Molecular Determinants for Functional Differences between Alanine-Serine-Cysteine Transporter 1 and Other Glutamate Transporter Family Members

The ASCTs (alanine, serine, and cysteine transporters) belong to the solute carrier family 1 (SLC1), which also includes the human glutamate transporters (excitatory amino acid transporters, EAATs) and the prokaryotic aspartate transporter Glt_Ph. Despite the high degree of amino acid sequence identity between family members, ASCTs function quite differently from the EAATs and Glt_Ph. The aim of this study was to mutate ASCT1 to generate a transporter with functional properties of the EAATs and Glt_Ph, to further our understanding of the structural basis for the different transport mechanisms of the SLC1 family. We have identified three key residues involved in determining differences between ASCT1, the EAATs and Glt_Ph. ASCT1 transporters containing the mutations A382T, T459R, and Q386E were expressed in Xenopus laevis oocytes, and their transport and anion channel functions were investigated. A382T and T459R altered the substrate selectivity of ASCT1 to allow the transport of acidic amino acids, particularly l-aspartate. The combination of A382T and T459R within ASCT1 generates a transporter with a similar profile to that of Glt_Ph, with preference for l-aspartate over l-glutamate. Interestingly, the amplitude of the anion conductance activated by the acidic amino acids does not correlate with rates of transport, highlighting the distinction between these two processes. Q386E impaired the ability of ASCT1 to bind acidic amino acids at pH 5.5; however, this was reversed by the additional mutation A382T. We propose that these residues differences in TM7 and TM8 combine to determine differences in substrate selectivity between members of the SLC1 family.     

The high‐energy transition state of the glutamate transporter homologue GltPh

Membrane transporters mediate cellular uptake of nutrients, signaling molecules, and drugs. Their overall mechanisms are often well understood, but the structural features setting their rates are mostly unknown. Earlier single‐molecule fluorescence imaging of the archaeal model glutamate transporter homologue Glt_Ph from Pyrococcus horikoshii suggested that the slow conformational transition from the outward‐ to the inward‐facing state, when the bound substrate is translocated from the extracellular to the cytoplasmic side of the membrane, is rate limiting to transport. Here, we provide insight into the structure of the high‐energy transition state of Glt_Ph that limits the rate of the substrate translocation process. Using bioinformatics, we identified Glt_Ph gain‐of‐function mutations in the flexible helical hairpin domain HP2 and applied linear free energy relationship analysis to infer that the transition state structurally resembles the inward‐facing conformation. Based on these analyses, we propose an approach to search for allosteric modulators for transporters.     

Transport Rates of a Glutamate Transporter Homologue Are Influenced by the Lipid Bilayer

The aspartate transporter from Pyrococcus horikoshii (Glt_Ph) is a model for the structure of the SLC1 family of amino acid transporters. Crystal structures of Glt_Ph provide insight into mechanisms of ion coupling and substrate transport; however, structures have been solved in the absence of a lipid bilayer so they provide limited information regarding interactions that occur between the protein and lipids of the membrane. Here, we investigated the effect of the lipid environment on aspartate transport by reconstituting Glt_Ph into liposomes of defined lipid composition where the primary lipid is phosphatidylethanolamine (PE) or its methyl derivatives. We showed that the rate of aspartate transport and the transmembrane orientation of Glt_Ph were influenced by the primary lipid in the liposomes. In PE liposomes, we observed the highest transport rate and showed that 85% of the transporters were orientated right-side out, whereas in trimethyl PE liposomes, 50% of transporters were right-side out, and we observed a 4-fold reduction in transport rate. Differences in orientation can only partially explain the lipid composition effect on transport rate. Crystal structures of Glt_Ph revealed a tyrosine residue (Tyr-33) that we propose interacts with lipid headgroups during the transport cycle. Based on site-directed mutagenesis, we propose that a cation-π interaction between Tyr-33 and the lipid headgroups can influence conformational flexibility of the trimerization domain and thus the rate of transport. These results provide a specific example of how interactions between membrane lipids and membrane-bound proteins can influence function and highlight the importance of the role of the membrane in transporter function.     

Functional Characterization of a Na+-dependent Aspartate Transporter from Pyrococcus horikoshii

Excitatory amino acid transporters (EAATs) are crucial in maintaining extracellular levels of glutamate, the most abundant excitatory neurotransmitter, below toxic levels. The recent three-dimensional crystal structure of Glt_Ph, an archaeal homolog of the EAATs, provides elegant structural details of this family of proteins, yet we know little about the mechanism of the bacterial transporter. Conflicting reports in the literature have described Glt_Ph as an aspartate transporter driven by Na⁺ or a glutamate transporter driven by either Na⁺ or H⁺. Here we use purified protein reconstituted into liposomes to thoroughly characterize the ion and substrate dependence of the Glt_Ph transport. We confirm that Glt_Ph is a Na⁺-dependent transporter that is highly selective for aspartate over other amino acids, and we show that transport is coupled to at least two Na⁺ ions. In contrast to the EAATs, transport via Glt_Ph is independent of H⁺ and K⁺. We propose a kinetic model of transport in which at least two Na⁺ ions are coupled to the cotransport of each aspartate molecule by Glt_Ph, and where an ion- and substrate-free transporter reorients to complete the transport cycle.     

Molecular Determinants of Substrate Specificity in Sodium-coupled Glutamate Transporters

Crystal structures of the archaeal homologue Glt_Ph have provided important insights into the molecular mechanism of transport of the excitatory neurotransmitter glutamate. Whereas mammalian glutamate transporters can translocate both glutamate and aspartate, Glt_Ph is only one capable of aspartate transport. Most of the amino acid residues that surround the aspartate substrate in the binding pocket of Glt_Ph are highly conserved. However, in the brain transporters, Thr-352 and Met-362 of the reentrant hairpin loop 2 are replaced by the smaller Ala and Thr, respectively. Therefore, we have studied the effects of T352A and M362T on binding and transport of aspartate and glutamate by Glt_Ph. Substrate-dependent intrinsic fluorescence changes were monitored in transporter constructs containing the L130W mutation. Glt_Ph-L130W/T352A exhibited an ∼15-fold higher apparent affinity for l-glutamate than the wild type transporter, and the M362T mutation resulted in an increased affinity of ∼40-fold. An even larger increase of the apparent affinity for l-glutamate, around 130-fold higher than that of wild type, was observed with the T352A/M362T double mutant. Radioactive uptake experiments show that Glt_Ph-T352A not only transports aspartate but also l-glutamate. Remarkably, Glt_Ph-M362T exhibited l-aspartate but not l-glutamate transport. The double mutant retained the ability to transport l-glutamate, but its kinetic parameters were very similar to those of Glt_Ph-T352A alone. The differential impact of mutation on binding and transport of glutamate suggests that hairpin loop 2 not only plays a role in the selection of the substrate but also in its translocation.     

Mechanism of Transport Modulation by an Extracellular Loop in an Archaeal Excitatory Amino Acid Transporter (EAAT) Homolog

Secondary transporters in the excitatory amino acid transporter family terminate glutamatergic synaptic transmission by catalyzing Na⁺-dependent removal of glutamate from the synaptic cleft. Recent structural studies of the aspartate-specific archaeal homolog, Glt_Ph, suggest that transport is achieved by a rigid body, piston-like movement of the transport domain, which houses the substrate-binding site, between the extracellular and cytoplasmic sides of the membrane. This transport domain is connected to an immobile scaffold by three loops, one of which, the 3–4 loop (3L4), undergoes substrate-sensitive conformational change. Proteolytic cleavage of the 3L4 was found to abolish transport activity indicating an essential function for this loop in the transport mechanism. Here, we demonstrate that despite the presence of fully cleaved 3L4, Glt_Ph is still able to sample conformations relevant for transport. Optimized reconstitution conditions reveal that fully cleaved Glt_Ph retains some transport activity. Analysis of the kinetics and temperature dependence of transport accompanied by direct measurements of substrate binding reveal that this decreased transport activity is not due to alteration of the substrate binding characteristics but is caused by the significantly reduced turnover rate. By measuring solute counterflow activity and cross-link formation rates, we demonstrate that cleaving 3L4 severely and specifically compromises one or more steps contributing to the movement of the substrate-loaded transport domain between the outward- and inward-facing conformational states, sparing the equivalent step(s) during the movement of the empty transport domain. These results reveal a hitherto unknown role for the 3L4 in modulating an essential step in the transport process.     

The twisting elevator mechanism of glutamate transporters reveals the structural basis for the dual transport-channel functions

Glutamate transporters facilitate the removal of this excitatory neurotransmitter from the synapse. Increasing evidence indicates that this process is linked to intrinsic chloride channel activity that is thermodynamically uncoupled from substrate transport. A recent cryo-EM structure of Glt_Ph – an archaeal homolog of the glutamate transporters – in an open channel state has shed light on the structural basis for channel opening formed at the interface of two domains within the transporter which is gated by two clusters of hydrophobic residues. These transporters cycle through several conformational states during the transport process, including the chloride conducting state, which appears to be stabilised by protein–membrane interactions and membrane deformation. Several point mutations that perturb the chloride conductance can have detrimental effects and are linked to the pathogenesis of the neurological disorder, episodic ataxia type 6.     

The archaeal glutamate transporter homologue GltPh shows heterogeneous substrate binding

Integral membrane glutamate transporters couple the concentrative substrate transport to ion gradients. There is a wealth of structural and mechanistic information about this protein family. Recent studies of an archaeal homologue, Glt_Ph, revealed transport rate heterogeneity, which is inconsistent with simple kinetic models; however, its structural and mechanistic determinants remain undefined. Here, we demonstrate that in a mutant Glt_Ph, which exclusively populates the outward-facing state, at least two substates coexist in slow equilibrium, binding the substrate with different apparent affinities. Wild type Glt_Ph shows similar binding properties, and modulation of the substate equilibrium correlates with transport rates. The low-affinity substate of the mutant is transient following substrate binding. Consistently, cryo-EM on samples frozen within seconds after substrate addition reveals the presence of structural classes with perturbed helical packing of the extracellular half of the transport domain in regions adjacent to the binding site. By contrast, an equilibrated structure does not show such classes. The structure at 2.2-Å resolution details a pattern of waters in the intracellular half of the domain and resolves classes with subtle differences in the substrate-binding site. We hypothesize that the rigid cytoplasmic half of the domain mediates substrate and ion recognition and coupling, whereas the extracellular labile half sets the affinity and dynamic properties.     

Free energy simulations of ligand binding to the aspartate transporter Glt(Ph)

Glutamate/Aspartate transporters cotransport three Na⁺ and one H⁺ ions with the substrate and countertransport one K⁺ ion. The binding sites for the substrate and two Na⁺ ions have been observed in the crystal structure of the archeal homolog Glt_Ph, while the binding site for the third Na⁺ ion has been proposed from computational studies and confirmed by experiments. Here we perform detailed free energy simulations of Glt_Ph, giving a comprehensive characterization of the substrate and ion binding sites, and calculating their binding free energies in various configurations. Our results show unequivocally that the substrate binds after the binding of two Na⁺ ions. They also shed light into Asp/Glu selectivity of Glt_Ph, which is not observed in eukaryotic glutamate transporters.     

Amino Acid Transporters and Exchangers from the SLC1A Family: Structure,Mechanism and Roles in Physiology and Cancer

The Solute Carrier 1A (SLC1A) family includes two major mammalian transport systems—the alanine serine cysteine transporters (ASCT1-2) and the human glutamate transporters otherwise known as the excitatory amino acid transporters (EAAT1-5). The EAATs play a critical role in maintaining low synaptic concentrations of the major excitatory neurotransmitter glutamate, and hence they have been widely researched over a number of years. More recently, the neutral amino acid exchanger, ASCT2 has garnered attention for its important role in cancer biology and potential as a molecular target for cancer therapy. The nature of this role is still being explored, and several classes of ASCT2 inhibitors have been developed. However none have reached sufficient potency or selectivity for clinical use. Despite their distinct functions in biology, the members of the SLC1A family display structural and functional similarity. Since 2004, available structures of the archaeal homologues Glt_Ph and Glt_Tk have elucidated mechanisms of transport and inhibition common to the family. The recent determination of EAAT1 and ASCT2 structures may be of assistance in future efforts to design efficacious ASCT2 inhibitors. This review will focus on ASCT2, the present state of knowledge on its roles in tumour biology, and how structural biology is being used to progress the development of inhibitors.

     

Low Affinity and Slow Na+ Binding Precedes High Affinity Aspartate Binding in the Secondary-active Transporter GltPh

Glt_Ph from Pyrococcus horikoshii is a homotrimeric Na⁺-coupled aspartate transporter. It belongs to the widespread family of glutamate transporters, which also includes the mammalian excitatory amino acid transporters that take up the neurotransmitter glutamate. Each protomer in Glt_Ph consists of a trimerization domain involved in subunit interactions and a transport domain containing the substrate binding site. Here, we have studied the dynamics of Na⁺ and aspartate binding to Glt_Ph. Tryptophan fluorescence measurements on the fully active single tryptophan mutant F273W revealed that Na⁺ binds with low affinity to the apoprotein (K_d 120 mm), with a particularly low k_on value (5.1 m⁻¹s⁻¹). At least two sodium ions bind before aspartate. The binding of Na⁺ requires a very high activation energy (E_a 106.8 kJ mol⁻¹) and consequently has a large Q₁₀ value of 4.5, indicative of substantial conformational changes before or after the initial binding event. The apparent affinity for aspartate binding depended on the Na⁺ concentration present. Binding of aspartate was not observed in the absence of Na⁺, whereas in the presence of high Na⁺ concentrations (above the K_d for Na⁺) the dissociation constants for aspartate were in the nanomolar range, and the aspartate binding was fast (k_on of 1.4 × 10⁵ m⁻¹s⁻¹), with low E_a and Q₁₀ values (42.6 kJ mol⁻¹ and 1.8, respectively). We conclude that Na⁺ binding is most likely the rate-limiting step for substrate binding.     

A Conserved Methionine Residue Controls the Substrate Selectivity of a Neuronal Glutamate Transporter

Glutamate transporters located in the brain maintain low synaptic concentrations of the neurotransmitter by coupling its flux to that of sodium and other cations. In the binding pocket of the archeal homologue Glt_Ph, a conserved methionine residue has been implicated in the binding of the benzyl moiety of the nontransportable substrate analogue threo-β-benzyloxyaspartate. To determine whether the corresponding methionine residue of the neuronal glutamate transporter EAAC1, Met-367, fulfills a similar role, M367L, M367C, and M367S mutants were expressed in HeLa cells and Xenopus laevis oocytes to monitor radioactive transport and transport currents, respectively. The apparent affinity of the Met-367 mutants for d-aspartate and l-glutamate, but not for l-aspartate, was 10–20-fold reduced as compared with wild type. Unlike wild type, the magnitude of I_max was different for each of the three substrates. d-Glutamate, which is also a transportable substrate of EAAC1, did not elicit any detectable response with M367C and M367S but acted as a nontransportable substrate analogue in M367L. In the mutants, substrates inhibited the anion conductance as opposed to the stimulation observed with wild type. Remarkably, the apparent affinity of the blocker d,l-threo-β-benzyloxyaspartate in the mutants was similar to that of wild type EAAC1. Our results are consistent with the idea that the side chain of Met-367 fulfills a steric role in the positioning of the substrate in the binding pocket in a step subsequent to its initial binding.     

The mechanism of substrate release by the aspartate transporter GltPh: insights from simulations

Glutamate transporters regulate excitatory amino acid neurotransmission across neuronal and glial cell membranes by coupling the translocation of their substrate (aspartate or glutamate) into the intracellular (IC) medium to the energetically favorable transport of sodium ions or other cations. The first crystallographically resolved structure of this family, the archaeal aspartate transporter, Glt(Ph), has served as a structural paradigm for elucidating the mechanism of substrate translocation by these transporters. Two helical hairpins, HP2 and HP1, at the core domains of the three subunits that form this membrane protein have been proposed to act as the respective extracellular and IC gates for substrate intake and release. Molecular dynamics simulations using the outward-facing structure have confirmed that the HP2 loop acts as an EC gate. The mechanism of substrate release at atomic scale, however, remained unknown due to the lack of structural data until the recent determination of the inward-facing structure of Glt(Ph). In the present study, we use this recently resolved structure to simulate the release of substrate to the cytoplasm and the roles of HP1 and HP2 in this process. The highly flexible HP2 loop is observed to serve as an activator (or initiator) prompting the release of a gatekeeper Na(+) to the cytoplasm and promoting the influx of water molecules from the cytoplasm, which effectively disrupt substrate-protein interactions and drive the dislodging of the substrate from its binding site. The completion of substrate release and exit, however, entails the opening of the highly stable HP1 loop as well. Overall, the unique conformational flexibility of the HP2 loop, the dissociation of a Na(+), the hydration of binding pocket, and final yielding of the HP1 loop 3-Ser motif emerge as the successive events controlling the release of the bound substrate to the cell interior by glutamate transporters.     

The Hydroxyl Side Chain of a Highly Conserved Serine Residue Is Required for Cation Selectivity and Substrate Transport in the Glial Glutamate Transporter GLT-1/SLC1A2

Glutamate transporters maintain synaptic concentration of the excitatory neurotransmitter below neurotoxic levels. Their transport cycle consists of cotransport of glutamate with three sodium ions and one proton, followed by countertransport of potassium. Structural studies proposed that a highly conserved serine located in the binding pocket of the homologous Glt_Ph coordinates l-aspartate as well as the sodium ion Na1. To experimentally validate these findings, we generated and characterized several mutants of the corresponding serine residue, Ser-364, of human glutamate transporter SLC1A2 (solute carrier family 1 member 2), also known as glutamate transporter GLT-1 and excitatory amino acid transporter EAAT2. S364T, S364A, S364C, S364N, and S364D were expressed in HEK cells and Xenopus laevis oocytes to measure radioactive substrate transport and transport currents, respectively. All mutants exhibited similar plasma membrane expression when compared with WT SLC1A2, but substitutions of serine by aspartate or asparagine completely abolished substrate transport. On the other hand, the threonine mutant, which is a more conservative mutation, exhibited similar substrate selectivity, substrate and sodium affinities as WT but a lower selectivity for Na⁺ over Li⁺. S364A and S364C exhibited drastically reduced affinities for each substrate and enhanced selectivity for l-aspartate over d-aspartate and l-glutamate, and lost their selectivity for Na⁺ over Li⁺. Furthermore, we extended the analysis of our experimental observations using molecular dynamics simulations. Altogether, our findings confirm a pivotal role of the serine 364, and more precisely its hydroxyl group, in coupling sodium and substrate fluxes.     

Biochemical Characterization of the C4-Dicarboxylate Transporter DctA from Bacillus subtilis

Bacterial secondary transporters of the DctA family mediate ion-coupled uptake of C₄-dicarboxylates. Here, we have expressed the DctA homologue from Bacillus subtilis in the Gram-positive bacterium Lactococcus lactis. Transport of dicarboxylates in vitro in isolated membrane vesicles was assayed. We determined the substrate specificity, the type of cotransported ions, the electrogenic nature of transport, and the pH and temperature dependence patterns. DctA was found to catalyze proton-coupled symport of the four C₄-dicarboxylates from the Krebs cycle (succinate, fumurate, malate, and oxaloacetate) but not of other mono- and dicarboxylates. Because (i) succinate-proton symport was electrogenic (stimulated by an internal negative membrane potential) and (ii) the divalent anionic form of succinate was recognized by DctA, at least three protons must be cotransported with succinate. The results were interpreted in the light of the crystal structure of the homologous aspartate transporter Glt_Ph from Pyrococcus horikoshii.The DctA family is one of several diverse families of secondary transporters that catalyze the uptake of C₄-dicarboxylates from the Krebs cycle in bacteria (16, 27). In Escherichia coli, DctA mediates the uptake of succinate, fumurate, and malate under aerobic conditions; genomic disruption of dctA in E. coli prevents growth with malate or fumarate as the sole carbon source, and the mutant grows poorly on succinate (5). Similarly, a dctA knockout mutant of Bacillus subtilis cannot grow with succinate or fumarate as the sole carbon source (1). DctA plays a major role in the symbiotic relationship between nitrogen-fixing rhizobia (43) and root nodule-forming plants (30, 37, 38). Transport assays with Sinorhizobium meliloti cells showed previously that in addition to succinate, malate, and fumarate, orotate is transported and that a range of other substrates such as succinamic acid and succinamide may be transported, because they inhibit the transport of orotate (42). In Corynebacterium glutamicum, malate transport by DctA is inhibited by α-ketoglutarate, oxaloacetate, and glyoxylate, indicating that these compounds may be substrates also (41).DctA transporters belong to a large family of secondary transporters (the DAACS [dicarboxylate/amino acid:cation symporter] family), which also comprises well-characterized glutamate/aspartate transporters and neutral amino acid transporters (32, 33). While DctA-type dicarboxylate transporters are found only in bacteria, glutamate/aspartate transporters of the DAACS family are found both in prokaryotes (e.g., GltT in Bacillus stearothermophilus, GltP in E. coli, and Glt_Ph in Pyrococcus horikoshii [2, 7, 34]) and in higher eukarya, where they play a pivotal role in the reuptake of the excitatory neurotransmitter glutamate from the synaptic cleft (4). Neutral amino acid (alanine, serine, and threonine) transporters are found in mammals (see, e.g., references 36 and 44) as well as bacteria (17).Secondary transporters of the DAACS family use (electro)chemical gradients of cations across the membrane to drive transport. The type of cotransported ions varies among family members: eukaryotic glutamate transporters couple the transport of glutamate to the symport of one proton and three sodium ions and the antiport of one potassium ion (24, 45). Bacterial and archaeal glutamate transporters utilize either sodium ions or protons for symport (2) and are independent of potassium ions (28, 31). The bacterial and mammalian neutral amino acid transporters are sodium ion coupled. Glutamate/aspartate transporters and bacterial serine/threonine transporters (SstTs) are electrogenic, but mammalian neutral amino acid transporters are obligate electroneutral amino acid antiporters (44).Insight into the structure-function relationships of the DAACS family members has greatly increased since crystal structures of the P. horikoshii aspartate transporter Glt_Ph have been determined (2, 29, 40). The protein consists of eight membrane-spanning helices and two reentrant regions (helical hairpins HP1 and HP2) (40). The C-terminal part of the protein (helices 7 and 8 and HP1 and HP2) is most strongly conserved with respect to other family members and binds the substrate and cotransported ions, with HP1 and HP2 functioning as lids that allow alternating access to the substrate- and ion-binding sites from either side of the membrane (3, 29). Glt_Ph forms a homotrimeric complex in which each protomer functions independently of the other subunits (11, 12, 18, 19, 23). The fold and oligomeric state are likely to be conserved throughout the family.Whereas the transport mechanisms of bacterial glutamate and neutral amino acid transporters of the DAACS family have been studied extensively in vitro, the C₄-dicarboxylate transporters of the DAACS family (DctA proteins) have been studied using whole cells only. To fully characterize these transporters, in vitro activity assays using either membrane vesicles or proteoliposomes containing purified protein are necessary. In such assays, the internal and external buffer compositions can be controlled, thus allowing manipulation of the chemical ion gradients and the electrical potential across the membrane. Here, we present the first biochemical characterization of a DctA family member in membrane vesicles. We have studied the DctA homologue from B. subtilis, which is annotated as DctP (1) but which we propose to rename DctA to reflect the homology to other DctA proteins. B. subtilis DctA (DctA_Bs) has 30 to 32% sequence identity to the aspartate transporter Glt_Ph and human excitatory amino acid transporter (EAAT) family members, over 40% sequence identity to the characterized bacterial glutamate transporters from E. coli and B. stearothermophilus, and 41 and 56% identity to DctA homologues from C. glutamicum and E. coli, respectively. We determined the substrate specificity of DctA_Bs, the type of cotransported ions, the electrogenic nature of transport, and the pH and temperature dependence patterns.     

Dynamics of the extracellular gate and ion-substrate coupling in the glutamate transporter

Glutamate transporters (GluTs) are the primary regulators of extracellular concentration of the neurotransmitter glutamate in the central nervous system. In this study, we have investigated the dynamics and coupling of the substrate and Na⁺ binding sites, and the mechanism of cotransport of Na⁺ ions, using molecular dynamics simulations of a membrane-embedded model of GluT in its apo (empty form) and various Na⁺- and/or substrate-bound states. The results shed light on the mechanism of the extracellular gate and on the sequence of binding of the substrate and Na⁺ ions to GluT during the transport cycle. The results suggest that the helical hairpin HP2 plays the key role of the extracellular gate for the substrate binding site, and that the opening and closure of the gate is controlled by substrate binding. GluT adopts an open conformation in the absence of the substrate exposing the binding sites of the substrate and Na⁺ ions to the extracellular solution. Based on the calculated trajectories, we propose that Na1 is the first element to bind GluT, as it is found to be important for the completion of the substrate binding site. The subsequent binding of the substrate, in turn, is shown to result in an almost complete closure of the extracellular gate and the formation of the Na2 binding site. Finally, binding of Na2 locks the extracellular gate and completes the formation of the occluded state of GluT.     

The Domain Interface of the Human Glutamate Transporter EAAT1 Mediates Chloride Permeation

The concentration of glutamate within the glutamatergic synapse is tightly regulated by the excitatory amino-acid transporters (EAATs). In addition to their primary role of clearing extracellular glutamate, the EAATs also possess a thermodynamically uncoupled Cl⁻ conductance. Several crystal structures of an archaeal EAAT homolog, Glt_Ph, at different stages of the transport cycle have been solved. In a recent structure, an aqueous cavity located at the interface of the transport and trimerization domains has been identified. This cavity is lined by polar residues, several of which have been implicated in Cl⁻ permeation. We hypothesize that this cavity opens during the transport cycle to form the Cl⁻ channel. Residues lining this cavity in EAAT1, including Ser-366, Leu-369, Phe-373, Arg-388, Pro-392, and Thr-396, were mutated to small hydrophobic residues. Wild-type and mutant transporters were expressed in Xenopus laevis oocytes and two-electrode voltage-clamp electrophysiology, and radiolabeled substrate uptake was used to investigate function. Significant alterations in substrate-activated Cl⁻ conductance were observed for several mutant transporters. These alterations support the hypothesis that this aqueous cavity at the interface of the transport and trimerization domains is a partially formed Cl⁻ channel, which opens to form a pore through which Cl⁻ ions pass. This study enhances our understanding as to how glutamate transporters function as both amino-acid transporters and Cl⁻ channels.     

Proximity of transmembrane segments 5 and 8 of the glutamate transporter GLT-1 inferred from paired cysteine mutagenesis

Background

GLT-1 is a glial glutamate transporter which maintains low synaptic concentrations of the excitatory neurotransmitter enabling efficient synaptic transmission. Based on the crystal structure of the bacterial homologue Glt_Ph, it has been proposed that the reentrant loop HP2, which connects transmembrane domains (TM) 7 and 8, moves to open and close access to the binding pocket from the extracellular medium. However the conformation change between TM5 and TM8 during the transport cycle is not clear yet. We used paired cysteine mutagenesis in conjunction with treatments with Copper(II)(1,10-Phenanthroline)₃ (CuPh), to verify the predicted proximity of residues located at these structural elements of GLT-1.

Methodology/Principal Findings

To assess the proximity of transmembrane domain (TM) 5 relative to TM8 during transport by the glial glutamate transporter GLT-1/EAAT2, cysteine pairs were introduced at the extracellular ends of these structural elements. A complete inhibition of transport by Copper(II)(1,10-Phenanthroline)₃ is observed in the double mutants I295C/I463C and G297C/I463C, but not in the corresponding single mutants. Glutamate and potassium, both expected to increase the proportion of inward-facing transporters, significantly protected against the inhibition of transport activity of I295C/I463C and G297C/I463C by CuPh. Transport by the double mutants I295C/I463C and G297C/I463C also was inhibited by Cd²⁺.

Conclusions/Significance

Our results suggest that TM5 (Ile-295, Gly-297) is in close proximity to TM8 (Ile-463) in the mammalian transporter, and that the spatial relationship between these domains is altered during the transport cycle.     

Functional Defects in the External and Internal Thin Gates of the γ-Aminobutyric Acid (GABA) Transporter GAT-1 Can Compensate Each Other

The GABA transporter GAT-1 belongs to the neurotransmitter:sodium:symporters which are crucial for synaptic transmission. GAT-1 mediates electrogenic transport of GABA together with sodium and chloride. Structure-function studies indicate that the bacterial homologue LeuT, which possess extra- and intracellular thin gates, is an excellent model for this class of neurotransmitter transporters. We recently showed that a conserved aspartate residue of GAT-1, Asp-451, whose LeuT equivalent participates in its thin extracellular gate, is functionally irreplaceable in GAT-1. Only the D451E mutant exhibited residual transport activity but with an elevated apparent sodium affinity as a consequence of an increased proportion of outward-facing transporters. Because during transport the opening and closing of external and internal gates should be tightly coupled, we have addressed the question of whether mutations of the intracellular thin gate residues Arg-44 and Asp-410 can compensate for the effects of their extracellular counterparts. Mutation of Asp-410 to glutamate resulted in impaired transport activity and a reduced apparent affinity for sodium. However, the transport activity of the double mutant D410E/D451E was increased by approximately 10-fold of that of each of the single mutants. Similar compensatory effects were also seen when other combinations of intra- and extracellular thin gate mutants were analyzed. Moreover, the introduction of D410E into the D451E background resulted in lower apparent sodium affinity than that of D451E alone. Our results indicate that a functional interaction of the external and internal gates of GAT-1 is essential for transport.     

The Split Personality of Glutamate Transporters: A Chloride Channel and a Transporter

Transporters and ion channels are conventionally categorised into distinct classes of membrane proteins. However, some membrane proteins have a split personality and can function as both transporters and ion channels. The excitatory amino acid transporters (EAATs) in particular, function as both glutamate transporters and chloride (Cl^?) channels. The EAATs couple the transport of glutamate to the co-transport of three Na⁺ ions and one H⁺ ion into the cell, and the counter-transport of one K⁺ ion out of the cell. The EAAT Cl^? channel is activated by the binding of glutamate and Na⁺, but is thermodynamically uncoupled from glutamate transport and involves molecular determinants distinct from those responsible for glutamate transport. Several crystal structures of an EAAT archaeal homologue, Glt_Ph, at different stages of the transport cycle, alongside numerous functional studies and molecular dynamics simulations, have provided extensive insights into the mechanism of substrate transport via these transporters. However, the molecular determinants involved in Cl^? permeation, and the mechanism by which this channel is activated are not entirely understood. Here we will discuss what is currently known about the molecular determinants involved in EAAT-mediated Cl^? permeation and the mechanisms that underlie their split personality.