Sci. Transl. Med. 2019; 11eaaw6435Crossref PubMed Scopus (3) Google Scholar], which is currently under clinical evaluation for the treatment of social anxiety symptoms in post-traumatic disorder [36.Mithoefer M.C. et al.3,4-methylenedioxymethamphetamine (MDMA)-assisted psychotherapy for post-traumatic stress disorder in military veterans, firefighters, and police officers: a randomised, double-blind, dose-response, phase 2 clinical trial.Lancet Psychiatry. 2018; 5: 486-497Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar] and autism [37.Danforth A.L. et al.Reduction in social anxiety after MDMA-assisted psychotherapy with autistic adults: a randomized, double-blind, placebo-controlled pilot study.Psychopharmacology. 2018; 235: 3137-3148Crossref PubMed Scopus (13) Google Scholar]. However, efflux can also happen in a pharmacologically independent way. During ischemia, ATP levels decrease and affect the function of the Na+/K+-ATPase. This leads to an increase of extracellular K+, which in turn reverses glutamate transport and thereby triggers neuronal death due to excitotoxicity [38.Rossi D.J. et al.Glutamate release in severe brain ischaemia is mainly by reversed uptake.Nature. 2000; 403: 316-321Crossref PubMed Scopus (1157) Google Scholar]. In the case of early-onset Parkinsonism, a coding variant has been identified, the DAT-Asp421Asn, characterized by a constitutive, anomalous dopamine efflux [39.Hansen F.H. et al.Missense dopamine transporter mutations associate with adult parkinsonism and ADHD.J. Clin. Invest. 2014; 124: 3107-3120Crossref PubMed Scopus (59) Google Scholar]. A similar alteration in the coding variant DAT-T356M has also been identified in autism [40.Hamilton P.J. et al.De novo mutation in the dopamine transporter gene associates dopamine dysfunction with autism spectrum disorder.Mol. Psychiatry. 2013; 18: 1315-1323Crossref PubMed Scopus (0) Google Scholar,41.DiCarlo G.E. et al.Autism-linked dopamine transporter mutation alters striatal dopamine neurotransmission and dopamine-dependent behaviors.J. Clin. Invest. 2019; 129: 3407-3419Crossref PubMed Scopus (4) Google Scholar]. Taken together, these studies suggest that NTT-mediated efflux could be potentially targeted for the development of future medications. However, future research will be required to expand on this important function of NTTs. The complexity of the NTT–ligand interaction can be gauged by several examples: amphetamine, for instance, is a substrate of monoamine transporters [31.Sitte H.H. Freissmuth M. Amphetamines, new psychoactive drugs and the monoamine transporter cycle.Trends Pharmacol. Sci. 2015; 36: 41-50Abstract Full Text Full Text PDF PubMed Google Scholar]; however, amphetamine