Figure - available from: Journal of General Physiology (JGP)
This content is subject to copyright.
![Amino acid sequence alignment of representative members of the glutamate transporter family and substrate transport by replacement mutants at these positions. (A) Amino acid sequence alignment of the representative members of the glutamate transporter family. EAAC, excitatory amino acid carrier; GLT, glutamate transporter; ASCT, alanine serine cysteine transporter; DctA Ec, dicarboxylate transporter Escherichia coli; DctA Rle, dicarboxylate transporter Rhizobium leguminosarum. (B) Uptake of d-[³H]-aspartate in Xenopus laevis oocytes expressing mutants in the stretch ⁴⁴⁴DRFRTVV⁴⁵⁰ (EAAC1 numbering) was done as described under Materials and Methods. Except for arginine-447, each residue of this stretch in EAAC1 was replaced by the equivalent residue of DctA Ec. Aspartate-444 of EAAC1 was changed not only to serine, but also to the other residues indicated. The values are corrected for those obtained with uninjected oocytes and are given as percent of WT uptake. Data shown are mean ± SEM, n = 3. (C) Oocytes expressing WT or the mutants D444S, D444C, and D444E were voltage clamped and gravity perfused with ND96 recording solution (see Materials and Methods) with and without 2 mM L-aspartate. The voltage was stepped from −25 mV to voltages between −100 and +40 mV, in increments of 10 mV. Each potential was held clamped for 250 ms, and the steady-state current from 210 to 240 ms at each potential was averaged. The current in the absence of L-aspartate was subtracted from that in its presence (I) and plotted against the holding potential (Vhold). The values shown are mean ± SEM from three oocytes from different batches that had similar expression levels.](publication/6300038/figure/fig1/AS:11431281177953080@1690664824179/Amino-acid-sequence-alignment-of-representative-members-of-the-glutamate-transporter.jpeg)
Amino acid sequence alignment of representative members of the glutamate transporter family and substrate transport by replacement mutants at these positions. (A) Amino acid sequence alignment of the representative members of the glutamate transporter family. EAAC, excitatory amino acid carrier; GLT, glutamate transporter; ASCT, alanine serine cysteine transporter; DctA Ec, dicarboxylate transporter Escherichia coli; DctA Rle, dicarboxylate transporter Rhizobium leguminosarum. (B) Uptake of d-[³H]-aspartate in Xenopus laevis oocytes expressing mutants in the stretch ⁴⁴⁴DRFRTVV⁴⁵⁰ (EAAC1 numbering) was done as described under Materials and Methods. Except for arginine-447, each residue of this stretch in EAAC1 was replaced by the equivalent residue of DctA Ec. Aspartate-444 of EAAC1 was changed not only to serine, but also to the other residues indicated. The values are corrected for those obtained with uninjected oocytes and are given as percent of WT uptake. Data shown are mean ± SEM, n = 3. (C) Oocytes expressing WT or the mutants D444S, D444C, and D444E were voltage clamped and gravity perfused with ND96 recording solution (see Materials and Methods) with and without 2 mM L-aspartate. The voltage was stepped from −25 mV to voltages between −100 and +40 mV, in increments of 10 mV. Each potential was held clamped for 250 ms, and the steady-state current from 210 to 240 ms at each potential was averaged. The current in the absence of L-aspartate was subtracted from that in its presence (I) and plotted against the holding potential (Vhold). The values shown are mean ± SEM from three oocytes from different batches that had similar expression levels.
Source publication
In the central nervous system, electrogenic sodium- and potassium-coupled glutamate transporters terminate the synaptic actions of this neurotransmitter. In contrast to acidic amino acids, dicarboxylic acids are not recognized by glutamate transporters, but the related bacterial DctA transporters are capable of transporting succinate and other dica...
Citations
... Consistently, the D444S/T448A EAAT3 mutant shows impaired potassium binding and transports dicarboxylates instead of glutamate 47 . Mutations of D444 to any amino acid, including glutamate, abolish transport currents, and the T448A mutation alone decreases uptake by 80% 48 . ...
Excitatory amino acid transporters (EAATs) uptake glutamate into glial cells and neurons. EAATs achieve million-fold transmitter gradients by symporting it with three sodium ions and a proton, and countertransporting a potassium ion via an elevator mechanism. Despite the availability of structures, the symport and antiport mechanisms still need to be clarified. We report high-resolution cryo-EM structures of human EAAT3 bound to the neurotransmitter glutamate with symported ions, potassium ions, sodium ions alone, or without ligands. We show that an evolutionarily conserved occluded translocation intermediate has a dramatically higher affinity for the neurotransmitter and the countertransported potassium ion than outward- or inward-facing transporters and plays a crucial role in ion coupling. We propose a comprehensive ion coupling mechanism involving a choreographed interplay between bound solutes, conformations of conserved amino acid motifs, and movements of the gating hairpin and the substrate-binding domain.
... The ion is coordinated by carbonyl oxygen atoms of residues in the HP2 (408, 409, and 411) and HP1 (331) tips and sidechains of D444 and T448 in TM8 ( Figure 3A) and takes the place of the substrate amino group, also coordinated by D444 ( Figure 3C, D) Consistently, D444S/T448A EAAT3 mutant shows impaired potassium binding and transports dicarboxylates instead of glutamate (Wang et al., 2013). Mutations of D444 to any amino acid, including glutamate, abolish transport currents, and T448A mutation alone decreases uptake by 80 % (Teichman and Kanner, 2007). ...
... EAAT3 is a neuronal and epithelial subtype of glutamate transporters, perhaps the best functionally characterized of all EAATs (Bendahan et al., 2000;Furuta et al., 1997;Grewer et al., 2003;Grewer et al., 2000;Holmseth et al., 2012;Kanai and Hediger, 1992;Rosental et al., 2011;Teichman and Kanner, 2007;Watts et al., 2014;Watzke et al., 2001;Zerangue and Kavanaugh, 1996a;Zhang et al., 2007). Our eight hEAAT3 structures provide the basis for substrate recognition and selectivity and the thermodynamic coupling of substrate uptake to trans-membrane movements of three sodium ions, a proton, and a potassium ion. ...
Excitatory amino acid transporters (EAATs) pump glutamate into glial cells and neurons. EAATs achieve million-fold transmitter gradients by symporting it with three sodium ions and a proton and counter-transporting a potassium ion via an elevator mechanism. Despite the availability of structures, the symport and antiport mechanisms remain unclear. We report high-resolution Cryo-EM structures of human EAAT3 bound to the neurotransmitter glutamate with symported ions, potassium ions, sodium ions alone, or in the absence of ligands. We show that an evolutionarily conserved occluded translocation intermediate has a dramatically higher affinity for the neurotransmitter and the counter-transported potassium ion than outward- or inward-facing transporters and plays a crucial role in ion coupling. We propose a comprehensive ion coupling mechanism involving a choreographed interplay between bound solutes, conformations of conserved amino acid motifs, and movements of the gating hairpin and the substrate-binding domain.
... From left to right: Aspartate binding in the original binding pocket (Fig 2B and 2C), binding at an intermediate site with partialopen loop (Fig 2D). The first conformation compares very well with the binding position and coordination of aspartate in the Glt ph crystal structure [34,35], suggesting that the accelerated binding protocol can provide meaningful results on the actual physical binding pose of the substrate. Asp-394 is known to coordinate the positively-charged amino group of aspartate, most likely through electrostatic interaction, however, an additional syn-conformation (see below) of the substrate was formed in the simulation, which could be an intermediate on the pathway to the stable configuration found in the crystal structure, pointing to flexibility of the ligand before stable binding is attained. ...
... In the structure of the archaeal glutamate transporter homologue Glt ph , the substrate, aspartate, is buried in the transport domain, closed off from the extracellular solution by HP2. Several charged conserved residues contribute to the binding of substrate (aspartate) [33,34]. A ...
... Other conserved residues close to the binding pocket predicted to contribute to substrate binding are Asp-394, Ala-358 from TM8, Thr-314 from TM7, Val-355 from HP2, Arg-276, and Ser-278 from HP1. Also, Gly-354 and Gly-357 located in HP2 work as a recognition group [33,34,[42][43][44] for binding with substrate and forming the closed (occluded) state. In previous simulation studies [18], HP2 loop has been shown to undergo a large scale movement to accomplish the gating function of the binding pocket. ...
Glutamate transporters are essential for removing the neurotransmitter glutamate from the synaptic cleft. Glutamate transport across the membrane is associated with elevator-like structural changes of the transport domain. These structural changes require initial binding of the organic substrate to the transporter. Studying the binding pathway of ligands to their protein binding sites using molecular dynamics (MD) simulations requires micro-second level simulation times. Here, we used three methods to accelerate aspartate binding to the glutamate transporter homologue Gltph and to investigate the binding pathway. 1) Two methods using user-defined forces to prevent the substrate from diffusing too far from the binding site. 2) Conventional MD simulations using very high substrate concentrations in the 0.1 M range. The final, substrate bound states from these methods are comparable to the binding pose observed in crystallographic studies, although they show more flexibility in the side chain carboxylate function. We also captured an intermediate on the binding pathway, where conserved residues D390 and D394 stabilize the aspartate molecule. Finally, we investigated glutamate binding to the mammalian glutamate transporter, excitatory amino acid transporter 1 (EAAT1), for which a crystal structure is known, but not in the glutamate-bound state. Overall, the results obtained in this study reveal new insights into the pathway of substrate binding to glutamate transporters, highlighting intermediates on the binding pathway and flexible conformational states of the side chain, which most likely become locked in once the hairpin loop 2 closes to occlude the substrate.
... Many amino acid residues important for the interaction with sodium (18,19), potassium (5,20) and glutamate (21,22) in eukaryotic transporters are conserved in the archaeal homologues, where these residues are oriented towards the binding pocket. Very recently, direct support for the transferability of information between the bacterial and the human transporters has been obtained through the similarity to the crystal structures of a thermo-stabilized form of the human glutamate transporter EAAT1 (23). ...
In the brain, glutamate transporters terminate excitatory neurotransmission by removing this neurotransmitter from the synapse via cotransport with three sodium ions into the surrounding cells. Structural studies have identified the binding sites of the three sodium ions in glutamate transporters. The residue side-chains directly interact with the sodium ions at the Na1 and Na3 sites and are fully conserved from archaeal to eukaryotic glutamate transporters. The Na2 site is formed by three main-chain oxygens on the extracellular reentrant hairpin loop HP2 and one on transmembrane helix 7. A glycine residue on HP2 is located closely to the three main-chain oxygens in all glutamate transporters, except for the astroglial transporter GLT-1, which has a serine residue at that position. Unlike for wild type GLT-1, substitution of the serine residue to glycine enables sustained glutamate transport also when sodium is replaced by lithium. Here, using functional and simulation studies, we studied the role of this serine/glycine switch on cation selectivity of substrate transport. Our results indicate that the side-chain oxygen of the serine residues can form a hydrogen bond with a main-chain oxygen on transmembrane helix 7. This leads to an expansion of the Na2 site such that water can participate in sodium coordination at Na2. Furthermore, we found other molecular determinants of cation selectivity on the nearby HP1 loop. We conclude that subtle changes in the composition of the two reentrant hairpin loops determine the cation specificity of acidic amino acid transport by glutamate transporters.
... GltPh and human EAATs share~36% sequence identity. Sequence conservation is higher for residues implicated in substrate and ion binding [14][15][16][17][18]. The first crystal structure of the trimeric GltPh was solved in the outward-occluded state [10], followed by structures determined in three additional conformations [11][12][13]19]: outward-open, inwardoccluded and intermediate, revealing a large scale translational motion during substrate transport [12]. ...
Glutamate homeostasis in the brain is maintained by glutamate transporter mediated accumulation. Impaired transport is associated with several neurological disorders, including stroke and amyotrophic lateral sclerosis. Crystal structures of the homolog transporter GltPh from Pyrococcus horikoshii revealed large structural changes. Substrate uptake at the atomic level and the mechanism of ion gradient conversion into directional transport remained enigmatic. We observed in repeated simulations that two local structural changes regulated transport. The first change led to formation of the transient Na2 sodium binding site, triggered by side chain rotation of T308. The second change destabilized cytoplasmic ionic interactions. We found that sodium binding to the transiently formed Na2 site energized substrate uptake through reshaping of the energy hypersurface. Uptake experiments in reconstituted proteoliposomes confirmed the proposed mechanism. We reproduced the results in the human glutamate transporter EAAT3 indicating a conserved mechanics from archaea to humans.
... It too has an uncoupled Cl À conductance, but is not coupled to the cotransport of H þ or the countertransport of K þ ( Fig. 1 A) (14,15). Many residues that have been implicated in substrate and ion binding and/or translocation in the EAATs are conserved throughout the family (16)(17)(18)(19)(20). Glt Ph exists as a trimer, where three identical protomers come together to form an extracellular facing bowl-like structure that sits within the cell membrane. ...
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, GltPh, 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.
... The Na1 and Na2 ions share the same binding sites as the Glt Ph 2NWX crystal structure, and the Na3 site binding site was determined from MD simulations and mutagenesis experiments (6). The coordination of the glutamate substrate is very similar to that of aspartate in Glt Ph , which is stable with a protonated E374, and is in good agreement with the mutagenesis experiments (21,23,36). ...
The uptake of glutamate in nerve synapses is carried out by the excitatory amino acid transporters (EAATs), involving the cotransport of a proton and three Na(+) ions and the countertransport of a K(+) ion. In this study, we use an EAAT3 homology model to calculate the pKa of several titratable residues around the glutamate binding site to locate the proton carrier site involved in the translocation of the substrate. After identifying E374 as the main candidate for carrying the proton, we calculate the protonation state of this residue in different conformations of EAAT3 and with different ligands bound. We find that E374 is protonated in the fully bound state, but removing the Na2 ion and the substrate reduces the pKa of this residue and favors the release of the proton to solution. Removing the remaining Na(+) ions again favors the protonation of E374 in both the outward- and inward-facing states, hence the proton is not released in the empty transporter. By calculating the pKa of E374 with a K(+) ion bound in three possible sites, we show that binding of the K(+) ion is necessary for the release of the proton in the inward-facing state. This suggests a mechanism in which a K(+) ion replaces one of the ligands bound to the transporter, which may explain the faster transport rates of the EAATs compared to its archaeal homologs.
... In order to facilitate homology modeling of EAATs from Glt Ph , we list in Table 2 all the functionally important residues close to the binding site, which are identified from the crystal structures and the mutation experiments in Table 1. Considering the conserved residues first, there seems to be good agreement between the crystal structure data and mutagenesis experiments, e.g., identification of D444 and R447 in EAAT3 as being involved in the coordination of glutamate a-amino and side-chain carboxyl groups, respectively [21,23]. There is, however a discrepancy related to D455 in EAAT3, which corresponds to D405 in Glt Ph . ...
Excitatory amino acid transporters (EAATs) are membrane proteins that enable sodium-coupled uptake of glutamate and other amino acids into neurons. Crystal structures of the archaeal homolog GltPh have been recently determined both in the inward- and outward-facing conformations. Here we construct homology models for the mammalian glutamate transporter EAAT3 in both conformations and perform molecular dynamics simulations to investigate its similarities and differences from GltPh. In particular, we study the coordination of the different ligands, the gating mechanism and the location of the proton and potassium binding sites in EAAT3. We show that the protonation of the E374 residue is essential for binding of glutamate to EAAT3, otherwise glutamate becomes unstable in the binding site. The gating mechanism in the inward-facing state of EAAT3 is found to be different from that of GltPh, which is traced to the relocation of an arginine residue from the HP1 segment in GltPh to the TM8 segment in EAAT3. Finally, we perform free energy calculations to locate the potassium binding site in EAAT3, and find a high-affinity site that overlaps with the Na1 and Na3 sites in GltPh.
... This unusual topology is in excellent agreement with that inferred from biochemical studies on the brain transporters (20 -22). Moreover, many of the amino acid residues of the brain transporters that have been inferred to be important in the interaction with sodium (23,24), potassium (7,25), and glutamate (26,27) are facing toward the binding pocket. Thus, the Glt Ph structures represent excellent models for the brain transporters. ...
Excitatory amino acid transporters remove synaptically released glutamate and maintain its concentrations below neurotoxic levels. EAATs also mediate a thermodynamically uncoupled substrate-gated anion conductance that may modulate cell excitability. A structure of an archeal homologue, which reflects an early intermediate on the proposed substrate translocation path, has been suggested to be similar to an anion conducting conformation. To probe this idea by functional studies, we have introduced two cysteine residues in the neuronal glutamate transporter EAAC1 at positions predicted to be close enough to form a disulfide bond only in outward-facing and early intermediate conformations of the homologue. Upon treatment of Xenopus laevis oocytes expressing the W441C/K269C double mutant with dithiothreitol, radioactive transport was stimulated >2-fold but potently inhibited by low micromolar concentrations of the oxidizing reagent copper(II)(1,10-phenanthroline)3. The substrate-induced currents by the untreated double mutant, reversed at approximately -20 mV, close to the reversal potential of chloride, but treatment with dithiothreitol resulted in transport currents with the same voltage dependence as the wild type. It appears therefore that in the oocyte expression system the introduced cysteine residues in many of the mutant transporters are already cross-linked and are only capable of mediating the substrate-gated anion conductance. Reduction of the disulfide bond now allows these transporters to execute the full transport cycle. Our functional data support the idea that the anion conducting conformation of the neuronal glutamate transporter is associated with an early step of the transport cycle.
... The trimer shape of the Glt Ph crystal formed a bowl or extracellular solvent-accessible basin and the co-crystallization of bound substrates confirmed the location of the hypothesized binding site. The binding site is adjacent to TM7, at the tips of HP2 and HP1 and the substrate was coordinated by several residues previously demonstrated to have a role in substrate interactions (27,(73)(74)(75)(76)(77)(78)(79)(80)(81). The conformational state that seemed to be displayed by this snapshot was the outward conformation with the binding site occluded by HP2. ...
Excitatory amino acid transporters or EAATs are the major transport mechanism for extracellular glutamate in the nervous system. This family of five carriers not only displays an impressive ability to regulate ambient extracellular glu concentrations but also regulate the temporal and spatial profile of glu after vesicular release. This dynamic form of regulation mediates several characteristic of synaptic, perisynaptic, and spillover activation of ionotropic and metabotropic receptors. EAATs function through a secondary active, electrogenic process but also possess a thermodynamically uncoupled ligand gated anion channel activity, both of which have been demonstrated to play a role in regulation of cellular activity. This review will highlight the inception of EAATs as a focus of research, the transport and channel functionality of the carriers, and then describe how these properties are used to regulate glutamatergic neurotransmission.
