The ion-coupling mechanism of human excitatory amino acid transporters.

Excitatory amino acid transporters (EAATs) maintain glutamate gradients in the brain essential for neurotransmission and to prevent neuronal death. They use ionic gradients as energy source and co-transport transmitter into the cytoplasm with Na+ and H+ , while counter-transporting K+ to re-initiate the transport cycle. However, the molecular mechanisms underlying ion-coupled transport remain incompletely understood. Here, we present 3D X-ray crystallographic and cryo-EM structures, as well as thermodynamic analysis of human EAAT1 in different ion bound conformations, including elusive counter-transport ion bound states. Binding energies of Na+ and H+ , and unexpectedly Ca2+ , are coupled to neurotransmitter binding. Ca2+ competes for a conserved Na+ site, suggesting a regulatory role for Ca2+ in glutamate transport at the synapse, while H+ binds to a conserved glutamate residue stabilizing substrate occlusion. The counter-transported ion binding site overlaps with that of glutamate, revealing the K+ -based mechanism to exclude the transmitter during the transport cycle and to prevent its neurotoxic release on the extracellular side.

Glutamate transporters: Critical components of glutamatergic transmission.

Glutamate is the major excitatory neurotransmitter in the vertebrate central nervous system. Once released, it binds to specific membrane receptors and transporters activating a wide variety of signal transduction cascades, as well as its removal from the synaptic cleft in order to avoid its extracellular accumulation and the overstimulation of extra-synaptic receptors that might result in neuronal death through a process known as excitotoxicity. Although neurodegenerative diseases are heterogenous in clinical phenotypes and genetic etiologies, a fundamental mechanism involved in neuronal degeneration is excitotoxicity. Glutamate homeostasis is critical for brain physiology and Glutamate transporters are key players in maintaining low extracellular Glutamate levels. Therefore, the characterization of Glutamate transporters has been an active area of glutamatergic research for the last 40 years. Transporter activity its regulated at different levels: transcriptional and translational control, transporter protein trafficking and membrane mobility, and through extensive post-translational modifications. The elucidation of these mechanisms has emerged as an important piece to shape our current understanding of glutamate actions in the nervous system.

Observing spontaneous, accelerated substrate binding in molecular dynamics simulations of glutamate transporters.

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.

Glutamate transporters have a chloride channel with two hydrophobic gates.

Glutamate is the most abundant excitatory neurotransmitter in the central nervous system, and its precise control is vital to maintain normal brain function and to prevent excitotoxicity1. The removal of extracellular glutamate is achieved by plasma-membrane-bound transporters, which couple glutamate transport to sodium, potassium and pH gradients using an elevator mechanism2-5. Glutamate transporters also conduct chloride ions by means of a channel-like process that is thermodynamically uncoupled from transport6-8. However, the molecular mechanisms that enable these dual-function transporters to carry out two seemingly contradictory roles are unknown. Here we report the cryo-electron microscopy structure of a glutamate transporter homologue in an open-channel state, which reveals an aqueous cavity that is formed during the glutamate transport cycle. The functional properties of this cavity, combined with molecular dynamics simulations, reveal it to be an aqueous-accessible chloride permeation pathway that is gated by two hydrophobic regions and is conserved across mammalian and archaeal glutamate transporters. Our findings provide insight into the mechanism by which glutamate transporters support their dual function, and add information that will assist in mapping the complete transport cycle shared by the solute carrier 1A transporter family.

Paired-Cysteine Scanning Reveals Conformationally Sensitive Proximity between the TM4b-4c Loop and TM8 of the Glutamate Transporter EAAT1.

Excitatory amino acid transporters (EAATs) take up the neurotransmitter glutamate from the synaptic cleft and maintain glutamate concentrations below neurotoxic levels. Recently, the crystal structures of thermostable EAAT1 variants have been reported; however, little is understood regarding the functional mechanism of the transmembrane domain (TM) 4b-4c loop, which contains more than 50 amino acids in mammalian EAATs that are absent in prokaryotic homologues. To explore the spatial position and function of TM4 during the transport cycle, we introduced pairwise cysteine substitutions between the TM4b-4c loop and TM8 in a cysteine-less version of EAAT1, CL-EAAT1. We observed pronounced inhibition of transport by Cu(II)(1,10-phenanthroline)3 (CuPh) for doubly substituted V238C/I469C and A243C/I469C variants, but not for corresponding singly substituted CL-EAAT1 or for more than 20 other double-cysteine variants. Dithiothreitol treatment partially restored the uptake activity of the CuPh-treated V238C/I469C and A243C/I469C doubly substituted variants, confirming that the effects of CuPh on these variants were due to the formation of intramolecular disulfide bonds. Glutamate, KCl, and d,l-threo-ß-benzyloxy-aspartate weakened CuPh inhibition of the V238C/I469C variant, but only KCl weakened CuPh inhibition of the V243C/I469C variant, suggesting that the TM4b-4c loop and TM8 are separated from each other in the inward-facing conformations of EAAT1. Our results suggest that the TM4b-4c loop and TM8 are positioned in close proximity during the transport cycle and are less closely spaced in the inward-facing conformation.

Consensus designs and thermal stability determinants of a human glutamate transporter.

Human excitatory amino acid transporters (EAATs) take up the neurotransmitter glutamate in the brain and are essential to maintain excitatory neurotransmission. Our understanding of the EAATs' molecular mechanisms has been hampered by the lack of stability of purified protein samples for biophysical analyses. Here, we present approaches based on consensus mutagenesis to obtain thermostable EAAT1 variants that share up to ~95% amino acid identity with the wild type transporters, and remain natively folded and functional. Structural analyses of EAAT1 and the consensus designs using hydrogen-deuterium exchange linked to mass spectrometry show that small and highly cooperative unfolding events at the inter-subunit interface rate-limit their thermal denaturation, while the transport domain unfolds at a later stage in the unfolding pathway. Our findings provide structural insights into the kinetic stability of human glutamate transporters, and introduce general approaches to extend the lifetime of human membrane proteins for biophysical analyses.

A novel mutation in SLC1A3 causes episodic ataxia.

Episodic ataxias (EAs) are rare channelopathies characterized by recurrent ataxia and vertigo, having eight subtypes. Mutated genes were found in four of these eight subtypes (EA1, EA2, EA5, and EA6). To date, only four missense mutations in the Solute Carrier Family 1 Member 3 gene (SLC1A3) have been reported to cause EA6. SLC1A3 encodes excitatory amino-acid transporter 1, which is a trimeric transmembrane protein responsible for glutamate transport in the synaptic cleft. In this study, we found a novel missense mutation, c.383T>G (p.Met128Arg) in SLC1A3, in an EA patient by whole-exome sequencing. The modeled structural analysis suggested that p.Met128Arg may affect the hydrophobic transmembrane environment and protein function. Analysis of the pathogenicity of all mutations found in SLC1A3 to date using multiple prediction tools showed some advantage of using the Mendelian Clinically Applicable Pathogenicity (M-CAP) score. Various types of SLC1A3 variants, including nonsense mutations and indels, in the ExAC database suggest that the loss-of-function mechanism by SLC1A3 mutations is unlikely in EA6. The current mutation (p.Med128Arg) presumably has a gain-of-function effect as described in a previous report.

Impaired K+ binding to glial glutamate transporter EAAT1 in migraine.

SLC1A3 encodes the glial glutamate transporter hEAAT1, which removes glutamate from the synaptic cleft via stoichiometrically coupled Na+-K+-H+-glutamate transport. In a young man with migraine with aura including hemiplegia, we identified a novel SLC1A3 mutation that predicts the substitution of a conserved threonine by proline at position 387 (T387P) in hEAAT1. To evaluate the functional effects of the novel variant, we expressed the wildtype or mutant hEAAT1 in mammalian cells and performed whole-cell patch clamp, fast substrate application, and biochemical analyses. T387P diminishes hEAAT1 glutamate uptake rates and reduces the number of hEAAT1 in the surface membrane. Whereas hEAAT1 anion currents display normal ligand and voltage dependence in cells internally dialyzed with Na+-based solution, no anion currents were observed with internal K+. Fast substrate application demonstrated that T387P abolishes K+-bound retranslocation. Our finding expands the phenotypic spectrum of genetic variation in SLC1A3 and highlights impaired K+ binding to hEAAT1 as a novel mechanism of glutamate transport dysfunction in human disease.

Substrate transport and anion permeation proceed through distinct pathways in glutamate transporters.

Advances in structure-function analyses and computational biology have enabled a deeper understanding of how excitatory amino acid transporters (EAATs) mediate chloride permeation and substrate transport. However, the mechanism of structural coupling between these functions remains to be established. Using a combination of molecular modeling, substituted cysteine accessibility, electrophysiology and glutamate uptake assays, we identified a chloride-channeling conformer, iChS, transiently accessible as EAAT1 reconfigures from substrate/ion-loaded into a substrate-releasing conformer. Opening of the anion permeation path in this iChS is controlled by the elevator-like movement of the substrate-binding core, along with its wall that simultaneously lines the anion permeation path (global); and repacking of a cluster of hydrophobic residues near the extracellular vestibule (local). Moreover, our results demonstrate that stabilization of iChS by chemical modifications favors anion channeling at the expense of substrate transport, suggesting a mutually exclusive regulation mediated by the movement of the flexible wall lining the two regions.

Structure and allosteric inhibition of excitatory amino acid transporter 1.

Human members of the solute carrier 1 (SLC1) family of transporters take up excitatory neurotransmitters in the brain and amino acids in peripheral organs. Dysregulation of the function of SLC1 transporters is associated with neurodegenerative disorders and cancer. Here we present crystal structures of a thermostabilized human SLC1 transporter, the excitatory amino acid transporter 1 (EAAT1), with and without allosteric and competitive inhibitors bound. The structures reveal architectural features of the human transporters, such as intra- and extracellular domains that have potential roles in transport function, regulation by lipids and post-translational modifications. The coordination of the allosteric inhibitor in the structures and the change in the transporter dynamics measured by hydrogen-deuterium exchange mass spectrometry reveal a mechanism of inhibition, in which the transporter is locked in the outward-facing states of the transport cycle. Our results provide insights into the molecular mechanisms underlying the function and pharmacology of human SLC1 transporters.

The amino acid transporter, SLC1A3, is plasma membrane-localised in adipocytes and its activity is insensitive to insulin.

The hormone insulin coordinates the catabolism of nutrients by protein phosphorylation. Phosphoproteomic analysis identified insulin-responsive phosphorylation of the Glu/Asp transporter SLC1A3/EAAT1 in adipocytes. The role of SLC1A3 in adipocytes is not well-understood. We show that SLC1A3 is localised to the plasma membrane and the major regulator of acidic amino acid uptake in adipocytes. However, its localisation and activity were unaffected by insulin or mutation of the insulin-regulated phosphosite. The latter was also observed using a heterologous expression system in Xenopus laevis oocytes. Thus, SLC1A3 maintains a constant import of acidic amino acids independently of nutritional status in adipocytes.

Late-onset episodic ataxia associated with SLC1A3 mutation.

Episodic ataxia type 6 (EA6) is caused by mutations in SLC1A3 that encodes excitatory amino acid transporter 1 (EAAT1), a glial glutamate transporter. EAAT1 regulates the extent and durations of glutamate-mediated signal by the clearance of glutamate after synaptic release. In addition, EAAT1 also has an anion channel activity that prevents additional glutamate release. We identified a missense mutation in SLC1A3 in a family with EA. The proband exhibited typical EA2-like symptoms such as recurrent ataxia, slurred speech with a duration of several hours, interictal nystagmus and response to acetazolamide, but had late-onset age of sixth decade. Whole-exome sequencing detected a heterozygous c.1177G>A mutation in SLC1A3. This mutation predicted a substitution of isoleucine for a highly conserved valine residue in the seventh transmembrane domain of EAAT1. The mutation was not present in 100 controls, a large panel of in-house genome data and various mutation databases. Most functional prediction scores revealed to be deleterious. Same heterozygous mutation was identified in one clinically affected family member and two asymptomatic members. Our data expand the mutation spectrum of SLC1A3 and the clinical phenotype of EA6.

Investigating substrate-induced motion between the scaffold and transport domains in the glutamate transporter EAAT1.

Excitatory amino acid transporter 1 plays an important role in keeping the synaptic glutamate concentration below neurotoxic levels by translocating this neurotransmitter into the cell. Both reentrant hairpin loops, HP1 and -2, have been shown to take part in binding the substrate and the more deeply buried sodium ion, and might therefore be a part of the intra- or extracellular gate of the transporter. However, the shape of the motion of either loop relative to transmembrane domain (TM) 4 during the transport cycle has not yet been fully resolved. Using copper(II) (1,10-phenanthroline)3 (CuPh) for cross-linking cysteine pairs, we found strong inhibition of transport when A243C (TM4) was combined with S366C (HP1), I453C (HP2), or T456C (HP2). These findings were reinforced by the impact of cadmium on transport activity, and both approaches consistently showed that proximity was exclusively intramonomeric. Under conditions that promote the inward-facing state, inhibition by CuPh in A243C/S366C was reduced, while the opposite was seen when the outward-facing one was stabilized, suggesting that the two positions are farther apart in the former conformation than in the latter. Surprisingly, maximal cross-linking of A243C with I453C or T456C was not observed under conditions that promote the inward-facing state. Altogether, our data suggest that the transporter may undergo complex relative movement between these positions on TM4 and HP1/HP2 during the transport cycle.

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, 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.

Cysteine mutagenesis reveals alternate proximity between transmembrane domain 2 and hairpin loop 1 of the glutamate transporter EAAT1.

Excitatory amino acid transporter 1 (EAAT1) plays an important role in restricting the neurotoxicity of glutamate. Previous structure-function studies have provided evidence that reentrant helical hairpin loop (HP) 1 has predominant function during the transport cycle. The proposed internal gate HP1 is packed against transmembrane domain (TM) 2 and TM5 in its closed state, and two residues located in TM2 and HP2 of EAAT1 are in close proximity. However, the spatial relationship between TM2 and HP1 during the transport cycle remains unknown. In this study, we used chemical cross-linking of introduced cysteine pair (V96C and S366C) in a cysteine-less version of EAAT1 to assess the proximity of TM2 and HP1. Here, we show that inhibition of transport by copper(II)(1,10-phenanthroline)3 (CuPh) and cadmium ion (Cd(2+)) were observed in the V96C/S366C mutant. Glutamate or potassium significantly protected against the inhibition of transport activity of V96C/S366C by CuPh, while TBOA potentiated the inhibition of transport activity of V96C/S366C by CuPh. We also checked the kinetic parameters of V96C/S366C treated with or without CuPh in the presence of NaCl, NaCl + L-glutamate, NaCl + TBOA, and KCl, respectively. The sensitivity of V96C and S366C to membrane-impermeable sulfhydryl reagent MTSET [(2-trimethylammonium) methanethiosulfonate] was attenuated by glutamate or potassium. TBOA had no effect on the sensitivity of V96C and S366C to MTSET. These data suggest that the spatial relationship between Val-96 of TM2 and Ser-366 of HP1 is altered in the transport cycle.

SLC1 glutamate transporters.

The plasma membrane transporters for the neurotransmitter glutamate belong to the solute carrier 1 family. They are secondary active transporters, taking up glutamate into the cell against a substantial concentration gradient. The driving force for concentrative uptake is provided by the cotransport of Na(+) ions and the countertransport of one K(+) in a step independent of the glutamate translocation step. Due to eletrogenicity of transport, the transmembrane potential can also act as a driving force. Glutamate transporters are expressed in many tissues, but are of particular importance in the brain, where they contribute to the termination of excitatory neurotransmission. Glutamate transporters can also run in reverse, resulting in glutamate release from cells. Due to these important physiological functions, glutamate transporter expression and, therefore, the transport rate, are tightly regulated. This review summarizes recent literature on the functional and biophysical properties, structure-function relationships, regulation, physiological significance, and pharmacology of glutamate transporters. Particular emphasis is on the insight from rapid kinetic and electrophysiological studies, transcriptional regulation of transporter expression, and reverse transport and its importance for pathophysiological glutamate release under ischemic conditions.

Mutating a conserved proline residue within the trimerization domain modifies Na+ binding to excitatory amino acid transporters and associated conformational changes.

Excitatory amino acid transporters (EAATs) are crucial for glutamate homeostasis in the mammalian central nervous system. They are not only secondary active glutamate transporters but also function as anion channels, and different EAATs vary considerably in glutamate transport rates and associated anion current amplitudes. A naturally occurring mutation, which was identified in a patient with episodic ataxia type 6 and that predicts the substitution of a highly conserved proline at position 290 by arginine (P290R), was recently shown to reduce glutamate uptake and to increase anion conduction by hEAAT1. We here used voltage clamp fluorometry to define how the homologous P259R mutation modifies the functional properties of hEAAT3. P259R inverts the voltage dependence, changes the sodium dependence, and alters the time dependence of hEAAT3 fluorescence signals. Kinetic analysis of fluorescence signals indicate that P259R decelerates a conformational change associated with sodium binding to the glutamate-free mutant transporters. This alteration in the glutamate uptake cycle accounts for the experimentally observed changes in glutamate transport and anion conduction by P259R hEAAT3.

Intracellular pH regulation in unstimulated Calliphora salivary glands is Na+ dependent and requires V-ATPase activity.

Salivary gland cells of the blowfly Calliphora vicina have a vacuolar-type H(+)-ATPase (V-ATPase) that lies in their apical membrane and energizes the secretion of a KCl-rich primary saliva upon stimulation with serotonin (5-hydroxytryptamine). Whether and to what extent V-ATPase contributes to intracellular pH (pH(i)) regulation in unstimulated gland cells is unknown. We used the fluorescent dye BCECF to study intracellular pH(i) regulation microfluorometrically and show that: (1) under resting conditions, the application of Na(+)-free physiological saline induces an intracellular alkalinization attributable to the inhibition of the activity of a Na(+)-dependent glutamate transporter; (2) the maintenance of resting pH(i) is Na(+), Cl(-), concanamycin A and DIDS sensitive; (3) recovery from an intracellular acid load is Na(+) sensitive and requires V-ATPase activity; (4) the Na(+)/H(+) antiporter is not involved in pH(i) recovery after a NH(4)Cl prepulse; and (5) at least one Na(+)-dependent transporter and the V-ATPase maintain recovery from an intracellular acid load. Thus, under resting conditions, the V-ATPase and at least one Na(+)-dependent transporter maintain normal pH(i) values of pH 7.5. We have also detected the presence of a Na(+)-dependent glutamate transporter, which seems to act as an acid loader. Despite this not being a common pH(i)-regulating transporter, its activity affects steady-state pH(i) in C. vicina salivary gland cells.