SLC6 Research Articles

Insights into transport mechanism from LeuT engineered to transport tryptophan

LeuT is a bacterial homologue of the neurotransmitter:sodium symporter (NSS) family and, being the only NSS member to have been structurally characterized by X-ray crystallography, is a model protein for studying transporter structure and mechanism. Transport activity in LeuT was hypothesized to require structural transitions between open-to-out and occluded conformations dependent upon protein:ligand binding complementarity. Here, using crystallographic and functional analysis, we show that binding site modification produces changes in both structure and activity that are consistent with complementarity-dependent structural transitions to the occluded state. The mutation I359Q converts the activity of tryptophan from inhibitor to transportable substrate. This mutation changes the local environment of the binding site, inducing the bound tryptophan to adopt a different conformer than in the wild-type complex. Instead of trapping the transporter open, tryptophan binding now allows the formation of an occluded state. Thus, transport activity is correlated to the ability of the ligand to promote the structural transition to the occluded state, a step in the transport cycle that is dependent on protein:ligand complementarity in the central binding site.

To be, or not to be two sites: that is the question about LeuT substrate binding

Transport proteins of the neurotransmitter sodium symporter (NSS) family regulate the extracellular concentration of several neurotransmitters in the central nervous system. The only member of this family for which atomic-resolution structural data are available is the prokaryotic homologue LeuT. This protein has been used as a model system to study the molecular mechanism of transport of the NSS family. In this Journal Club, we discuss two strikingly different LeuT transport mechanisms: one involving a single high-affinity substrate binding site and one recently proposed alternative involving two high-affinity substrate binding sites that are allosterically coupled.

Discovery of novel selective serotonin reuptake inhibitors through development of a protein-based pharmacophore.

The serotonin transporter (SERT), a member of the neurotransmitter sodium symporter (NSS) family, is responsible for the reuptake of serotonin from the synaptic cleft to maintain neurotransmitter homeostasis. SERT is established as an important target in the treatment of anxiety and depression. Because a high-resolution crystal structure is not available, a computational model of SERT was built based upon the X-ray coordinates of the leucine transporter LeuT, a bacterial NSS homologue. The model was used to develop the first SERT structure-based pharmacophore. Virtual screening (VS) of a small molecule structural library using the generated SERT computational model yielded candidate ligands of diverse scaffolds. Pharmacological analysis of the VS hits identified two SERT-selective compounds, potential lead compounds for further SERT-related medication development.

The Role of Local Hydration and Hydrogen-Bonding Dynamics in Ion and Solute Release from Ion-Coupled Secondary Transporters

Recent progress in crystallographic studies of sodium-coupled secondary transporters has revealed striking similarities in the structural organization of ion and solute binding. Previous reports suggested that the Na2 sodium binding site in the neurotransmitter sodium symporter (NSS) leucine transporter (LeuT) is conserved across sodium/proton coupled secondary transporters of many distantly related families. This site is implicated in the conformational dynamics controlled by the binding and release of both translocated solute and ion(s) through a mechanism that largely remains unknown. In this study, we used extensive equilibrium molecular dynamics simulations, potential of mean force (PMF) computations, and quasi-harmonic analysis of the LeuT transporter with and without sodium ion bound at the Na2 site to delineate the role of this site in the conformational dynamics of the protein. PMF computations show that in presence of the sodium ion in Na2 the conserved T354 residue is locked into a single rotameric state in contrast to two degenerate states available in the absence of ion in Na2. Molecular dynamics (MD) simulations suggest the formation of a stable water wire from the cytoplasm to the Na2 site in the occluded state. It is plausible that local hydration plays an important role in transport cycle facilitating release of the ion from Na2. An unbinding of the ion from the Na2 site leads to a tightening of the extracellular thin gates and a destabilization of the intracellular thin gate and thus may promote an unbinding of the cotransported substrate. The study lends additional support to the hypothesis that one of the main drivers in the transport cycle of Na-coupled secondary transporters is the binding of the Na2 ion that controls dynamical equilibrium between an inward-facing to an outward-facing conformation.

Dynamics of the Outward and Inward Facing States of the Serotonin Transporter

The serotonin transporter (SERT), a member of the neurotransmitter:sodium symporter (NSS) family, is responsible for clearing neuronal synapses of serotonin, and a major drug target in psychopharmacology. Since no structures have been reported for SERT, structural information is deduced from the crystal structure of LeuT, a prokaryotic NSS homologue. SERT is believed to function by alternating between outward-facing (OF) and inward-facing (IF) states, hence a description of these and intermediate states is vital to the understanding of its transport cycle. To date, studies of SERT structure and dynamics have been restricted to models of the OF state, that in which LeuT is crystallized. We have generated models of substrate/ion-bound as well as free states of SERT, using the OF crystal structure, and our recently published IF models of LeuT. We performed comparative modeling for model generation, docking to position the substrate and extensive optimization of the protein sidechains to generate these models. These SERT models were then simulated in the presence of membrane, water and ions, to characterize the dynamics of the transporter in different states. The simulations reveal differential water permeation behavior among the OF and IF states, and are discussed in view of the known channel-like behavior in SERT. Ion binding patterns hint at a putative binding site for K+, which is known to assist in SERT function. We describe major differences in local and global conformational behavior between the OF and IF states, which provide clues to the transition between these states, and the transport cycle. To our knowledge, these studies provide the first description of dynamics of SERT in the IF state, and a comparative view of the dynamics of the OF and IF as well as substrate/ion bound and free states of SERT.

Neurotransmitter/sodium symporter orthologue LeuT has a single high-affinity substrate site

Neurotransmitter/sodium symporters (NSSs) couple the uptake of neurotransmitter with one or more sodium ions1–3, removing neurotransmitter from the synaptic cleft. NSSs are essential to the function of chemical synapses, are associated with multiple neurological diseases and disorders4, and are the targets of therapeutic and illicit drugs5. LeuT, a prokaryotic orthologue of the NSS family, is a model transporter for understanding the relationships between molecular mechanism and atomic structure in a broad range of sodium-dependent and sodium-independent secondary transporters6–13. At present there is a controversy over whether there are one or two high-affinity substrate binding sites in LeuT. The first-reported crystal structure of LeuT, together with subsequent functional and structural studies, provided direct evidence for a single, high-affinity, centrally located substrate-binding site, defined as the S1 site14,15. Recent binding, flux and molecular simulation studies, however, have been interpreted in terms of a model where there are two high-affinity binding sites: the central, S1, site and a second, the S2 site, located within the extracellular vestibule16. Furthermore, it was proposed that the S1 and S2 sites are allosterically coupled such that occupancy of the S2 site is required for the cytoplasmic release of substrate from the S1 site16. Here we address this controversy by performing direct measurement of substrate binding to wild-type LeuT and to S2 site mutants using isothermal titration calorimetry, equilibrium dialysis and scintillation proximity assays. In addition, we perform uptake experiments to determine whether the proposed allosteric coupling between the putative S2 site and the S1 site manifests itself in the kinetics of substrate flux. We conclude that LeuT harbours a single, centrally located, high-affinity substrate-binding site and that transport is well described by a simple, single-substrate kinetic mechanism.

Modeling and Dynamics of the Inward-Facing State of a Na+/Cl− Dependent Neurotransmitter Transporter Homologue

The leucine transporter (LeuT) has recently commanded exceptional attention due mainly to two distinctions; it provides the only crystal structures available for a protein homologous to the pharmacologically relevant neurotransmitter: sodium symporters (NSS), and, it exhibits a hallmark 5-TM inverted repeat (“LeuT-fold”), a fold recently discovered to also exist in several secondary transporter families, underscoring its general role in transporter function. Constructing the transport cycle of “LeuT-fold” transporters requires detailed structural and dynamic descriptions of the outward-facing (OF) and inward-facing (IF) states, as well as the intermediate states. To this end, we have modeled the structurally unknown IF state of LeuT, based on the known crystal structures of the OF state of LeuT and the IF state of vSGLT, a “LeuT-fold” transporter. The detailed methodology developed for the study combines structure-based alignment, threading, targeted MD and equilibrium MD, and can be applied to other proteins. The resulting IF-state models maintain the secondary structural features of LeuT. Water penetration and solvent accessibility calculations show that TM1, TM3, TM6 and TM8 line the substrate binding/unbinding pathway with TM10 and its pseudosymmetric partner, TM5, participating in the extracellular and intracellular halves of the lumen, respectively. We report conformational hotspots where notable changes in interactions occur between the IF and OF states. We observe Na2 exiting the LeuT-substrate- complex in the IF state, mainly due to TM1 bending. Inducing a transition in only one of the two pseudosymmetric domains, while allowing the second to respond dynamically, is found to be sufficient to induce the formation of the IF state. We also propose that TM2 and TM7 may be facilitators of TM1 and TM6 motion. Thus, this study not only presents a novel modeling methodology applied to obtain the IF state of LeuT, but also describes structural elements involved in a possibly general transport mechanism in transporters adopting the “LeuT-fold”.

Postendocytic Sorting of Constitutively Internalized Dopamine Transporter in Cell Lines and Dopaminergic Neurons

The dopamine transporter (DAT) mediates reuptake of released dopamine and is the target for psychostimulants, such as cocaine and amphetamine. DAT undergoes marked constitutive endocytosis, but little is known about the fate and sorting of the endocytosed transporter. To study DAT sorting in cells lines, we fused the one-transmembrane segment protein Tac to DAT, thereby generating a transporter (TacDAT) with an extracellular antibody epitope suited for trafficking studies. TacDAT was functional and endocytosed constitutively in HEK293 cells. According to an ELISA-based assay, TacDAT intracellular accumulation was increased by the lysosomal protease inhibitor leupeptin and by monensin, an inhibitor of lysosomal degradation and recycling. Monensin also reduced TacDAT surface expression consistent with partial recycling. In both HEK293 cells and in the dopaminergic cell line 1Rb3An27, constitutively internalized TacDAT displayed primary co-localization with the late endosomal marker Rab7, less co-localization with the "short loop" recycling marker Rab4, and little co-localization with the marker of "long loop" recycling endosomes, Rab11. Removal by mutation of N-terminal ubiquitination sites did not affect this sorting pattern. The sorting pattern was distinct from a bona fide recycling membrane protein, the beta(2)-adrenergic receptor, that co-localized primarily with Rab11 and Rab4. Constitutively internalized wild type DAT probed with the fluorescently tagged cocaine analogue JHC 1-64, exhibited the same co-localization pattern as TacDAT in 1Rb3An27 cells and in cultured midbrain dopaminergic neurons. We conclude that DAT is constitutively internalized and sorted in a ubiquitination-independent manner to late endosomes/lysosomes and in part to a Rab4 positive short loop recycling pathway.

Structural Analysis of the Extracellular Entrance to the Serotonin Transporter Permeation Pathway

Neurotransmitter transporters are responsible for removal of biogenic amine neurotransmitters after release into the synapse. These transporters are the targets for many clinically relevant drugs, such as antidepressants and psychostimulants. A high resolution crystal structure for the monoamine transporters has yet to be solved. We have developed a homology model for the serotonin transporter (SERT) based on the crystal structure of the leucine transporter (LeuT(Aa)) from Aquifex aeolicus. The objective of the present studies is to identify the structural determinants forming the entrance to the substrate permeation pathway based on predictions from the SERT homology model. Using the substituted cysteine accessibility method, we identified residues predicted to reside at the entrance to the substrate permeation pathway that were reactive with methanethiosulfonate (MTS) reagents. Of these residues, Gln(332) in transmembrane helix (TMH) VI was protected against MTS inactivation in the presence of serotonin. Surprisingly, the reactivity of Gln(332) to MTS reagents was enhanced in the presence of cocaine. Bifunctional MTS cross-linkers also were used to examine the distances between helices predicted to form the entrance into the substrate and ion permeation pathway. Our studies suggest that substrate and ligand binding may induce conformational shifts in TMH I and/or VI, providing new opportunities to refine existing homology models of SERT and related monoamine transporters.

Binding and Orientation of Tricyclic Antidepressants within the Central Substrate Site of the Human Serotonin Transporter

Tricyclic antidepressants (TCAs) have been used for decades, but their orientation within and molecular interactions with their primary target is yet unsettled. The recent finding of a TCA binding site in the extracellular vestibule of the bacterial leucine transporter 11 A above the central site has prompted debate about whether this vestibular site in the bacterial transporter is applicable to binding of antidepressants to their relevant physiological target, the human serotonin transporter (hSERT). We present an experimentally validated structural model of imipramine and analogous TCAs in the central substrate binding site of hSERT. Two possible binding modes were observed from induced fit docking calculations. We experimentally validated a single binding mode by combining mutagenesis of hSERT with uptake inhibition studies of different TCA analogs according to the paired mutation ligand analog complementation paradigm. Using this experimental method, we identify a salt bridge between the tertiary aliphatic amine and Asp(98). Furthermore, the 7-position of the imipramine ring is found vicinal to Phe(335), and the pocket lined by Ala(173) and Thr(439) is utilized by 3-substituents. These protein-ligand contact points unambiguously orient the TCA within the central binding site and reveal differences between substrate binding and inhibitor binding, giving important clues to the inhibition mechanism. Consonant with the well established competitive inhibition of uptake by TCAs, the resulting binding site for TCAs in hSERT is fully overlapping with the serotonin binding site in hSERT and dissimilar to the low affinity noncompetitive TCA site reported in the leucine transporter (LeuT).

Bivalent phenethylamines as novel dopamine transporter inhibitors: evidence for multiple substrate-binding sites in a single transporter.

Bivalent ligands--compounds incorporating two receptor-interacting moieties linked by a flexible chain--often exhibit profoundly enhanced binding affinity compared with their monovalent components, implying concurrent binding to multiple sites on the target protein. It is generally assumed that neurotransmitter sodium symporter (NSS) proteins, such as the dopamine transporter (DAT), contain a single domain responsible for recognition of substrate molecules. In this report, we show that molecules possessing two substrate-like phenylalkylamine moieties linked by a progressively longer aliphatic spacer act as progressively more potent DAT inhibitors (rather than substrates). One compound bearing two dopamine (DA)-like pharmacophoric 'heads' separated by an 8-carbon linker achieved an 82-fold gain in inhibition of [(3)H] 2beta-carbomethoxy-3beta-(4-fluorophenyl)-tropane (CFT) binding compared with DA itself; bivalent compounds with a 6-carbon linker and heterologous combinations of DA-, amphetamine- and beta-phenethylamine-like heads all resulted in considerable and comparable gains in DAT affinity. A series of short-chain bivalent-like compounds with a single N-linkage was also identified, the most potent of which displayed a 74-fold gain in binding affinity. Computational modelling of the DAT protein and docking of the two most potent bivalent (-like) ligands suggested simultaneous occupancy of two discrete substrate-binding domains. Assays with the DAT mutants W84L and D313N--previously employed by our laboratory to probe conformation-specific binding of different structural classes of DAT inhibitors--indicated a bias of the bivalent ligands for inward-facing transporters. Our results strongly indicate the existence of multiple DAT substrate-interaction sites, implying that it is possible to design novel types of DAT inhibitors based upon the 'multivalent ligand' strategy.

The Allosteric Role of Ion Binding in the Functional Mechanisms of Transporters With LeuT Fold

Recent crystallographic studies revealed that five transporter families without much sequence similarities among them have similar structure folds to LeuT, a bacterial neurotransmitter:sodium symporter (NSS). The LeuT fold is characterized by an internal two-fold structural pseudosymmetry. Interestingly, the transport cycle of at least some members of each of these families is dependent on a sodium gradient across the membrane. Remarkably, the role of sodium is mimicked by a proton in others. We report our computational findings focusing on the LeuT conformations with various combinations of bound substrate and ions, performed in the context of on-going collaborative studies utilizing electron paramagnetic resonance (EPR) spectroscopy and single-molecule fluorescence (smFRET) to identify dynamic details of the mechanism.

Substrate-dependent proton antiport in neurotransmitter:sodium symporters

SUMMARYNeurotransmitter:sodium symporters (NSS), targets for psychostimulants and therapeutic drugs, play a critical role in neurotransmission. Whereas eukaryotic NSS exhibit Cl−-dependent transport, bacterial NSS feature Cl−-independent substrate transport. Recently we showed in LeuT and Tyt1 that mutation of an acidic side chain near one of the Na+-binding sites renders substrate binding and/or transport Cl− dependent. We reasoned that the negative charge - provided either by Cl− or by the transporter itself - is required for substrate translocation. Here we show that Tyt1 reconstituted in proteoliposomes is strictly dependent on the Na+ gradient and is stimulated by an inside negative membrane potential and by an inversely-oriented H+ gradient. Remarkably, Na+/substrate symport elicited H+ efflux, indicative of Na+/substrate symport-coupled H+ antiport. Mutations that render the transport phenotype Cl−-dependent essentially abolish the pH dependence. We propose unifying features of charge balance by all NSS members with similar mechanistic features but with different molecular solutions.

Emerging structure–function relationships defining monoamine NSS transporter substrate and ligand affinity

Monoamine transporters are a group of transmembrane neurotransmitter sodium symporter (NSS) transporters that play a crucial role in regulating biogenic monoamine concentrations at peripheral and central synapses. Given the key role played by serotonin, dopamine and noradrenaline in addictive and disease states, structure–function studies have been conducted to help guide the development of improved central nervous system therapeutics. Extensive pharmacological, immunological and biochemical studies, in conjunction with three-dimensional homology modeling, have been performed to structurally and functionally characterise the monoamine transporter substrate permeation pathway, substrate selectivity, and binding sites for ions, substrates and inhibitors at the molecular level. However, only recently has it been possible to start to construct an accurate molecular interaction network for the monoamine transporters and their corresponding substrates and inhibitors. Crystal structures of Aquifex aeolicus leucine transporter (LeuT Aa), a homologous protein to monoamine transporters that has been experimentally demonstrated to share similar structural folds with monoamine transporters, have been determined in complex with amino acids and inhibitors. The molecular interactions of leucine and tricyclic antidepressants (TCA) has supported many of the predictions based on the mutational studies. Models constructed from LeuT Aa are now allowing a rational approach to further clarify the molecular determinants of NSS transporter–ligand complexes, and potentially the ability to better manipulate drug specificity and affinity. In this review, we compare the structure–function relationships of other SLC6 NSS family transporters with monoamine transporters, and discuss possible mechanisms involved in substrate binding and transport, and modes of inhibition by TCAs.

Subcellular Localization of the Neuronal Glycine Transporter GLYT2 in Brainstem

The neuronal glycine transporter GLYT2 belongs to the neurotransmitter:sodium:symporter (NSS) family and removes glycine from the synaptic cleft, thereby aiding the termination of the glycinergic signal and achieving the reloading of the presynaptic terminal. The task fulfilled by this transporter is fine tuned by regulating both transport activity and intracellular trafficking. Different stimuli such as neuronal activity or protein kinase C (PKC) activation can control GLYT2 surface levels although the intracellular compartments where GLYT2 resides are largely unknown. Here, by biochemical and immunological techniques in combination with electron and confocal microscopy, we have investigated the subcellular distribution of GLYT2 in rat brainstem tissue, and characterized the vesicles that contain the transporter. GLYT2 is shown to be present in small and larger vesicles that contain the synaptic vesicle protein synaptophysin, the recycling endosome small GTPase Rab11, and in the larger vesicle population, the vesicular inhibitory amino acid transporter VIAAT. Rab5A, the GABA transporter GAT1, synaptotagmin2 and synaptobrevin2 (VAMP2) were not present. Coexpression of a Rab11 dominant negative mutant with recombinant GLYT2 impaired transporter trafficking and glycine transport. Dual immunogold labeling of brainstem synaptosomes showed a very close proximity of GLYT2 and Rab11. Therefore, the intracellular GLYT2 resides in a subset of endosomal membranes and may traffic around several compartments, mainly Rab11-positive endosomes.

Substrate–Na + complex formation: Coupling mechanism for γ-aminobutyrate symporters

Crystal structures of transmembrane transport proteins belonging to the important families of neurotransmitter-sodium symporters reveal how they transport neurotransmitters across membranes. Substrate-induced structural conformations of gated neurotransmitter-sodium symporters have been in the focus of research, however, a key question concerning the mechanism of Na + ion coupling remained unanswered. Homology models of human glial transporter subtypes of the major inhibitory neurotransmitter γ-aminobutyric acid were built. In accordance with selectivity data for subtype 2 vs. 3, docking and molecular dynamics calculations suggest similar orthosteric substrate (inhibitor) conformations and binding crevices but distinguishable allosteric Zn 2+ ion binding motifs. Considering the occluded conformational states of glial human γ-aminobutyric acid transporter subtypes, we found major semi-extended and minor ring-like conformations of zwitterionic γ-aminobutyric acid in complex with Na + ion. The existence of the minor ring-like conformation of γ-aminobutyric acid in complex with Na + ion may be attributed to the strengthening of the intramolecular H-bond by the electrostatic effect of Na + ion. Coupling substrate uptake into cells with the thermodynamically favorable Na + ion movement through substrate–Na + ion complex formation may be a mechanistic principle featuring transmembrane neurotransmitter-sodium symporter proteins.

Cloning and functional expression of the first eukaryotic Na+–tryptophan symporter, AgNAT6

The nutrient amino acid transporter (NAT) subfamily of the neurotransmitter sodium symporter family (NSS, also known as the solute carrier family 6, SLC6) represents transport mechanisms with putative synergistic roles in the absorption of essential and conditionally essential neutral amino acids. It includes a large paralogous expansion of insect-specific genes, with seven genes from the genome of the malaria mosquito, Anopheles gambiae. One of the An. gambiae NATs, AgNAT8, was cloned, functionally expressed and characterized in X. laevis oocytes as a cation-coupled symporter of aromatic amino acids, preferably l-phenylalanine, l-tyrosine and l-DOPA. To explore an evolutionary trend of NAT-SLC6 phenotypes, we have cloned and characterized AgNAT6, which represents a counterpart of AgNAT8 descending from a recent gene duplication (53.1% pairwise sequence identity). In contrast to AgNAT8, which preferably mediates the absorption of phenol-branched substrates, AgNAT6 mediates the absorption of indole-branched substrates with highest apparent affinity to tryptophan (K(0.5)(Trp)=1.3 micromol l(-1) vs K(0.5)(Phe)=430 micromol l(-1)) and [2 or 1 Na(+) or K(+)]:[aromatic substrate] stoichiometry. AgNAT6 is highly transcribed in absorptive and secretory regions of the alimentary canal and specific neuronal structures, including the neuropile of ventral ganglia and sensory afferents. The alignment of AgNATs and LeuT(Aa), a bacterial NAT with a resolved 3D structure, reveals three amino acid differences in the substrate-binding pocket that may be responsible for the indole- vs phenol-branch selectivity of AgNAT6 vs AgNAT8. The identification of transporters with a narrow selectivity for essential amino acids suggests that basal expansions in the SLC6 family involved duplication and retention of NATs, improving the absorption and distribution of under-represented essential amino acids and related metabolites. The identified physiological and expression profiles suggest unique roles of AgNAT6 in the active absorption of indole-branched substrates that are used in the synthesis of the neurotransmitter serotonin as well as the key circadian hormone and potent free-radical scavenger melatonin.

Conformational Cycle Of A Bacterial Homolog Of Human Neurotransmitter Sodium Symporters

Neurotransmitter:sodium symporters (NSS) control the magnitude and duration of synaptic signaling through active reuptake of specific neurotransmitters. While it is known that the transmembrane sodium gradient supplies the energy for transport, the coupling mechanism to substrate translocation from its primary binding site (S1) is poorly understood. Conformational rearrangements of extracellular and cytoplasmic regions of the protein are thought to regulate alternating access to S1, and substrate binding at a second site (S2) located near the extracellular region of the transporter, to act as a symport coupler (Shi et al 2008 Mol Cell 30, 667). We used spin labeling and EPR spectroscopy to investigate the conformational dynamics of a highly homologous bacterial member of the NSS family, the leucine transporter LeuT, for which the crystal structure is known (Yamashita et al 2005 Nature 437, 215). Changes in global and local structural constraints derived from the EPR analysis and induced by sodium and leucine binding were then correlated to conformational changes in the LeuT structure in proteoliposomes. Sodium binding was found to increase the distance between the probes as assessed from global rearrangements measured in the extracellular region of LeuT. In contrast, subsequent leucine binding in the presence of sodium was found to decrease the distance between the probes. Consistent with these observations, sodium binding increases spin label mobility and water accessibility at positions within the S2 site. Furthermore, leucine binding increases spin label order and decreases water accessibility. These results suggest a model in which sodium binding to LeuT primes the transporter for substrate binding in S1 by increasing the population of an “outward-facing” conformation, exposing the substrate permeation pathway. With a filled S1, the extracellular pathway constricts, consistent with the occluded state observed in the LeuT crystal structure.

Docking of Multiple Ligands into a New Homology Model of the Serotonin Transporter

The serotonin transporter (SERT) is a presynaptic membrane protein that terminates neuronal transmission of serotonin (5HT) by transporting its substrate into the emitting neuron. Recently new crystal structures of the Leucine Transporter (LeuT) with multiple bound ligands have been published [1]. LeuT is a bacterial sodium dependent amino acid transporter, which is a homologue of the mammalian neurotransmitter sodium symporter (NSS) family (tcdb-code 2.A.22, www.tcdb.org), such as SERT. In light of our studies on the molecular basis of inhibitor binding we constructed a new homology model of SERT and used this for docking studies of a series of pharmacologically active ligands.

A Competitive Inhibitor Traps LeuT in an Open-to-Out Conformation

Secondary transporters are workhorses of cellular membranes, catalyzing the movement of small molecules and ions across the bilayer and coupling substrate passage to ion gradients. However, the conformational changes that accompany substrate transport, the mechanism by which a substrate moves through the transporter, and principles of competitive inhibition remain unclear. We used crystallographic and functional studies on the leucine transporter (LeuT), a model for neurotransmitter sodium symporters, to show that various amino acid substrates induce the same occluded conformational state and that a competitive inhibitor, tryptophan (Trp), traps LeuT in an open-to-out conformation. In the Trp complex, the extracellular gate residues arginine 30 and aspartic acid 404 define a second weak binding site for substrates or inhibitors as they permeate from the extracellular solution to the primary substrate site, which demonstrates how residues that participate in gating also mediate permeation.