Search

The serotonin transporter, SERT, initiates the reuptake of extracellular serotonin in the synapse to terminate neurotransmission. Recently, the cryo-EM structures of SERT bound to ibogaine resolved in different states provided the first glimpse of functional conformations at atomistic resolution. However, the conformational dynamics and structural transitions to various intermediate states are not fully understood. Furthermore, while experimental SERT structures were complexed with drug molecules and inhibitors, the molecular basis of how the physiological substrate, serotonin, is recognized, bound, and transported remains unclear. In this study, we performed microsecond long simulations of the human SERT to investigate the structural dynamics to various intermediate states and elucidated the complete substrate import pathway. Using Markov state models, we characterized a sequential order of conformational driven ion-coupled substrate binding and transport events and calculated the free energy barriers of conformation transitions associated with the import mechanism. We identified a set of crucial residues that recognize the substrate at the extracellular surface of SERT and our biochemical screening results show that mutations causes dramatic reduction in transport function. Our simulations also revealed a third sodium ion binding site coordinated by Glu136 and Glu508 in a buried cavity which helps maintain the conserved fold adjacent to the orthosteric site for transport function. The mutation of these residues results in a complete loss of transport activity. Our study provides novel insights on the molecular basis of dynamics driven ion-substrate recognition and transport of SERT that can serve as a model for other closely related neurotransmitter transporters.

Sodium-coupled neurotransmitter transporters play a fundamental role in the termination of synaptic neurotransmission, which makes them a major drug target. The reconstitution of these secondary active transporters into liposomes has shed light on their molecular transport mechanisms. From the earliest days of the reconstitution technique up to today’s single-molecule studies, insights from live functioning transporters have been indispensable for our understanding of their physiological impact. The two classes of sodium-coupled neurotransmitter transporters, the neurotransmitter: sodium symporters and the excitatory amino acid transporters, have vastly different molecular structures, but complementary proteoliposome studies have sought to unravel their ion-dependence and transport kinetics. Furthermore, reconstitution experiments have been used on both protein classes to investigate the role of e.g. the lipid environment, of posttranslational modifications, and of specific amino acid residues in transport. Techniques that allow the detection of transport at a single-vesicle resolution have been developed, and single-molecule studies have started to reveal single transporter kinetics, which will expand our understanding of how transport across the membrane is facilitated at protein level. Here, we review a selection of the results and applications where the reconstitution of the two classes of neurotransmitter transporters has been instrumental.

Transporters of the solute carrier 6 (SLC6) family mediate the reuptake of neurotransmitters such as dopamine, norepinephrine, serotonin, GABA, and glycine. SLC6 family members are 12 transmembrane helix‐spanning proteins that operate using the transmembrane sodium gradient for transport. These transporters assume various quaternary arrangements ranging from monomers to complex stoichiometries with multiple subunits. Dopamine and serotonin transporter oligomerization has been implicated in trafficking of newly formed proteins from the endoplasmic reticulum to the plasma membrane with a pre‐fixed assembly. Once at the plasma membrane, oligomers are kept fixed in their quaternary assembly by interaction with phosphoinositides. While it remains unclear how oligomer formation precisely affects physiological transporter function, it has been shown that oligomerization supports the activity of release‐type psychostimulants. Most recently, single molecule microscopy experiments unveiled that the stoichiometry differs between individual members of the SLC6 family. The present overview summarizes our understanding of the influence of plasma membrane constituents on transporter oligomerization, describes the known interfaces between protomers and discusses open questions., We review the current knowledge on oligomerization of the solute carrier 6 (SLC6) family, which includes the transporters for the neurotransmitters dopamine, serotonin, and GABA. Early biochemical studies suggested dimer and tetramer configurations and identified several interaction motifs. Not all interfaces could be confirmed. Oligomers seem to become defined during transporter trafficking, are dependent on the plasma membrane PIP2 content, and affect substrate efflux. Simulations are starting to contribute to our understanding of transporter oligomerization, whereas recent single molecule experiments refined the oligomeric state by finding several non‐exchanging configurations for SERT, but only monomers and dimers for DAT.

Dopamine clearance in the brain is controlled by the dopamine transporter (DAT), a protein residing in the plasma membrane, which drives reuptake of extracellular dopamine into presynaptic neurons. Studies have revealed that the βγ subunits of heterotrimeric G proteins modulate DAT function through a physical association with the C-terminal region of the transporter. Regulation of neurotransmitter transporters by Gβγ subunits is unprecedented in the literature; therefore, it is interesting to investigate the structural details of this particular protein-protein interaction. Here, we refined the crystal structure of the Drosophila melanogaster DAT (dDAT), modeling de novo the N- and C-terminal domains; subsequently, we used the full-length dDAT structure to generate a comparative model of human DAT (hDAT). Both proteins were assembled with Gβ1γ2 subunits employing protein-protein docking, and subsequent molecular dynamics simulations were run to identify the specific interactions governing the formation of the hDAT:Gβγ and dDAT:Gβγ complexes. A [L/F]R[Q/E]R sequence motif containing the residues R588 in hDAT and R587 in dDAT was found as key to bind the Gβγ subunits through electrostatic interactions with a cluster of negatively charged residues located at the top face of the Gβ subunit. Alterations of DAT function have been associated with multiple devastating neuropathological conditions; therefore, this work represents a step toward better understanding DAT regulation by signaling proteins, allowing us to predict therapeutic target regions.

Objectives. To study the expression of various neurotransmitter transporters (glutamate, aspartate, lactate, choline) in cultures of astrocytes from different part of the brain (cortex, hippocampus, and brainstem) in rats aged three and 11 days. Materials and methods. An experimental study was performed using 24 Rattus norvegicus rats aged three (n = 12) and 11 days (n = 12). High-throughput sequencing results were analyzed. Results. Expression of the glutamate and aspartate transporters in the brainstem in three-day-old rats was greater than in other areas, though the reverse pattern was seen in rats aged 11 days. Expression of the lactate transporter with age was identical to that in the cortex. Conclusions. The data obtained here demonstrated the nature of neuroastrocyte relationships and the role of astrocytes in the process of signal transmission. The results of the study, carried out using original methods of investigating neurotransmitter transporters, allow them to be recommended for monitoring the processes of neurogenesis and neurohomeostasis, including in cerebrovascular and neurodegenerative diseases.

SUMMARYVesicular transporters (VTs) define the type of neurotransmitter that synaptic vesicles (SVs) store and release. While certain neurons in mammalian brain release multiple transmitters, the prevalence, physiology of such pluralism and if the release occurs from same or distinct vesicle pools is not clear. Using quantitative single vesicle imaging, we show that a small population of neuronal SVs indeed contain different VTs to accomplish corelease. Surprisingly, this population is highly diverse (27 types), expressing distinct dual transporters suggesting corelease of various combinations of neurotransmitters. Using glutamatergic vesicles as an example, we demonstrate that transporter colocalization not only determine the transmitter type but also influences the SV content and synaptic quantal size. Thus, presence of diverse transporters on the same vesicle is bona-fide and, depending on the VT types, this may act as one mechanism to regulate neurotransmitter type, content and release in space and time.

Regulation of the ability of a neurotransmitter [our focus: serotonin, norepinephrine, dopamine, acetylcholine, glycine, and gamma-aminobutyric acid (GABA)] to reach its receptor targets is regulated in part by controlling the access the neurotransmitter has to receptors. Transporters, located at both the cellular plasma membrane and in subcellular vesicles, carry a myriad of responsibilities that include enabling neurotransmitter release and controlling uptake of neurotransmitter back into a cell or vesicle. Driven largely by electrochemical gradients, these transporters move neurotransmitters. The regulation of the transporters themselves through changes in expression and/or posttranslational modification allows for fine-tuning of this system. Transporters have been best recognized as targets for psychoactive stimulants and remain a mainstay target of primarily central nervous system (CNS) acting drugs for treatment of debilitating diseases such as depression and anxiety. Studies reveal, however, that transporters are found and functional in tissues outside the CNS (gastrointestinal and cardiovascular tissues, for example). The importance of neurotransmitter transporters is underscored with discoveries that dysfunction of transporters can cause life-changing disease. This article provides a high-level review of major classes of both plasma membrane transporters and vesicular transporters. © 2021 American Physiological Society. Compr Physiol 11:2279-2295, 2021.

Neurotransmitter transporters limit spillover between synapses and maintain the extracellular neurotransmitter concentration at low yet physiologically meaningful levels. They also exert a key role in providing precursors for neurotransmitter biosynthesis. In many cases, neurons and astrocytes contain a large intracellular pool of transporters that can be redistributed and stabilized in the plasma membrane following activation of different signaling pathways. This means that the uptake capacity of the brain neuropil for different neurotransmitters can be dynamically regulated over the course of minutes, as an indirect consequence of changes in neuronal activity, blood flow, cell-to-cell interactions, etc. Here we discuss recent advances in the mechanisms that control the cell membrane trafficking and biophysical properties of transporters for the excitatory, inhibitory and modulatory neurotransmitters glutamate, GABA, and dopamine.

To the SLC6 family belong 20 human transporters that utilize the sodium electrochemical gradient to move biogenic amines, osmolytes, amino acids and related compounds into cells. They are classified into two functional groups, the Neurotransmitter transporters (NTT) and Nutrient amino acid transporters (NAT). Here we summarize how since their first cloning in 1998, the insect (Lepidopteran) Orthologs of the SLC6 family transporters have represented very important tools for investigating functional–structural relationships, mechanism of transport, ion and pH dependence and substate interaction of the mammalian (and human) counterparts. Supplementary Information The online version contains supplementary material available at 10.1007/s11064-021-03410-1.

The local lipid annulus, or “fingerprint”, of four SLC6 transporters (dDAT, hDAT, hSERT, and GlyT2) embedded in a complex neuronal membrane were compared and characterised using molecular dynamics. Our analysis included the development of new tools to improve membrane leaflet detection and the analysis of leaflet-dependent properties. Overall, the lipid fingerprints of the four transporters are comprised of similar lipids when grouped by headgroup or tail saturation. The enrichment and depletion of specific lipids, including sites of cholesterol contacts, varies between transporters. The subtle differences in lipid fingerprints results in varying membrane biophysical properties near the transporter. Our results highlight that the lipid-fingerprint of SLC6 transporters in complex membranes is highly dependent on membrane composition. Our results further characterize how the presence and identity of membrane proteins affects the complex interplay of lipid-protein interactions, influencing the local lipid environment and membrane biophysical properties.

Drosophila melanogaster express vesicular transporters for the storage of neurotransmitters acetylcholine, biogenic amines, GABA, and glutamate. The large array of powerful molecular-genetic tools available in Drosophila enhances the use of this model organism for studying transporter function and regulation.

Proteins are nature's biomolecular machines. Proteins, such as transporters, pumps and motors, have complex function/operating-machinery/mechanisms, comparable to the macro-scaled machines that we encounter in our daily life. These proteins, as it is for their macro-scaled counterparts, convert (part of) other/various forms of energy into work. In this study, we are performing the first law analysis on a set of proteins, including the dopamine transporter, glycine transporters I and II, glutamate transporter, sodium-potassium pump and Ca

The brain has specific properties that make it uniquely dependent upon transporters. This is the 3rd edition of a biennial special issue that originates from a scientific meeting devoted to studies of transporters and their relationship to brain function and to neurodevelopmental, neurologic, and psychiatric disorders. The field continues to rapidly evolve with advances in studies of structure that inform mechanism, with genetic analyses in humans revealing surprising aspects of biology, and with integrated cellular to whole animal analyses of the role of transporters in their control of physiology and pathophysiology. This special issue includes a sampling of review articles that address timely questions of the field followed by several primary research articles.

γ-Hydroxybutyric acid (GHB) is an endogenous compound proposed to act as a neurotransmitter. Na+ -dependent, high-affinity GHB transport has long been considered important evidence supporting this hypothesis. However, the molecular identity of such a high-affinity transporter remains unknown. In this study, we sought to identify and characterize GHB synaptic transport through a series of studies using both native and recombinant systems with the ultimate aim of providing evidence to clarify the proposed role of GHB as a neurotransmitter in the mammalian brain. Native [3 H]GHB transport was studied in isolated rat brain synaptosomes and compared to synaptic membranes. As a targeted approach, GHB was also screened against a panel of Na+ -dependent SLC6 neurotransmitter transporters recombinantly expressed in Xenopus laevis oocytes or tsA201 cells. Finally, the low-affinity GHB transporters, MCT1/2 and SMCT1, were probed as GHB transporters in L-[14 C]lactate uptake assays in synaptosomes. We found no evidence of high-affinity [3 H]GHB transport in purified rat brain cortical or striatal synaptosomes or at any of the 11 SLC6 transporters tested. Instead, our results indicate the binding of [3 H]GHB to an unidentified membrane component, distinct from any of the known GHB targets. In accordance with others, we found that GHB and the analog 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) can, in millimolar concentrations, inhibit L-[14 C]lactate uptake at MCT1 and/or MCT2 and that this also can occur in synaptosomes. In conclusion, through a variety of in vitro pharmacological studies, we were unsuccessful in identifying a specific synaptic high-affinity transporter for GHB. Our findings emphasize the need to reevaluate GHB's role as a potential neurotransmitter. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Mancozeb (MZ), an organic-metal fungicide used predominantly on vegetables and fruits, has been linked to neurodegeneration and behavioral disruptions in a variety of organisms, including humans. Both γ-aminobutyric acid and dopamine neurons appear to be more vulnerable to MZ exposure than other neuronal populations. Based on these observations, we hypothesized that MZ may be differentially transported into these cells through their presynaptic neurotransmitter transporters. To test this, we pretreated Caenorhabditis elegans with transporter antagonists followed by exposure to various concentrations of MZ. Potential neuroprotection was monitored via green fluorescence associated with various neuron populations in transgenic worm strains. Neurodegeneration associated with subacute MZ treatment (30 min) was not altered by transporter antagonist pretreatment. On the other hand, pretreatment with a dopamine transporter antagonist (GBR12909) appeared to protect dopaminergic neurons from chronic (24 h) MZ treatment. These results are consistent with other reports that dopamine transporter levels or activity may modulate toxicity for neurotoxicants.

Sodium (Na+) acts as an indispensable allosteric regulator of the activities of biologically important neurotransmitter transporters and G-protein coupled receptors (GPCRs), which comprise well-known drug targets for psychiatric disorders and addictive behavior. How selective these allosteric Na+-binding sites are for the cognate cation over abiogenic Li+, a first-line drug to treat bipolar disorder, is unclear. Here, we reveal how properties of the host protein and its binding cavity affect the outcome of the competition between Li+ and Na+ for allosteric binding sites in sodium transporters and receptors. We show that rigid Na+-sites that are crowded with multiple protein ligands are well-protected against Li+ attack, but their flexible counterparts or buried Na+-sites containing only one or two protein ligands are vulnerable to Li+ substitution. These findings suggest a novel possible mode of Li+ therapeutic action: By displacing Na+ bound by ≤2 protein ligands in buried GPCR sites and stabilizing the receptor's inactive state, Li+ could prohibit conformational changes to an active state, leading to lower cytosolic levels of activated guanine nucleotide-binding proteins, which are hyperactive/overexpressed in bipolar disorder patients.

The human dopamine (DA) transporter (hDAT) is a key regulator of neurotransmission and a target for antidepressants and addictive drugs. Despite the recent resolution of dDAT structures from Drosophila melanogaster, complete understanding of its mechanism of function and even information on its biological assembly is lacking. The resolved dDAT structures are monomeric, but growing evidence suggests that hDAT might function as a multimer, and its oligomerization may be relevant to addictive drug effects. Here, using structure-based computations, we examined the possible mechanisms of hDAT dimerization and its dynamics in a lipid bilayer. Using a combination of site-directed mutagenesis, DA-uptake, and cross-linking experiments that exploited the capacity of Cys-306 to form intermonomeric disulfide bridges in the presence of an oxidizing agent, we tested the effects of mutations at transmembrane segment (TM) 6 and 12 helices in HEK293 cells. The most probable structural model for hDAT dimer suggested by computations and experiments differed from the dimeric structure resolved for the bacterial homolog, LeuT, presumably because of a kink at TM12 preventing favorable monomer packing. Instead, TM2, TM6, and TM11 line the dimer interface. Molecular dynamics simulations of the dimeric hDAT indicated that the two subunits tend to undergo cooperative structural changes, both on local (extracellular gate opening/closure) and global (transition between outward-facing and inward-facing states) scales. These observations suggest that hDAT transport properties may be allosterically modulated under conditions promoting dimerization. Our study provides critical insights into approaches for examining the oligomerization of neurotransmitter transporters and sheds light on their drug modulation.

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.

That channels and transporters can influence the membrane morphology is increasingly recognized. Less appreciated is that the extent and free-energy cost of these deformations likely varies among different functional states of a protein, and thus, that they might contribute significantly to defining its mechanism. We consider the trimeric Na+-aspartate symporter GltPh, a homolog of an important class of neurotransmitter transporters, whose mechanism entails one of the most drastic structural changes known. Molecular simulations indicate that when the protomers become inward-facing, they cause deep, long-ranged, and yet mutually-independent membrane deformations. Using a novel simulation methodology, we estimate that the free-energy cost of this membrane perturbation is in the order of 6–7 kcal/mol per protomer. Compensating free-energy contributions within the protein or its environment must thus stabilize this inward-facing conformation for the transporter to function. We discuss these striking results in the context of existing experimental observations for this and other transporters.

Members of the solute carrier family 6 (SLC6) of sodium- and (sometimes chloride-) dependent neurotransmitter transporters [29, 22, 70] are primarily plasma membrane located and may be divided into four subfamilies that transport monoamines, GABA, glycine and neutral amino acids, plus the related bacterial NSS transporters [99]. The members of this superfamily share a structural motif of 10 TM segments that has been observed in crystal structures of the NSS bacterial homolog LeuTAa, a Na+-dependent amino acid transporter from Aquiflex aeolicus [126] and in several other transporter families structurally related to LeuT [45].

Neurotransmitter transporters are targets for a wide range of therapeutically useful drugs. This is because they have the capacity to selectively manipulate the dynamics of neurotransmitter concentrations and thereby enhance or diminish signalling through particular brain pathways. High affinity glycine transporters (GlyTs) regulate extracellular concentrations of glycine and provide novel therapeutic targets for neurological disorders.

Immunocytochemistry and Western blotting are still major methods for protein localization, but they rely on the specificity of the antibodies. Validation of antibody specificity remains challenging mostly because ideal negative controls are often unavailable. Further, immunochemical labeling patterns are also influenced by a number of other factors such as postmortem changes, fixation procedures and blocking agents as well as the general assay conditions (e.g., buffers, temperature, etc.). Western blotting similarly depends on tissue collection and sample preparation as well as the electrophoretic separation, transfer to blotting membranes and the immunochemical probing of immobilized molecules. Publication of inaccurate information on protein distribution has downstream consequences for other researchers because the interpretation of physiological and pharmacological observations depends on information on where ion channels, receptors, enzymes or transporters are located. Despite numerous reports, some of which are strongly worded, erroneous localization data are being published. Here we describe the extent of the problem and illustrate the nature of the pitfalls with examples from studies of neurotransmitter transporters. We explain the importance of supplementing immunochemical observations with other measurements (e.g., mRNA levels and distribution, protein activity, mass spectrometry, electrophysiological recordings, etc.) and why quantitative considerations are integral parts of the quality control. Further, we propose a practical strategy for researchers who plan to embark on a localization study. We also share our thoughts about guidelines for quality control. GLIA 2016;64:2045-2064.

A plasma membrane amino acid transporter B0,+ (ATB0,+), encoded by the SLC6A14 gene, is specific for neutral and basic amino acids. It is up-regulated in several types of malignant cancers. Neurotransmitter transporters of the SLC6 family interact with specific SEC24 proteins of the COPII complex along their pathway from the endoplasmic reticulum (ER) to Golgi. This study focused on the possible role of SEC24 proteins in ATB0,+ trafficking. Rat ATB0,+ was expressed in HEK293 cells, its localization and trafficking were examined by Western blot, deglycosylation, immunofluorescence (co-localization with ER and trans-Golgi markers) and biotinylation. The expression of ATB0,+ at the plasma membrane was decreased by dominant negative mutants of SAR1, a GTPase, whose activity triggers the formation of the COPII complex. ATB0,+ co-precipitated with SEC24C (but not with the remaining isoforms A, B and D). This interaction was confirmed by immunocytochemistry and the proximity ligation assay. Co-localization of SEC24C with endogenous ATB0,+ was also observed in MCF-7 breast cancer cells. Contrary to the endogenous transporter, part of the overexpressed ATB0,+ is directed to proteolysis, a process significantly reversed by a proteasome inhibitor bortezomib. Co-transfection with a SEC24C dominant negative mutant attenuated ATB0,+ expression at the plasma membrane, due to proteolytic degradation. These results support a hypothesis that lysine at position +2 downstream of the ER export "RI" motif on the cargo protein is crucial for SEC24C binding and for further trafficking to the Golgi. Moreover, there is an equilibrium between ER export and degradation mechanisms in case of overexpressed transporter.

Recent work demonstrated the propensity of dopamine transporters (DATs) to form trimers or higher oligomers, enhanced upon binding a furopyrimidine, AIM-100. AIM-100 binding promotes DAT endocytosis and thereby moderates dopaminergic transmission. Despite the neurobiological significance of these events, the molecular mechanisms that underlie the stabilization of DAT trimer and the key interactions that modulate the trimerization of DAT, and not serotonin transporter SERT, remain unclear. In the present study, we determined three structural models, termed trimer-W238, -C306 and -Y303, for possible trimerization of DATs . To this aim, we used structural data resolved for DAT and its structural homologs that share the LeuT fold, advanced computational modeling and simulations, site-directed mutagenesis experiments and live-cell imaging assays. The models are in accord with the versatility of LeuT fold to stabilize dimeric or higher order constructs. Selected residues show a high propensity to occupy interfacial regions. Among them, D231-W238 in the extracellular loop EL2, including the intersubunit salt-bridge forming pair D231/D232-R237 (not present in SERT) (in trimer-W238), the loop EL3 (trimers-C306 and -Y303), and W497 on the intracellularly exposed IL5 loop (trimer-C306) and its spatial neighbors (e.g. K525) near the C-terminus are computationally predicted and experimentally confirmed to play important roles in enabling the correct folding and/or oligomerization of DATs in the presence of AIM-100. The study suggests the possibility of controlling the effective transport of dopamine by altering the oligomerization state of DAT upon small molecule binding, as a possible intervention strategy to modulate dopaminergic signaling. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.

Glutamate is the major excitatory neurotransmitter in the vertebrate central nervous system. During synaptic activity, glutamate is released and binds to specific membrane receptors and transporters activating, in the one hand, a wide variety of signal transduction cascades, while in the other hand, its removal from the synaptic cleft. Extracellular glutamate concentrations are maintained within physiological levels mainly by glia glutamate transporters. Inefficient clearance of this amino acid is neurotoxic due to a prolonged hyperactivation of its postsynaptic receptors, exacerbating a wide array of intracellular events linked to an ionic imbalance, that results in neuronal cell death. This process is known as excitotoxicity and is the underlying mechanisms of an important number of neurodegenerative diseases. Therefore, it is important to understand the regulation of glutamate transporters function. The transporter activity can be regulated at different levels: gene expression, transporter protein targeting and trafficking, and post-translational modifications of the transporter protein. The identification of these mechanisms has paved the way to our current understanding the role of glutamate transporters in brain physiology and will certainly provide the needed biochemical information for the development of therapeutic strategies towards the establishment of novel therapeutic approaches for the treatment and/or prevention of pathologies associated with excitotoxicity insults. This article is part of the issue entitled ‘Special Issue on Neurotransmitter Transporters’.

The coupled transport of ions and substrates allows transporters to accumulate substrates using the energy in transmembrane ion gradients and electrical potentials. During transport, conformational changes that switch accessibility of substrate and ion binding sites from one side of the membrane to the other must be controlled so as to prevent uncoupled movement of ions or substrates. In the Neurotransmitter:Sodium Symporter (NSS) family, Na+ stabilizes the transporter in an outward-open state, thus decreasing the likelihood of uncoupled Na+ transport. In a step essential for coupled transport, substrate binding must overcome the effect of Na+, allowing intracellular substrate and Na+ release from an inward-open state. However, it is unclear which specific elements of the protein mediate this conformational response to substrate binding. Previously, we showed that in the prokaryotic NSS transporter LeuT, the effect of Na+ on conformation occurs at the Na2 site, where it influences conformation by fostering interaction between two domains of the protein (JBC 291: 1456, 2016). Here, we identify a conserved tyrosine residue in the substrate binding site required for substrate to enable conversion to inward-open states by establishing an interaction between the two transporter domains. We further identify additional interactions between the two transporter domains in the extracellular pathway that are required. Together with our previous work on the conformational effect of Na+, these results identify mechanistic components underlying ion-substrate coupling in NSS transporters.

This article presents detailed descriptions of procedures and troubleshooting tips for basic in vitro and ex vivo uptake assays for the functional characterization of glutamate transporters and the assessment of the effect of compounds that modulate their activity. Assays are performed in cell lines that transiently or stably express a particular transporter under investigation, in primary cultures of astrocytes, or in ex vivo synaptosomal preparations that endogenously express these transporters. Two main assays are described, including dose-response assays to measure potencies of test compounds for stimulation or inhibition of function (EC50 or IC50 values, respectively) and kinetic functional assays to calculate apparent affinity (KM ) and maximal velocity (Vmax ) of radiolabeled substrate uptake into the cells. The methods described use glutamate transporters as an example; however, the protocols can be adapted to other neurotransmitter transporters using their respective substrates (e.g., GABA uptake through GATs, serotonin uptake through SERTs, among others). © 2018 by John Wiley & Sons, Inc.

GAT1 is a member of the neurotransmitter:sodium: symporter family and mediates transport of GABA together with sodium and chloride in an electrogenic process enabling efficient synaptic transmission. Biochemical and modelling studies based on the structure of the bacterial homologue LeuT are consistent with a transport mechanism whereby the binding pocket is alternately accessible to either side of the membrane. This is achieved by the sequential opening and closing of extracellular and intracellular gates. The amino acid residues participating in the formation of these gates are highly conserved within the neurotransmitter:sodium: symporter family. Net flux requires that the gating mechanism is operative regardless if the binding pocket is loaded with substrate or empty. On the other hand, exchange of labelled for non-labelled substrate across the membrane only requires gating in the presence of substrate. To address the question if the gating requirements of the substrate-bound and empty transporters are similar or different, we analyzed the impact of mutation of intra- and extra-cellular gate residues on net GABA influx and on exchange by liposomes inlaid with the mutant transporters. Whereas net flux by all four internal gate mutants tested was severely abrogated, each exhibited significant levels of exchange. In contrast, two external gate mutants were impaired in both processes. Our results indicate that perturbation of the internal gate of GAT1 selectively impairs the gating mechanism of the empty transporter. This article is part of the issue entitled ‘Special Issue on Neurotransmitter Transporters’.

To investigate the expression of transporters of different neurotransmitters (glutamate, aspartate, lactate, choline) in the culture of astrocytes isolated from different regions of the brain (cortex, hippocampus and brainstem) in 3- and 11-day rats.An experimental study was performed on 24 3- (n=12) and 11-days (n=12) old rats (Rattus norvegicus). The results of high-performance sequencing were analyzed.The expression of glutamate and aspartate transporters in the brainstem of 3-day rats was higher than in other regions, however, an opposite effect was observed in 11-day rats. The expression of lactate transporters with age became identical to those of the cortex.The data demonstrate the particular qualities of neuro-astrocytic connections and the important role of astrocytes in signal transmission. Results of the study performed by using genetic methods developed by the authors for the study of neurotransmitter transporters make it possible to recommend these methods to control the neurogenesis and neurohomeostasis, including in cerebrovascular and neurodegenerative diseases.Цель исследования. Изучение экспрессии транспортеров различных нейромедиаторов (глутамат, аспартат, лактат, холин) в культурах астроцитов из разных областей головного мозга (кора, гиппокамп и ствол мозга) у крыс 3- и 11-дневного возраста. Материал и методы. Проведено экспериментальное исследование с участием 24 крыс Rattus norvegicus 3- (n=12) и 11-дневного (n=12) возраста. Анализировали данные высокопроизводительного секвенирования. Результаты. Экспрессия транспортеров глутамата и аспартата в стволе головного мозга у 3-дневных крыс выше, чем в других регионах, однако наблюдается обратное изменение у крыс в возрасте 11 дней. Показатели экспрессии транспортеров лактата с возрастом становятся идентичными таковым в коре. Заключение. Полученные данные демонстрируют характер нейроастроцитарных связей и роль астроцитов в процессе передачи сигнала. Результаты исследования, проведенного с использованием оригинальных методов изучения транспортеров нейротрансмиттеров, позволяют рекомендовать их для контроля процессов нейрогенеза и нейрогомеостаза, в том числе при цереброваскулярных и нейродегенеративных заболеваниях.

The human dopamine transporter (hDAT) is located on presynaptic neurons, where it plays an essential role in limiting dopaminergic signaling by temporarily curtailing high neurotransmitter concentration through rapid re-uptake. Transport by hDAT is energized by transmembrane ionic gradients. Dysfunction of this transporter leads to disease states, such as Parkinson’s disease, bipolar disorder or depression. It has been shown that hDAT and other members of the monoamine transporter family exist in oligomeric forms at the plasma membrane. Several residues are known to be involved in oligomerization, but interaction interfaces, oligomer orientation and the quarternary arrangement in the plasma membrane remain poorly understood. Here we examine oligomeric forms of hDAT using a direct approach, by following dimerization of two randomly-oriented hDAT transporters in 512 independent simulations, each being 2 μs in length. We employed the DAFT (docking assay for transmembrane components) approach, which is an unbiased molecular dynamics simulation method to identify oligomers, their conformations and populations. The overall ensemble of a total of >1 ms simulation time revealed a limited number of symmetric and asymmetric dimers. The identified dimer interfaces include all residues known to be involved in dimerization. Importantly, we find that the surface of the bundle domain is largely excluded from engaging in dimeric interfaces. Such an interaction would typically lead to inhibition by stabilization of one conformation, while substrate transport relies on a large scale rotation between the inward-facing and the outward-facing state., Author summary The human dopamine transporter efficiently removes the neurotransmitter dopamine from the synaptic cleft. Alteration of dopamine transporter function is associated with several neurological diseases, including mood disorders or attention-deficit hyperactivity disorder, but is also a major player in addiction and drug abuse. Functional studies have revealed that not only is transporter oligomerization involved in surface expression and endocytosis, but, more importantly, in reverse transport (efflux) of dopamine that is triggered by amphetamine-like drugs of abuse. Structural knowledge of transporter oligomerization is largely missing. We performed a large scale comprehensive computational study on transporter oligomerization to reveal dimer geometries and the residues involved in the interfaces. The dimer conformations we find in our dataset are fully consistent with all available experimental data in the literature, but also show novel interfaces. We further verified all dimer geometries by free energy calculations. Our results identified an unpredicted—but for the mechanism of substrate transport essential—property: the bundle domain, which moves during the transport cycle, is excluded from contributing to dimer interfaces, thereby allowing for unrestrained movements necessary to translocate substrates through the membrane.

Vesicular neurotransmitter transporters are responsible for the accumulation of neurotransmitters in secretory vesicles and play essential roles in chemical transmission. The SLC17 family contributes to sequestration of anionic neurotransmitters such as glutamate, aspartate, and nucleotides. Identification and subsequent cellular and molecular biological studies of SLC17 transporters unveiled the principles underlying the actions of these transporters. Recent progress in reconstitution methods in combination with postgenomic approaches has advanced studies on neurotransmitter transporters. This review summarizes the molecular properties of SLC17-type transporters and recent findings regarding the novel SLC18 transporter.

We present the dynamic mechanism of concerted motions in a full-length molecular model of the human dopamine transporter (hDAT), a member of the neurotransmitter/sodium symporter (NSS) family, involved in state-to-state transitions underlying function. The findings result from an analysis of unbiased atomistic molecular dynamics simulation trajectories (totaling >14 μs) of the hDAT molecule immersed in lipid membrane environments with or without phosphatidylinositol 4,5-biphosphate (PIP2) lipids. The N-terminal region of hDAT (N-term) is shown to have an essential mechanistic role in correlated rearrangements of specific structural motifs relevant to state-to-state transitions in the hDAT. The mechanism involves PIP2-mediated electrostatic interactions between the N-term and the intracellular loops of the transporter molecule. Quantitative analyses of collective motions in the trajectories reveal that these interactions correlate with the inward-opening dynamics of hDAT and are allosterically coupled to the known functional sites of the transporter. The observed large-scale motions are enabled by specific reconfiguration of the network of ionic interactions at the intracellular end of the protein. The isomerization to the inward-facing state in hDAT is accompanied by concomitant movements in the extracellular vestibule and results in the release of an Na(+) ion from the Na2 site and destabilization of the substrate dopamine in the primary substrate binding S1 site. The dynamic mechanism emerging from the findings highlights the involvement of the PIP2-regulated interactions between the N-term and the intracellular loop 4 in the functionally relevant conformational transitions that are also similar to those found to underlie state-to-state transitions in the leucine transporter (LeuT), a prototypical bacterial homologue of the NSS. This work was supported by the National Institutes of Health grants P01DA012408, R01DA035263, and U54GM087519. J.M. is supported by the “Caja Madrid” Graduate Fellowship and the “La Caixa” Graduate Fellowship. M.V.L. is supported by a Ruth L. Kirschstein National Research Service Award F31DA035533.

© 2015 Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. Solute carrier (SLC) transporters — a family of more than 300 membrane-bound proteins that facilitate the transport of a wide array of substrates across biological membranes — have important roles in physiological processes ranging from the cellular uptake of nutrients to the absorption of drugs and other xenobiotics. Several classes of marketed drugs target well-known SLC transporters, such as neurotransmitter transporters, and human genetic studies have provided powerful insight into the roles of more-recently characterized SLC transporters in both rare and common diseases, indicating a wealth of new therapeutic opportunities. This Review summarizes knowledge on the roles of SLC transporters in human disease, describes strategies to target such transporters, and highlights current and investigational drugs that modulate SLC transporters, as well as promising drug targets.

Background Much of the structure-based mechanistic understandings of the function of SLC6A neurotransmitter transporters emerged from the study of their bacterial LeuT-fold homologs. It has become evident, however, that structural differences such as the long N- and C-termini of the eukaryotic neurotransmitter transporters are involved in an expanded set of functional properties to the eukaryotic transporters. These functional properties are not shared by the bacterial homologs, which lack the structural elements that appeared later in evolution. However, mechanistic insights into some of the measured functional properties of the eukaryotic transporters that have been suggested to involve these structural elements are sparse or merely descriptive. Results To learn how the structural elements added in evolution enable mechanisms of the eukaryotic transporters in ways not shared with their bacterial LeuT-like homologs, we focused on the human dopamine transporter (hDAT) as a prototype. We present the results of a study employing large-scale molecular dynamics simulations and comparative Markov state model analysis of experimentally determined properties of the wild-type and mutant hDAT constructs. These offer a quantitative outline of mechanisms in which a rich spectrum of interactions of the hDAT N-terminus and C-terminus contribute to the regulation of transporter function (e.g., by phosphorylation) and/or to entirely new phenotypes (e.g., reverse uptake (efflux)) that were added in evolution. Conclusions The findings are consistent with the proposal that the size of eukaryotic neurotransmitter transporter termini increased during evolution to enable more functions (e.g., efflux) not shared with the bacterial homologs. The mechanistic explanations for the experimental findings about the modulation of function in DAT, the serotonin transporter, and other eukaryotic transporters reveal separate roles for the distal and proximal segments of the much larger N-terminus in eukaryotic transporters compared to the bacterial ones. The involvement of the proximal and distal segments — such as the role of the proximal segment in sustaining transport in phosphatidylinositol 4,5-bisphosphate-depleted membranes and of the distal segment in modulating efflux — may represent an evolutionary adaptation required for the function of eukaryotic transporters expressed in various cell types of the same organism that differ in the lipid composition and protein complement of their membrane environment. Electronic supplementary material The online version of this article (10.1186/s12915-018-0495-6) contains supplementary material, which is available to authorized users.

The endogenous neutral amino acid L-proline exhibits a variety of physiological and behavioral actions in the nervous system, highlighting the importance of accurately regulating its extracellular abundance. The L-proline transporter PROT (Slc6A7) is believed to control the spatial and temporal distribution of L-proline at glutamatergic synapses by rapid uptake of this amino acid into presynaptic terminals. Despite the importance of members of the Slc6 transporter family regulating neurotransmitter signaling and homeostasis in brain, evidence that PROT dysfunction supports risk for mental illness is lacking. Here we report the disruption of the PROT gene by homologous recombination. Mice defective in PROT displayed altered expression of glutamate transmission-related synaptic proteins in cortex and thalamus. PROT deficiency perturbed mouse behavior, such as reduced locomotor activity, decreased approach motivation and impaired memory extinction. Thus, our study demonstrates that PROT regulates behaviors that are needed to respond to environmental changes in vivo and suggests that PROT dysfunctions might contribute to mental disorders showing altered response choice following task contingency changes.

The efficacy of boron neutron capture therapy relies on the selective delivery of boron carriers to malignant cells. p-Boronophenylalanine (BPA), a boron delivery agent, has been proposed to be localized to cells through transporter-mediated mechanisms. In this study, we screened aromatic amino acid transporters to identify BPA transporters. Human aromatic amino acid transporters were functionally expressed in Xenopus oocytes and examined for BPA uptake and kinetic parameters. The roles of the transporters in BPA uptake were characterized in cancer cell lines. For the quantitative assessment of BPA uptake, HPLC was used throughout the study. Among aromatic amino acid transporters, ATB0,+, LAT1 and LAT2 were found to transport BPA with Km values of 137.4 ± 11.7, 20.3 ± 0.8 and 88.3 ± 5.6 μM, respectively. Uptake experiments in cancer cell lines revealed that the LAT1 protein amount was the major determinant of BPA uptake at 100 μM, whereas the contribution of ATB0,+ became significant at 1000 μM, accounting for 20–25% of the total BPA uptake in MCF-7 breast cancer cells. ATB0,+, LAT1 and LAT2 transport BPA at affinities comparable with their endogenous substrates, suggesting that they could mediate effective BPA uptake in vivo. The high and low affinities of LAT1 and ATB0,+, respectively, differentiate their roles in BPA uptake. ATB0,+, as well as LAT1, could contribute significantly to the tumor accumulation of BPA at clinical dose.

Background The mammalian neurotransmitter transporters are complex proteins playing a central role in synaptic transmission between neurons by rapid reuptake of neurotransmitters. The proteins which transport dopamine, noradrenaline and serotonin belong to the Neurotransmitter:Sodium Symporters (NSS). Due to their important role, dysfunctions are associated with several psychiatric and neurological diseases and they also serve as targets for a wide range of therapeutic and illicit drugs. Despite the central physiological and pharmacological importance, direct evidence on structure–function relationships on mammalian NSS proteins has so far been unsuccessful. The crystal structure of the bacterial NSS protein, LeuT, has been a turning point in structural investigations. Scope of review To provide an update on what is known about the binding sites for substrates and inhibitors in the LeuT. The different binding modes and binding sites will be discussed with special emphasis on the possible existence of a second substrate binding site. It is the goal to give an insight into how investigations on ligand binding in LeuT have provided basic knowledge about transporter conformations and translocation mechanism which can pave the road for a deeper understanding of drug binding and function of the mammalian transporters. Major conclusions The LeuT is a suitable model for the structural investigation of NSS proteins including the possible location of drug binding sites. It is still debated whether the LeuT is a suitable model for the molecular mechanisms behind substrate translocation. General significance Structure and functional aspects of NSS proteins are central for understanding synaptic transmission. With the purification and crystallization of LeuT as well as the dopamine transporter from Drosophila melanogaster, the application of biophysical methods such as fluorescence spectroscopy, neutron- or x-ray scattering and NMR for understanding its function becomes increasingly available. This article is part of a Special Issue entitled Structural biochemistry and biophysics of membrane proteins.

SLC6 neurotransmitter transporters facilitate the Na+- and Cl--dependent uptake of amino acids and amino acid derivatives into cells. Disrupting transport leads to a range of neurological disorders. However, the SLC6 substrate transport mechanism is a topic of ongoing debate. Here, we review the prominent SLC6 substrate transport mechanisms through the lens of molecular dynamics simulations. SLC6 transporters are membrane proteins, yet their transport mechanism(s) have largely been studied without considering the impacts of synaptic lipid composition, or endogenous lipid modulators, on transporter structure and function. In this review, we highlight the importance of studying membrane transporters in an appropriate membrane model, and present opportunities for the community to glean understanding and insight into SLC6 transporter structure and function-in particular transport mechanism(s)-when both membrane lipids and endogenous lipid modulators are considered.

Much of the structure-based mechanistic understandings of the function of SLC6A neurotransmitter transporters emerged from the study of their bacterial LeuT-fold homologs. It has become evident, however, that structural differences such as the long N- and C-termini of the eukaryotic neurotransmitter transporters impart an expanded set of functional properties to the eukaryotic transporters, which are not shared by the bacterial homologs that lack the structural elements that appeared later in evolution. However, mechanistic insights into some of the measured functional properties of the eukaryotic transporters, that have been suggested to involve these structural elements, are sparse. To learn how the structural elements added in evolution enable mechanisms of the eukaryotic transporters in ways not shared with their bacterial LeuT-like homologs, we focused on the human dopamine transporter (hDAT) as a prototype. We present the results of a study employing large-scale molecular dynamics simulations and comparative Markov State Model analysis of experimentally determined properties of the wild type and mutant hDAT constructs, which reveal a rich spectrum of interactions of the hDAT N-terminus and the mechanisms by which these contribute to regulation (e.g., by phosphorylation), or to entirely new phenotypes (e.g., reverse uptake – efflux) added in evolution. We reveal separate roles for the distal and proximal segments of the much larger N-terminus shared by the eukaryotic transporters compared to the bacterial ones, consistent with the proposal that the size of this region increased during evolution to enable more, and different, modes of regulation that are not shared with the bacterial homologs.

Selective serotonin reuptake inhibitors are clinically prescribed antidepressants that act by increasing the local concentration of neurotransmitter at synapses and in extracellular spaces via blockade of the serotonin transporter. Here we report x-ray structures of engineered thermostable variants of the human serotonin transporter bound to the antidepressants sertraline, fluvoxamine, and paroxetine. The drugs prevent serotonin binding by occupying the central substrate binding site and stabilizing the transporter in an outward-open conformation. These structures explain how residues within the central site orchestrate binding of chemically diverse inhibitors and mediate transporter-drug selectivity.

The recycling of neurotransmitters is essential for sustained synaptic transmission. In Drosophila, histamine recycling is required for visual synaptic transmission. Synaptic histamine is rapidly taken up by laminar glia, and is converted to carcinine. After delivered back to photoreceptors, carcinine is hydrolyzed to release histamine and β-alanine. This histamine is repackaged into synaptic vesicles, but it is unclear how the β-alanine is returned to the laminar glial cells. Here, we identified a new β-alanine transporter, which we named BalaT (Beta-alanine Transporter). Null balat mutants exhibited lower levels of β-alanine, as well as less β-alanine accumulation in the retina. Moreover, BalaT is expressed and required in retinal pigment cells for maintaining visual synaptic transmission and phototaxis behavior. These results provide the first genetic evidence that retinal pigment cells play a critical role in visual neurotransmission, and suggest that a BalaT-dependent β-alanine trafficking pathway is required for histamine homeostasis and visual neurotransmission. DOI: http://dx.doi.org/10.7554/eLife.29146.001, eLife digest Neurons transmit information around the body in the form of electrical signals, but these signals cannot cross the gaps between neurons. To send a message to its neighbor, a neuron releases a molecule known as a neurotransmitter into the gap between the cells. The neurotransmitter binds to proteins on the recipient neuron and triggers new electrical signals inside that cell. When the message has been received, the neurotransmitter molecules are returned to the first neuron so that they can be reused. This recycling is particularly important in the visual system, where neurons communicate via rapid-fire signaling. In fruit flies, for example, light-sensitive neurons in the eye known as photoreceptors release a neurotransmitter called histamine when they detect light. Supporting cells called laminar glia take up any leftover histamine and combine it with another molecule known as β-alanine to form a larger molecule. The photoreceptors absorb this larger molecule and break it back down into histamine and β-alanine. However, it is not clear how the β-alanine returns to the glia to allow this cycle to continue. Many molecules rely on so-called “transporter” proteins to help them move into or out of cells. To identify transporters that might help to move β-alanine, Han, Xiong et al. prepared a list of fruit fly genes that encode transporter proteins found inside the insect’s head. Testing the resulting proteins in a cultured cell system revealed that one of them was able to transport β-alanine. This protein, named BalaT, is found in another type of support cell called retinal pigment cells. Mutant flies that cannot produce BalaT are blind because their photoreceptors have problems in transmitting information to other neurons. Han, Xiong et al. propose that BalaT transports β-alanine from photoreceptors to retinal pigment cells, which then pass β-alanine on to the laminar glia. Follow-up studies are required to find out exactly how the laminar glia take up histamine. DOI: http://dx.doi.org/10.7554/eLife.29146.002

Excitatory amino acid transporters (EAATs) are secondary active transporters of L-glutamate and L- or D-aspartate. These carriers also mediate a thermodynamically uncoupled anion conductance that is gated by Na+ and substrate binding. The activation of the anion channel by binding of Na+ alone, however, has only been demonstrated for mammalian EAAC1 (EAAT3) and EAAT4. To date, no difference has been observed for the substrate dependence of anion channel gating between the glial, EAAT1 and EAAT2, and the neuronal isoforms EAAT3, EAAT4 and EAAT5. Here we describe a difference in the Na+-dependence of anion channel gating between glial and neuronal isoforms. Chloride flux through transporters without glutamate binding has previously been described as substrate-independent or “leak” channel activity. Choline or N-methyl-D-glucamine replacement of external Na+ ions significantly reduced or abolished substrate-independent EAAT channel activity in EAAT3 and EAAT4 yet has no effect on EAAT1 or EAAT2. The interaction of Na+ with the neuronal carrier isoforms was concentration dependent, consistent with previous data. The presence of substrate and Na+-independent open states in the glial EAAT isoforms is a novel finding in the field of EAAT function. Our results reveal an important divergence in anion channel function between glial and neuronal glutamate transporters and highlight new potential roles for the EAAT-associated anion channel activity based on transporter expression and localization in the central nervous system.

A ubiquitous feature of neurotransmitter transporters is the presence of short C-terminal PDZ binding motifs acting as important trafficking elements. Depending on their very C-terminal sequences, PDZ binding motifs are usually divided into at least three groups; however this classification has recently been questioned. To introduce a 3D aspect into transporter’s PDZ motif similarities, we compared their interactions with the natural collection of all 13 PDZ domains of the largest PDZ binding protein MUPP1. The GABA, glycine and serotonin transporters showed unique binding preferences scattered over one or several MUPP1 domains. On the contrary, the dopamine and norepinephrine transporter PDZ motifs did not show any significant affinity to MUPP1 domains. Interestingly, despite their terminal sequence diversity all three GABA transporter PDZ motifs interacted with MUPP1 domain 7. These results indicate that similarities in binding schemes of individual transporter groups might exist. Results also suggest the existence of variable PDZ binding modes, allowing several transporters to interact with identical PDZ domains and potentially share interaction partners in vivo.

ɣ-aminobutyric-acid (GABA) functions as the principal inhibitory neurotransmitter in the central nervous system. Imbalances in GABAergic neurotransmission are involved in the pathophysiology of various neurological diseases such as epilepsy, Alzheimer's disease and stroke. GABA transporters (GATs) facilitate the termination of GABAergic signaling by transporting GABA together with sodium and chloride from the synaptic cleft into presynaptic neurons and surrounding glial cells. Four different GATs have been identified that all belong to the solute carrier 6 (SLC6) transporter family: GAT1-3 (SLC6A1, SLC6A13, SLC6A11) and betaine/GABA transporter 1 (BGT1, SLC6A12). BGT1 has emerged as an interesting target for treating epilepsy due to animal studies that reported anticonvulsant effects for the GAT1/BGT1 selective inhibitor EF1502 and the BGT1 selective inhibitor RPC-425. However, the precise involvement of BGT1 in epilepsy remains elusive because of its controversial expression levels in the brain and the lack of highly selective and potent tool compounds. This review gathers the current structural and functional knowledge on BGT1 with emphasis on brain relevance, discusses all available compounds, and tries to shed light on the molecular determinants driving BGT1 selectivity. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.

Most antidepressants elicit their therapeutic benefits through selective blockade of Na+−Cl− - coupled neurotransmitters transporters. Here we report x-ray structures of the Drosophila melanogaster dopamine transporter in complexes with the polycyclic antidepressants nisoxetine or reboxetine. The inhibitors stabilize the transporter in an outward-open conformation by occupying the substrate binding site. These structures explain how interactions between the binding pocket and substituents on the aromatic rings of antidepressants modulate drug – transporter selectivity.

Neurotransmitter transporters are ion-coupled symporters that drive the uptake of neurotransmitters from neural synapses. In the past decade, the structure of a bacterial amino acid transporter, leucine transporter (LeuT), has given valuable insights into the understanding of architecture and mechanism of mammalian neurotransmitter transporters. Different conformations of LeuT, including a substrate-free state, inward-open state, and competitive and non-competitive inhibitor-bound states, have revealed a mechanistic framework for the transport and transport inhibition of neurotransmitters. The current review integrates our understanding of the mechanistic and pharmacological properties of eukaryotic neurotransmitter transporters obtained through structural snapshots of LeuT.

Structure determination of mammalian integral membrane proteins is challenging due to their instability upon detergent solubilisation and purification. Recent successes in the structure determination of G-protein-coupled receptors (GPCRs) resulted from the development of GPCR-specific protein engineering strategies. One of these, conformational thermostabilisation, could in theory facilitate structure determination of other membrane proteins by improving their tolerance to detergents and locking them in a specific conformation. We have therefore used this approach on the cocaine-sensitive rat serotonin transporter (SERT). Out of a panel of 554 point mutants throughout SERT, 10 were found to improve its thermostability. The most stabilising mutations were combined to make the thermostabilised mutants SAH6 (L99A + G278A + A505L) and SAH7 (L405A + P499A + A505L) that were more stable than SERT by 18 °C and 16 °C, respectively. Inhibitor binding assays showed that both of the thermostabilised SERT mutants bound [125I]RTI55 (β-CIT) with affinity similar to that of the wild-type transporter, although cocaine bound with increased affinity (17- to 56-fold) whilst ibogaine, imipramine and paroxetine all bound with lower affinity (up to 90-fold). Neither SAH6 nor SAH7 was capable of transporting [3H]serotonin into HEK293 cell lines stably expressing the mutants, although serotonin bound to them with an apparent Ki of 155 μM or 82 μM, respectively. These data combined suggest that SAH6 and SAH7 are thermostabilised in a specific cocaine-bound conformation, making them promising candidates for crystallisation. Conformational thermostabilisation is thus equally applicable to membrane proteins that are transporters in addition to those that are GPCRs., Graphical Abstract Highlights ► The serotonin transporter was thermostabilised to facilitate structural studies. ► Mutant SERT-SAH6 contained three point mutations that improved thermostability up to 18 °C. ► SERT-SAH6 preferentially bound cocaine compared to other inhibitors. ► SERT-SAH6 bound serotonin with high affinity but could not transport it. ► SERT-SAH6 is locked in a cocaine-bound conformation ideal for crystallisation.

Variations in the expression levels of the dopamine transporter (DAT) can influence responsiveness to psychostimulant drugs like cocaine. To better understand this relationship, we studied a new DAT-low expresser (DAT-LE) mouse model and performed behavioral and biochemical studies with it. Immunoblotting and [(3) H]WIN 35,428 binding analyses revealed that these mice express ∼35% of wildtype (WT) mouse striatal DAT levels. Compared to WT mice, DAT-LE mice were hyperactive in a novel open-field environment. Despite their higher basal locomotor activity, cocaine (10 or 20 mg/kg, i.p.) induced greater locomotor activation in DAT-LE mice than in WT mice. The maximal velocity (Vmax ) of DAT-mediated [(3) H]DA uptake into striatal synaptosomes was reduced by 46% in DAT-LE mice, as compared to WT. Overall, considering the reduced number of DAT binding sites (Bmax ) along with the reduced Vmax in DAT-LE mice, a 2-fold increase in DA uptake turnover rate (Vmax /Bmax ) was found, relative to WT mice. This suggests that neuroadaptive changes have occurred in the DAT-LE mice that would help to compensate for their low DAT numbers. Interestingly, these changes do not include a reduction in tyrosine hydroxylase levels, as was previously reported in DAT knockout homozygous and heterozygous animals. Further, these changes are not sufficient to prevent elevated novelty- and cocaine-induced locomotor activity. Hence, these mice represent a unique model for studying changes of in vivo DAT function and regulation that result from markedly reduced levels of DAT expression.

The human monoamine transporters (MATs) facilitate the reuptake of monoamine neurotransmitters from the synaptic cleft. MATs are linked to a number of neurological diseases and are the targets of both therapeutic and illicit drugs. Until recently, no high-resolution structures of the human MATs existed, and therefore, studies of this transporter family have relied on investigations of the homologues bacterial transporters such as the leucine transporter LeuT, which has been crystallized in several conformational states. A two-substrate transport mechanism has been suggested for this transporter family, which entails that high-affinity binding of a second substrate in an extracellular site is necessary for the substrate in the central binding site to be transported. Compelling evidence for this mechanism has been presented, however, a number of equally compelling accounts suggest that the transporters function through a mechanism involving only a single substrate and a single high-affinity site. To shed light on this apparent contradiction, we have performed extensive molecular dynamics simulations of LeuT in the outward-occluded conformation with either one or two substrates bound to the transporter. We have also calculated the substrate binding affinity in each of the two proposed binding sites through rigorous free energy simulations. Results show that substrate binding is unstable in the extracellular vestibule and the substrate binding affinity within the suggested extracellular site is very low (0.2 and 3.3 M for the two dominant binding modes) compared to the central substrate binding site (14 nM). This suggests that for LeuT in the outward-occluded conformation only a single high-affinity substrate binding site exists.