The dopamine transporter (DAT), the norepinephrine transporter (NET), and the serotonin transporter (SERT) are members of the solute carrier six (SLC6) family. DAT, NET, and SERT mediate reuptake of released dopamine, norepinephrine, and serotonin, respectively (Kristensen et al., 2011). By this action, they terminate monoaminergic signaling and—in concert with the vesicular monoamine transporters—replenish vesicular stores. These transporters are secondary active in nature; they utilize the free energy contained in the transmembrane Na⁺ gradient (established by the Na⁺/K⁺ pump) to drive concentrative monoamine uptake into cells in which they are expressed (Burtscher et al., 2019). SLC6 transporters have been postulated to operate via the alternate access mechanism (Jardetzky, 1966): they undergo a closed loop of partial reactions constituting a complete transport cycle (Rudnick and Sandtner, 2019). These partial reactions require conformational rearrangements and binding/unbinding of substrate and co-substrate ions. It is gratifying to note that crystal structures obtained from the prokaryotic homolog LeuT, Drosophila DAT, and human SERT itself support the general concept of alternate access (Yamashita et al., 2005; Penmatsa et al., 2013; Coleman et al., 2016). These crystal structures also reveal SERT and DAT to be closely related. This is evident from the root mean square deviation, which differs by only approximately 1 Å between the outward-facing structure of human SERT and Drosophila DAT. DAT, NET, and SERT also share a rich and partially overlapping pharmacology (Sitte and Freissmuth, 2015): there are many drugs that inhibit transporter function by either blocking or inducing reverse transport. These mechanisms account for therapeutics used for the treatment of neuropsychiatric disorders (major depression, general anxiety disorder, and attention-deficit hyperactivity disorder) and for many illicit drugs that are psychoactive and abused (Hasenhuetl et al., 2019; Niello et al., 2020).

Despite the similarity in structure and function, the three transporters differ in many more aspects than just ligand recognition: the transport stoichiometry of SERT and DAT/NET is considered to be electroneutral and electrogenic, respectively. The crystal structure of both hSERT and dDAT shows two bound Na⁺ ions. However, only one Na⁺ ion is thought to be released on the intracellular side in both the transporters (Rudnick and Sandtner, 2019). Cl^-, on the other hand, has been shown to play a modulatory role in the transport cycle of SERT and DAT, but Cl^- is not essential for the transport stoichiometry (Erreger et al., 2008; Hasenhuetl et al., 2016). However, it has long been known that SERT antiports K_in⁺, that is, intracellular K⁺ (Rudnick and Nelson, 1978); for DAT and NET, K_in⁺ is thought to be immaterial (Gu et al., 1994; Gu et al., 1996; Sonders et al., 1997; Erreger et al., 2008). If true, only SERT can utilize the chemical potential of the cellular K⁺ gradient to establish and maintain a substrate gradient. It has, therefore, remained enigmatic, why closely related transporters can differ so fundamentally in their stoichiometry and their kinetic decision points. The effects of K_in⁺, intracellular Na⁺ (Na_in⁺), and membrane voltage on the transport cycle of SERT have been recently analyzed in great detail (Hasenhuetl et al., 2016). However, much less is known on how these factors impinge on the transport cycle of DAT and NET (Galli et al., 1998; Sonders et al., 1997; Hoffman et al., 1999; Prasad and Amara, 2001; Erreger et al., 2008; Li et al., 2015). In this study, we investigated the role of intracellular cations and voltage on substrate transport through DAT, NET, and SERT. To this end, we simultaneously recorded substrate-induced currents and uptake of the fluorescent substrate APP⁺ (4-(4-dimethylamino)phenyl-1-methylpyridinium) into single HEK293 cells expressing DAT, NET, and SERT under voltage control. These measurements were conducted in the whole-cell patch-clamp configuration, which allowed for control of the intra- and extracellular ion composition via the electrode and bath solution, respectively. Our analysis revealed that K_in⁺ did bind to the inward-facing state of DAT and NET, but, in contrast to SERT, K_in⁺ was released prior to the return step from the substrate-free inward- to the substrate-free outward-facing conformations. We also found that substrate uptake by DAT and NET, unlike SERT, was voltage-dependent under physiological ionic gradients. Moreover, the absence of K_in⁺ had no appreciable effect on the transport rate of DAT and NET. The transient nature of K_in⁺ binding was incorporated into a refined kinetic model, which highlighted the differences between SERT and DAT/NET. Notably, this model allows for a unifying description, which attributes all existing functional differences between DAT, NET, and SERT to the difference in the handling of K_in⁺.

Fluorometric analyses of the SERT, DAT, and NET transport cycle provide mechanistic and functional insights into the modus operandi of these transporters by combining advantages of assays that employ radiolabeled ligands (used to determine global transporter turnover rates) with those that involve electrophysiology (which provide voltage control and unmatched temporal resolution) (Schwartz et al., 2006). Fluorescent substrates of monoamine transporters have previously been used to monitor transporter-mediated uptake in real time (Schwartz et al., 2003; Mason et al., 2005; Schwartz et al., 2005; Oz et al., 2010; Solis et al., 2012; Karpowicz et al., 2013; Wilson et al., 2014; Zwartsen et al., 2017; Cao et al., 2020). One such fluorescent substrate is APP⁺, a fluorescent analog of MPP⁺, a neurotoxin that targets monoaminergic neurons (Javitch et al., 1985; Pifl et al., 1991). Like MPP⁺, APP⁺ is also taken up by cells expressing DAT, NET, and SERT; its fluorescent properties are well understood (Solis et al., 2012; Karpowicz et al., 2013; Wilson et al., 2014). In the present study, we relied on APP⁺ to explore the transport cycle of DAT, NET, or SERT by single-cell analysis, which allowed for simultaneously recording cellular uptake by fluorescence and substrate-induced currents. It was also possible to control the concentrations of the relevant ions and the membrane potential with the unprecedented precision of the whole-cell patch-clamp configuration and to thereby examine their impact on transport rates. To the best of our knowledge, our experiments are the first to address the following question: why do the structurally similar DAT, NET, and SERT differ in transport kinetics and handling of co-substrate binding? It is evident that DAT and NET resemble SERT in most aspects. We show here that all major differences can be accounted for by the distinct handling of K_in⁺. (i) in SERT, physiological K_in⁺ concentrations accelerated the rate of substrate uptake; it was twofold faster than in the absence K_in⁺ (Figure 6G). In contrast, DAT and NET returned to the outward-facing state with the same rate regardless of whether K_in⁺was present or not (Figure 6E and F). Accordingly, K_in⁺ did not affect the rate of substrate uptake by DAT and NET (Figure 4A and B). (ii) The catalytic rate of SERT was independent of voltage in the presence of physiological ionic gradients (Figure 3L and Figure 5L). This was not the case for DAT and NET (Figure 3J and K and Figure 5J and K, respectively). (iii) In all three transporters, release of Na_in⁺ from the inward-facing conformation was electrogenic. In SERT, this electrogenic Na_in⁺ dissociation was canceled out by electrogenic K_in⁺ binding to the inward-open empty transporter and its subsequent antiport, thereby rendering the cycle completion rate voltage-independent. In DAT and NET, however, the cycle completion rate remained voltage-dependent despite the fact that K_in⁺ also bound in a voltage-dependent manner. (iv) K_in⁺ is also relevant to account for the distinct nature of the steady-state current component in SERT and DAT. The steady current component carried by the electroneutral SERT was produced by an uncoupled Na⁺ flux through a channel state that was in equilibrium with the K_in⁺-bound inward-facing conformation (Schicker et al., 2012). This uncoupling explains the existence of differences in the voltage dependence of SERT-mediated uptake and of steady-state currents (Figure 3L). DAT-mediated transport was accompanied by the translocation of coupled net positive charges in each cycle. Thus, DAT-mediated steady-state currents were originally modeled to be strictly coupled to substrate transport (Erreger et al., 2008). However, our data suggest that DAT also carries a previously suggested uncoupled current component (Sonders et al., 1997; Sitte et al., 1998; Carvelli et al., 2004), which adds to those associated with DAT-mediated ionic transport. This uncoupled current in DAT, just like in SERT, is contingent on the presence of intracellular K_in⁺.

The binding site for K_in⁺ in DAT, NET, and SERT is largely unknown. Binding of K_in⁺ to DAT at the Na₂ site was proposed earlier by a study that employed extensive molecular dynamic simulations to understand intracellular Na⁺ dissociation from DAT (Razavi et al., 2017). The amino acids that co-ordinate and form the Na₂ site are absolutely conserved in all three plasmalemmal monoamine transporters (Penmatsa et al., 2013; Coleman et al., 2016; Cheng and Bahar, 2019). In SERT, Na⁺ binding to Na₂ site is thought to trigger conformational transitions essential for physiological substrate transport (Felts et al., 2014). Dissociation of Na⁺ from the Na₂ site, in turn, leads to water flux from the cytosol that promotes transition of the transporter into the inward-facing conformation and subsequent substrate dissociation on the intracellular side (Cheng and Bahar, 2019). Our results highlight the fact that the voltage dependence of peak amplitudes is identical in the presence of high Na_in⁺ and high K_in⁺ (Figure 5J–L) in all three plasmalemmal monoamine transporters; these observations support the conjecture that K_in⁺ and Na_in⁺ binding occurs at the Na₂ site in a mutually exclusive manner. Interestingly, differences between DAT/NET and SERT are further substantiated by the ability of SERT to bind to intracellular Li⁺. The exact nature of this interaction is unknown and necessitates an in-depth investigation that is beyond the scope of this study.

The most parsimonious explanation for all differences between SERT, NET, and DAT was to posit that K_in⁺ is antiported by SERT but not by DAT and NET. All three transporters carry a negative charge through the membrane on return from the substrate-free inward- to the substrate-free outward-facing conformation (presumably a negatively charged amino-acid). In the case of SERT, however, the charge on the transporter is neutralized by the counter-transported K_in⁺. Because the return step is slow and therefore rate-limiting, it determines the voltage dependence of substrate uptake. We note that the lack of voltage dependence, which we observed in the SERT-mediated uptake, is not contingent on its assumed electroneutrality. Even an electroneutral transporter can support voltage-dependent uptake if the rate-limiting step is associated with charge movement. The K_in⁺-binding site in SERT, alternatively, can also accept protons (Keyes and Rudnick, 1982). Hence protons—as an alternative co-substrate that is antiported—support the return step from the inward- to the outward-facing substrate-free conformation (Hasenhuetl et al., 2016). The alternative is to postulate, based on recent evidence in LeuT (Billesbølle et al., 2016), that antiport of K_in⁺ is a general feature of all SLC6 transporters. However, this can be refuted for DAT and NET for the following reasons: the presence or absence of K_in⁺ did not change their catalytic rates (Figure 6E and F). Thus, in the absence of K_in⁺, the transporters seem to return from the substrate-free inward- to the substrate-free outward-facing conformation. In this case, however, the transporters carried one positive charge less through the membrane. This change in ion translocation must, therefore, translate into a concomitant, substantial change in the voltage dependence of substrate transport, that is, the voltage dependence of transport ought to be much steeper in the absence than in the presence of K_in⁺. This was not observed (Figure 4A and B). Additionally, H⁺ failed to accelerate the catalytic rate of DAT (data not shown) and the slope of the IV curve for the peak current remained steep (Figure 5—figure supplement 2). These observations indicate that protons (like K_in⁺) cannot be antiported by DAT.

In monoamine transporters, there is a continuum between full substrates, partial substrates, atypical inhibitors, and typical inhibitors (Hasenhuetl et al., 2019; Bhat et al., 2019). Interestingly, APP⁺ is a full substrate of DAT: the currents, which were elicited by APP⁺, were indistinguishable to those induced by the cognate substrate dopamine and other full substrates such as D-amphetamine (Erreger et al., 2008). In contrast, APP⁺ elicited the peak current but failed to induce the steady current through SERT, which was readily seen in the presence of 5-HT. In oocytes expressing SERT, APP⁺-elicited currents reached only ~20% of the amplitudes of the 5-HT-induced currents (Solis et al., 2012). In SERT-expressing HEK293 cells, the cognate substrate elicits currents of a magnitude in the low pA range. Hence, transport-associated currents induced by APP⁺ are expected to be lost in the noise. Taken together these observations, APP⁺ is a poor substrate for SERT: its actions can be rationalized by assuming that it traps transporters in one or several conformational intermediates, which are exited with a rate slower than the return step. Therefore, DAT and SERT diverge in their handling of APP⁺: while APP⁺ had similar K_M values for DAT and SERT, the catalytic rate of the transport cycle was equivalent to that of the cognate substrate in DAT, but substantially lower in SERT.

Originally, NET expressed in HEK293 cells was reported to support both a peak and a steady current, when challenged with a substrate (Galli et al., 1995; Sommerauer et al., 2012). However, in the present study, we only observed the peak current with our superfusion system, which allowed for a rapid exchange of solutions: neither APP⁺ nor the cognate substrate norepinephrine elicited a steady current. The absence of the steady current can be attributed to the very slow catalytic rate of NET: it is evident that NET, in contrast to DAT and SERT, returns on a timescale of seconds from the inward- to the outward-facing state. NET dwells in the inward-facing state with a lifetime of τ = ~7 s (Figure 6F), which is >10–20 times longer than the dwell time of DAT and SERT (DAT, τ = ~0.3 s; SERT, τ = ~0.6 s; Figure 6E and G). Turnover numbers determined by maximal uptake rates divided by maximal binding sites were also reported as five times lower in NET than in DAT expressed in COS-7 and human neuroblastoma cells (Pifl et al., 1996). Thus, this very slow turnover explains the absence of coupled or uncoupled NET-mediated currents in spite of the proposed electrogenic stoichiometry (Gu et al., 1996).

Our analysis provides a unifying concept of substrate transport through all three monoamine transporters, that is, they are equivalent in all aspects of their transport cycle but one: in SERT, the binding site for K_in⁺ remains intact upon conversion of the transporter from the inward- to the outward-facing conformation. In contrast, this binding site is less stable in DAT and NET. The resulting loss in affinity leads to the shedding of K_in⁺ prior to the return step. The energetics associated with this change in affinity has little to no bearing on the overall spontaneity of forward substrate transport in DAT (Figure 7—figure supplement 1). The repercussions of this subtle difference are profound: SERT and DAT/NET differ (i) in their voltage dependence of substrate uptake, (ii) in the nature of the substrate-induced current, and (iii) in the energy sources tapped for concentrative substrate transport. If DAT and NET do not antiport K_in⁺, its concentrative power must be independent of the existing K⁺ gradient across the plasma membrane. On the other hand, if the stoichiometry of DAT and NET is electrogenic, a change in membrane voltage is predicted to increase or decrease substrate uptake at steady state depending on the direction of the voltage change. In this context, it is important to note that the experiments conducted in the present study all report on substrate transport at pre-steady state. Additional insights on whether or not DAT/NET can antiport K_in⁺ can come from experiments conducted at the thermodynamic equilibrium. Such experiments need to be performed by, for instance, employing a vesicular membrane preparation that contains reconstituted DAT or NET. Such preparations allow for the control of the inner and outer ion composition while preventing the substrate to escape from the vesicular confinement. However, steady-state assessment of transporter-mediated substrate uptake is hindered by the fact that all three monoamine transporters can also transport substrate in the absence of K_in⁺. These observations are difficult to reconcile with the concept of transport by fixed stoichiometry. We, therefore, surmise that DAT, NET, and SERT operate with a mixed stoichiometry. Based on our data we conclude that DAT and NET are less likely than SERT to antiport K_in⁺, because we cannot rule out that they can occasionally carry the K_in⁺ ion through the membrane. Conversely, SERT antiports K_in⁺ in the majority of its cycles but may return empty in some instances. We thus believe that the differences between these three transporters with respect to their handling of K_in⁺ represent a continuum, as opposed to divergence, in ionic coupling and kinetic decision points during substrate transport. In this context, it is worth pointing out that the family of SLC1 glutamate transporters, another important family of neurotransmitter transporters, comprises members that are categorized as either (i) K⁺-dependent, that is, they utilize K_in⁺ as an obligatory co-substrate or (ii) K⁺-independent, that is, they do not require K_in⁺ for completion of the transport cycle (Alleva and Machtens, 2021). However, K_in⁺ was shown to bind to the same binding sites in both subfamilies (Wang et al., 2019; Kortzak et al., 2019; Wang et al., 2020). We note that the presence of K_in⁺ is not obligatory for any representative of the monoamine transporters. Even SERT, which antiports K_in⁺, can transport substrate in the absence of K⁺; this occurs at a lower velocity (Figure 6G and Hasenhuetl et al., 2016). It is not clear whether K_in⁺-independent SLC1 transporters can still utilize the K⁺ gradient to fuel concentrative uptake. If these transporters do, they would mechanistically resemble SERT, if not we would expect them to operate like DAT and NET.

The differences in K_in⁺ handling by SERT and by DAT and NET and concomitant differences in voltage-dependence profiles may have a distinct physiological impact and may have arisen due to evolutionary optimization and adaptation to physiological requirements. An analysis based on the alignment of the primary sequences of DAT, NET, and SERT reveals that SERT is the most divergent member of the three monoamine transporters. SERT is expressed, in both neurons and non-excitable cells, for example, in the trophoblast of the placenta (Balkovetz et al., 1989; Kliman et al., 2018; Rudnick and Sandtner, 2019). The membrane potential in these cells can range from at least 0 mV to −84 mV, with further variations under different biological processes (Carstensen et al., 1973; Friedhoff and Sonenberg, 1983; Greenwood et al., 1996; Birdsey et al., 1997; Kirby et al., 2003). Thus, SERT must operate under very different transmembrane voltages. It is, therefore, conceivable that the voltage independence of SERT is an evolutionary adaptation to maintain effective forward transport over a large variation in membrane potential. DAT, on the other hand, has been previously predicted to exploit membrane hyperpolarization caused by activated autoreceptors and open G protein-coupled inwardly rectifying potassium channel to augment dopamine reuptake and synaptic clearance (Sonders et al., 1997). Hence, there seems to be a trade-off in plasmalemmal monoamine transporter function. Because of electrogenic binding and subsequent counter-transport of K_in⁺, SERT operates in the forward transport mode with a constant rate regardless of membrane potential, but it cannot exploit the membrane potential to fuel its concentrative power. In contrast, DAT and NET can harvest the energy of the transmembrane potential to fuel its concentrative power. As a trade-off, the substrate uptake rate of DAT and NET is voltage-dependent and strongly reduced or increased upon membrane depolarization or hyperpolarization, respectively.

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 2, 3, 5, and 6 in the following DOI published by Dryad: https://doi.org/10.5061/dryad.6q573n5z8.

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Author details

1. Shreyas Bhat

   Institute of Pharmacology and the Gaston H. Glock Research Laboratories for Exploratory Drug Development, Center of Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review and editing

   Contributed equally with

   Marco Niello

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7019-9180

2. Marco Niello

   Institute of Pharmacology and the Gaston H. Glock Research Laboratories for Exploratory Drug Development, Center of Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Writing - original draft

   Contributed equally with

   Shreyas Bhat

   Competing interests

   No competing interests declared

3. Klaus Schicker

   Division of Neurophysiology and Neuropharmacology, Centre for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria

   Contribution

   Conceptualization, Formal analysis, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

4. Christian Pifl

   Center for Brain Research, Medical University of Vienna, Vienna, Austria

   Contribution

   Conceptualization, Resources, Writing - review and editing

   Competing interests

   No competing interests declared

5. Harald H Sitte

   Institute of Pharmacology and the Gaston H. Glock Research Laboratories for Exploratory Drug Development, Center of Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria

   Contribution

   Resources, Supervision, Funding acquisition, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1339-7444

6. Michael Freissmuth

   Institute of Pharmacology and the Gaston H. Glock Research Laboratories for Exploratory Drug Development, Center of Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Writing - review and editing

   Competing interests

   No competing interests declared

7. Walter Sandtner

   Institute of Pharmacology and the Gaston H. Glock Research Laboratories for Exploratory Drug Development, Center of Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria

   Contribution

   Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Methodology, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   walter.sandtner@meduniwien.ac.at

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3637-260X

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