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. Author manuscript; available in PMC: 2020 Mar 25.
Published in final edited form as: Curr Opin Struct Biol. 2019 Mar 25;54:161–170. doi: 10.1016/j.sbi.2019.03.011

Insights into the mechanism and pharmacology of neurotransmitter sodium symporters

Vikas Navratna a, Eric Gouaux a,b
PMCID: PMC6592764  NIHMSID: NIHMS1523975  PMID: 30921707

Abstract

Neurotransmitter sodium symporters (NSS) belong to the SLC6 family of solute carriers and play an essential role in neurotransmitter homeostasis throughout the body. In the past decade, structural studies employing bacterial orthologues of NSSs have provided insight into the mechanism of neurotransmitter transport. While the overall architecture of SLC6 transporters is conserved amongst species, in comparison to the bacterial homologs, the eukaryotic SLC6 family members harbor differences in amino acid sequence and molecular structure, which underpins their functional and pharmacological diversity, as well as their ligand specificity. Here we review the structures and mechanisms of eukaryotic NSSs, focusing on the molecular basis for ligand recognition and on transport mechanism.

Introduction

Neurotransmitter sodium symporters (NSS) that include the dopamine transporter (DAT), serotonin transporter (SERT), norepinephrine transporter (NET), glycine transporter (GlyT), and γ-aminobutyric acid (GABA) transporter (GAT) are secondary active transporters that belong to the SLC6 family of solute carriers, and are involved in neurotransmission and neurotransmitter homeostasis [1]. NSS have been the principal drug targets for an array of psychostimulants and antidepressants [2]. Dysregulation of NSS function has been implicated in attention deficit hyperactivity disorder (ADHD), bipolar affective disorder, autism, anxiety, clinical depression, addiction, schizophrenia, epilepsy, dopamine transporter deficiency syndrome, and Parkinson’s disease [3-5].

NSSs recycle synaptic neurotransmitters by transporting them back to the pre-synaptic neuron in a sodium and chloride dependent manner [5-7]. The binding of neurotransmitter to the outward-open transporter triggers a series of conformational changes in which the transporter isomerizes to an inward-open conformation, and subsequently, releases the neurotransmitter into pre-synaptic neuron. Following release, the transporter reorients to the outward-open conformation [5-8]. Conceptual insights into this kind of alternating access mechanism employed by membrane transporters was proposed over 50 years ago [9,10], and the molecular underpinnings of this process have been elucidated over the past two decades by x-ray crystallography using bacterial orthologues [11,12].

Bacterial orthologues that include LeuT from Aquifex aeolicus and MhsT from Bacillus halodurans have proven to be robust vehicles for investigation of NSS structure and mechanism [13-21]. In addition, structures of LeuT have also provided insights into the molecular principles of ligand binding. In LeuT, antidepressants bind within the extracellular vestibule (EV) and not to the central substrate site (CS), consistent with a non-competitive mechanism of inhibition [16]. Upon engineering the residues from the CS of eukaryotic NSS into LeuT, the inhibitors bind to the CS [21]. However, because LeuT is distantly related to the eukaryotic NSSs, use of LeuT as a model to study eukaryotic transporter – ligand interaction has substantial limitations.

Architecture and sequence conservation in members of SLC6 family

Members of the SLC6 family adopt a conserved LeuT fold, with substantial sequence conservation in key regions of structure and in the sodium ion binding sites (Fig. 1a-1e) [12,16,17]. The conformationally dynamic transmembrane helices (TMs), such as the core domain (TMs 1, 2, 6, and 7) and TM5, are more conserved than the rigid scaffold domain (TMs 3, 4, 8, and 9) and TM10 (Fig. 1b). The functional and pharmacological diversity seen in NSSs, despite a conserved architecture, is due to their amino acid sequence variation. The termini of eukaryotic NSS are longer than their prokaryotic counterparts and are involved in regulation of transport, substrate binding, efflux, post-translational modification and trafficking [22-25]. The N-terminus in the Drosophila DAT (dDAT) and human SERT (hSERT) crystal structures begins with a conserved membrane-proximal RXXW motif, which interacts with intracellular loops (ILs) 3, 4, and 5 and regulates transporter conformation, substrate transport, and amphetamine induced substrate efflux [26-29]. dDAT and hSERT structures also show a ten-residue helical ‘latch’ on the C-terminus, which interacts with IL1, and modulates protein folding, trafficking and transport (Fig. 1b) [30-33].

Figure 1: Architecture and sequence conservation.

Figure 1:

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(a) Topology of LeuT fold, a characteristic (5+5) inverted repeat, where the TMs 1-5 are related to TMs 6-10 by an anti-parallel pseudo 2-fold axis. The sodium and substrate binding sites are highlighted as blue spheres and purple stick respectively. (b) Architecture of hSERT color coded, based on the sequence conservation across human members of 19 SLC6 subfamilies, using ConSurf server. The (c) central substrate site, (d) allosteric site, (e) ion-binding site, (f) putative non-conserved zinc site, and (g) cholesterol-binding sites of NSS are mapped on to hSERT structure.

Extracellular loop 6 (EL6) and extracellular portions of TMs 11 and 12, which sculpt a portion of the allosteric site (AS) in hSERT, are the least conserved regions of the SLC6 family following the termini (Fig. 1b and 1d), and variation in the sequence and geometry of this region dictates allosteric ligand specificity in eukaryotes [30]. TM12 is variable across the SLC6 family, except for a highly conserved Pro residue midway along the helix in eukaryotic NSS, which kinks TM12, splaying the intracellular portion away from the transporter and exposing the regions of TM3 and IL1 that are closer to the C-terminal latch (Fig. 1b) [30,33]. The C-terminal end of TM12 in eukaryotes contains motifs that are essential for cell surface expression and trafficking [2,31]. We speculate that the kink in TM12 of the eukaryotes aids in positioning of the C-terminal latch in an orientation suitable for interaction with IL1.

Central substrate and ion-binding sites

The CS is approximately halfway across the membrane bilayer and is ensconced by conserved hydrophobic and polar residues from TMs 1, 3, 6, 8 and 10 (Fig. 1b and 1c). The residues that coordinate ions necessary for substrate transport and binding are highly conserved and are contributed primarily by core domain and TM8 (Fig. 1e). The sodium ions also stabilize the non-helical regions of TM1 and TM6 [2,17]. Sodium in the Na1 site of hSERT is positioned in octahedral coordination sphere by residues from TMs 1a, 1b, 6a, and 7. The residues from TMs 1a, 1b, and 8 coordinate a second sodium ion (Na2 site) in trigonal bipyramidal fashion. The outward-to-inward conformational transition in prokaryotes is demonstrated to be dependent on sodium, particularly of the Na2 site [34-36]. The eukaryotic NSSs, unlike prokaryotes, also require chloride ion (Cl−) to function. However, the significance of Cl− and its coordination site were identified in LeuT by homology modeling, molecular dynamics (MD) simulations and site directed mutagenesis studies [7,37]. The structures of dDAT and hSERT show a Cl− ion in a tetrahedral site coordinated by residues from TMs 2, 6a, and 7 (Fig. 1e). A tetrahedral zinc binding site has been identified in hDAT using homology modeling and site directed mutagenesis, and is coordinated by His193 and Asp206 from EL2 and, His375 and E396 from EL4 [38]. Zinc stabilizes the outward-open conformation of hDAT, and thus can inhibit transporter activity [39]. However, this zinc site is not conserved in dDAT or hSERT (Fig. 1f).

Cholesterol-binding site

Cholesterol modulates activity by stabilizing an outward-open conformation of NSS transporters [40,41]. In dDAT, a cholesterol molecule was modeled in a shallow cavity (site1) created by TMs 1a, 5, and 7. Cholesterol hemisuccinate (CHS), a structural analog of cholesterol, was found at a second cavity (site 2) formed by TMs 2, 7, and 11 [33,40]. Using homology modeling, MD simulation, and cholesterol-binding motif analysis, five putative cholesterol-binding sites were identified in NSSs. Among them, the site corresponding to site 1 in dDAT structure was found to be the most conserved (Fig. 1g) [42]. In hSERT, a single CHS molecule was modeled near the extracellular side of TM12 (Fig. 1g) [30].

Allosteric site (AS), a potential drug target

Site directed mutagenesis experiments within the EV aided in locating the AS in hSERT, and binding of ligands at the hSERT AS modulates transport and ligand binding kinetics [43,44]. As shown by x-ray crystallographic studies, residues from TMs 1b, 6a, 10, 11, EL4, EL6, and a portion of EL2 comprise the AS (Fig. 1b, 1d, and 2), and sequence variation in this region determines allosteric ligand specificity [30]. The amino acid variation in TM10 and difference in positioning of residues in TM1b make the AS in hSERT chemically and geometrically different than dDAT, leading to differences in ligand specificity (Fig. 2b). In fact, the modulatory effect of potential allosteric agents of DAT and NET was negligible on SERT [45,46]. Allosteric modulators of NSS sterically hinder the pathway to and from the CS, thereby modulating the off-rate of the CS ligand. For example, excess of cold citalopram slows the dissociation of radioactive citalopram from the CS of hSERT [30]. Similarly, excess of cold MRS7292, an adenosine based allosteric modulator of hDAT, prevents the dissociation of radioactive WIN35428, a cocaine analog, from the CS [47].

Figure 2: Comparison of structure and allosteric site of dDAT and hSERT.

Figure 2:

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(a) Superposition of dDAT and hSERT structures. The Cα RMSD of superposition of TMs 1-8 of dDAT and hSERT is 0.5 Å, and for regions comprised by ELs 2, 4 and 6, and TMs 9-12 it is 1.2 Å (b) Comparison of chemical nature and geometry of allosteric site of dDAT and hSERT. Polar positive, polar negative, polar neutral, non-polar aliphatic, non-polar aromatic and other residues are colored in blue, red, green, gray, yellow and purple respectively. (c) Superposition of AS of hSERT bound to maltose (blue) and (S)-citalopram (green). (d) Comparison of the boundaries of allosteric site in DA and DCP bound dDAT structures.

Structures of hSERT have been solved in complex with four widely prescribed selective serotonin reuptake inhibitors (SSRIs). The AS of hSERT-citalopram complex was occupied by (S)-citalopram (PDBID: 5I73). In the paroxetine-hSERT structure a maltose group from the detergent was modeled into the AS (PDBID: 5I6X). A comparison of sites occupied by (S)-citalopram and maltose suggests binding site plasticity (Fig. 2c). The moderate resolution of the sertraline and fluvoxamine complexes (PDBIDs: 6AWO and 6AWP) hindered the modeling of drugs at AS [30]. All the structures of hSERT are in outward-open conformation, thus the fate of AS in various transporter conformations is unknown. However, comparing the dopamine (DA) and 3, 4-dichlorophenethylamine (DCP) bound dDAT structures reveals that, in the DCP bound partially-occluded state of dDAT, the AS appears to be constricted, and the pathway from AS to CS seems shut due to Phe319 flip (Fig. 2d). These observations suggest that the AS could collapse upon outward-to-inward transition. A bulky high-affinity allosteric site ligand could act as a wedge and sterically prevent outward-to-inward transition, thereby acting as an inhibitor of transport.

Comparison of dDAT and hSERT structures

The overall architecture of hSERT is similar to dDAT (RMSD 0.7 Å/ 272 Cα) (Fig. 2a). EL2 in hSERT is longer than dDAT, and it folds back onto the extracellular pocket like a lid, and together with EL4 and EL6 it defines a portion of the AS (Fig. 1b). Compared to dDAT, TM9 and TM12 are closer to one another in hSERT. TM12a is pressed towards TM10 and TM11, reducing the kink, subsequently ‘pushing’ EL6 closer to EL2 by ~4 Å (Fig. 2a) [30,33]. These subtle differences on the extracellular side combined with minor sequence variations in the central site forms the basis of pharmacological diversity between dDAT and hSERT. Compared to hSERT, dDAT is a promiscuous hybrid transporter that transports DA, norepinephrine (NE), and tyramine, and it binds several inhibitors of monoamine transporters with high affinity. As a result, dDAT has been extensively used as a model to study pharmacology of eukaryotic NSS.

Role of the central site residues on inhibitor specificity

The CS acts as a pliable pouch, and is mainly sculpted by residues from TMs 1, 3, 6, 8, and 10. Based on the nature of interactions with ligands the CS is partitioned into three subsites (A-C) (Fig. 3a) [21,48]. Upon binding the CS the amine group of ligand occupies a tripod grip formed by conserved Phe and Asp from subsite A and a Phe residue from subsite C (Phe43, Asp46, and Phe 319 of dDAT; Phe95, Asp98, and Phe325 of hSERT). The conserved Asp from subsite A also forms a hydrogen bond with the conserved Tyr of subsite B (Tyr124 of dDAT; Tyr176 of hSERT), and plays a crucial role in substrate recognition (Fig. 3a). The sequence variation in subsite B appears to play an important role in ligand specificity. For example, substitution of non-conserved polar residues in subsite B of dDAT with polar residues of hNET residues resulted in enhanced nisoxetine-binding affinity [21,30,33,49-52].

Figure 3: Pharmacological diversity of NSS.

Figure 3:

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(a) Snapshots of the CS bound to substrates, substrate analogs, psychostimulants, and antidepressants. Subsites A-C are indicated by dotted lines. The residues of TMs 1, 6, and 8 form subsite A. Subsite B is comprised of residues from TMs 3 and 8. Subsite C is comprised by TM6 and TM10 residues. The residues from TM8, which also coordinate sodium at Na2 site, are less susceptible to movement, and hence are hidden in the figure. The residues in other regions, mainly subsite C, adjust side chain orientation to accommodate different ligands. (b) Superposition of active sites of DA and DCP bound dDAT. The TMs 1a, 6b, 2, and a portion of TMs 10 and 3, close-in on the bound DCP. To accommodate these movements, extracellular side of TM11 moves away from the CS. (c) Superposition of CS of S439 and T439 variants of hSERT bound to paroxetine. The ligand molecules in all panels are shown as ball and stick models.

Substrate and substrate analogs

All dDAT-ligand structures are in an outward-open conformation except in the presence of DCP. DCP is a substrate analog that locks dDAT in a partially-occluded-outward-facing conformation. Consistent with the substrate-bound occluded state in LeuT, the Phe319 residue in the DCP bound structure is flipped towards Tyr124, constricting the access to the CS (Fig. 2d and 3b). In all other dDAT and hSERT structures the distance between the side chains of these Tyr and Phe residues is ~ 10 Å, suggesting that the bulky inhibitors interfere with the closure of this gate and thereby lock transporters in an outward-open conformation (Fig. 3a). CHS, Fab, or the detergent used in crystallization could have prevented the complete transition to an occluded conformation in the presence of DCP. In the DA bound structure, the hydrogen bonding interactions made by hydroxyl groups of the catechol ring with TM3 may cause DA to shift away from the tripod ‘grip’ and farther into the subsite B. Asp46 side chain reorientation maintains the interaction with the amine group of DA, suggesting that TM3 of subsite B together with Asp of TM1 dictates substrate recognition (Fig. 3b). Amphetamine and methamphetamine act as generic substrates of monoamine transporters. Upon binding, they modify the conformation of transporters and induce substrate efflux [53,54]. In the structure, amphetamines occupy a pose similar to inhibitors (Fig. 3a).

Psychostimulants and antidepressants

Cocaine and its analogs (β-CFT and RTI-55) show a similar binding pose (Fig. 3a). The benzoate or halophenyl groups occupy subsite B. The bulky tropane, interacting with subsite A and C, acts like a wedge preventing the closure of the extracellular gate, thereby locking the transporter in an outward-open conformation. The tricyclic group of the tricyclic antidepressant (TCA) nortriptyline makes a network of interactions with subsite B and C. The bulkiness of the group recruits additional residues from TM10 (Ala479) into subsite C. The long alkyl chain, similar to tropane in cocaine analogs, acts as a wedge preventing the closure of the extracellular gate (Fig. 3a). The norepinephrine reuptake inhibitors (NRIs), such as nisoxetine and reboxetine, have discontinuous aromatic groups, unlike the TCAs. Both nisoxetine and reboxetine have a phenoxy group that occupies subsite B. Due to the steric effect of the ethoxy group in reboxetine, Phe325 assumes a pose similar to the nortriptyline bound structure. The bulky morpholine ring prevents closure of the extracellular gate (Fig. 3a). The chemical structures of SSRIs are fairly diverse, and similar to NRIs, yet they differ from TCAs by not having the bulky tricyclic group responsible for broad spectrum specificity of TCAs. This steric difference in SSRIs coupled with sequence variation in subsite B of SERT makes them selective SERT drugs. The positioning of bulky aromatic groups closer to the interface of subsite B and C, in both citalopram and paroxetine bound structures, results in Phe341 shifting ‘down’, away from Phe335 (Fig. 3a and 3c). In all inhibitor bound structures the position of Phe335 is identical, except in the case of sertraline. The tetrahydronaphthalene of sertraline reorients Phe335 to favor edge-face interaction (Fig. 3a).

In hSERT-SSRI structures, except paroxetine, the electronegative halogenated aromatic rings of all inhibitors occupy subsite B, similar to cocaine analogs. The binding pose of paroxetine, however, is reversed such that the piperidine, benzodioxol, and fluorophenyl moieties of the drug occupy subsites A, B, and C respectively (Fig. 3c). The binding pose of paroxetine in hSERT has been studied extensively because of its extraordinarily high affinity. Based on homology modeling, MD simulations, mutagenesis, radioligand binding and transport assays, it has been suggested that paroxetine binds to SERT with the fluorophenyl group in subsite B [51,55]. It has been argued that the thermostabilizing hSERT mutation T439S in the hSERT CS could have been responsible for the differential positioning of paroxetine in the crystal structure. However, the structure of hSERT with reverted T439S still shows a similar binding pose of paroxetine [50]. Incidentally, the binding pose of paroxetine at the CS of LeuBAT, a hybrid leucine-biogenic amine transporter, is similar to hSERT [21]. In addition, binding and transport experiments involving SERT containing genetically engineered photo-cross-linkers in the CS indicate a binding pose that is in agreement with the crystal structure [56]. Solving a higher resolution structure of hSERT, perhaps using iodinated or brominated derivative of paroxetine to obtain an anomalous difference signal, could resolve this issue [55].

Structural basis of alternating access mechanism

The transporter community has employed structural and biophysical approaches on bacterial orthologues such as LeuT and MhsT to study conformational dynamics of transport. Crystal structures are available for the following states – outward-open, outward-occluded, inward-occluded, inward-open, and sodium-free-outward-return conformations [13,17,19,20]. In the outward-open conformation, the CS is accessible to the synaptic space via a cone-shaped extracellular pathway in the EV skirted by helices TM1b, 6a, 3 and 10 (Fig. 1b and 4) [13,14,16]. Upon binding the substrate, TMs 1b and 6a ‘move in’ towards the center of transporter [17]. Although the access to the CS closes, in this outward-occluded conformation, the EV remains partially open. Sodium sites at the pivot points of TM1b and 6a maintain the coordination sphere throughout these conformational changes. In the inward-occluded conformation, TMs 1b, 2, 6a and 7 cluster together and seal the access to the vestibule [19]. The strain from the closure of the vestibule leads to unwinding of the intracellular half of TM5, exposing the Na2 site to the cytoplasm. It is proposed that a coordinated unwinding of intracellular parts of TMs 5 and 1 release sodium from the Na2 site. The structure of MhsT only shows unwound TM5 (Fig. 4) [19]. TM1a unwinding has been inferred using hydrogen/deuterium exchange mass spectrometry (HDX-MS) experiments [57]. The HDX-MS experiments also suggest that partial unwinding in TM7 followed by release of sodium and substrate enhances the propensity of inward-open conformation [57,58]. One of the characteristics of inward-open state is the protuberance of TM1a, allowing access to the CS from the intracellular side. TM6b motion is not as large as TM1a. On the extracellular side, motion in TM7 closes the pathway further [13]. The inward-open to outward-open transition occurs through transient occluded-return states. The outward-oriented-return state, devoid of substrate and sodium, resembles the outward-open state [13,20]. However, there is a flip in orientation of the conserved Leu25 situated in the linker between TM1b and 1a, such that it occupies the CS [20]. In presence of sodium, Leu25 moves away from CS allowing the stabilization of outward-open state.

Figure 4: Structure based schematic of alternating-access mechanism in bacterial orthologues of NSS.

Figure 4:

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Inward-occluded conformation depicted in the figure is from MhsT, while all other conformations are from LeuT. A consensus which emerged in all these structures suggests that the core domain along with TM5 undergoes significant conformational changes during the outward-inward transition. Noteworthy of these changes are closure of the EV due to a concerted motion between TMs 1b, 2, 6a and 7, and projecting out of TM1a to provide access to the CS from intracellular side. TMs 3, 4, 8, and 9-12 are collectively denoted as an orange block.

Conclusion and future perspective

The differences in sequence length and conservation in termini and the extracellular face of human NSS compared to their insect and bacterial orthologues underscores a necessity for studies to understand the role of these differences in transport and regulation. The structures of bacterial NSS orthologues provide a framework of the entire transport mechanism except the inward-oriented sodium free return state. However, the transient nature of several states required manipulation of expression construct and conditions to crystallize the proteins in a desired conformational state. In this context, it is essential to employ tools and methods that would enable structural characterization of various conformations without having to genetically engineer the expression construct for stability. Structures of human NSS in multiple conformations will advance our knowledge of the transport mechanism, fate of the AS in the transport cycle, Cl− ion dependency of conformational dynamics, effect of post-translational modifications such as phosphorylation on dynamics of the eukaryotic transporters, and allow for development of conformation-dependent psychopharmaceuticals.

Acknowledgements

We thank J. Coleman, F. Jalali-Yazdi, and D. Yang for discussion and valuable inputs on the manuscript, L. Vaskalis for assistance with figures, and H. Owen for help with manuscript preparation. This work was supported by the National Institute of Mental Health of the NIH under award number R37MH070039. E.G. is an investigator with the Howard Hughes Medical Institute.

Footnotes

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References and recommended reading

The manuscripts of special (•) and outstanding (••) interest have been highlighted.

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