Glutamate Transporter Homologue Research Articles

Modulation mechanism of a glutamate transporter homolog by fatty acids

Modulation mechanism of a glutamate transporter homolog by fatty acids

Mutation in glutamate transporter homologue GltTk provides insights into pathologic mechanism of episodic ataxia 6

Episodic ataxias (EAs) are rare neurological conditions affecting the nervous system and typically leading to motor impairment. EA6 is linked to the mutation of a highly conserved proline into an arginine in the glutamate transporter EAAT1. In vitro studies showed that this mutation leads to a reduction in the substrates transport and an increase in the anion conductance. It was hypothesised that the structural basis of these opposed functional effects might be the straightening of transmembrane helix 5, which is kinked in the wild-type protein. In this study, we present the functional and structural implications of the mutation P208R in the archaeal homologue of glutamate transporters GltTk. We show that also in GltTk the P208R mutation leads to reduced aspartate transport activity and increased anion conductance, however a cryo-EM structure reveals that the kink is preserved. The arginine side chain of the mutant points towards the lipidic environment, where it may engage in interactions with the phospholipids, thereby potentially interfering with the transport cycle and contributing to stabilisation of an anion conducting state.

Exploring the transport cycle of DgoT, a bacterial homolog of human vesicular glutamate transporters.

The solute carrier 17 (SLC17) family belongs to the major facilitator superfamily (MFS) and encompasses vesicular glutamate transporters (VGLUTs), inorganic phosphate transporters, the lysosomal H+/sialic acid cotransporter sialin, and the vesicular nucleotide transporter (VNUT). SLC17 members mediate symport or exchange of various substrates with H+ at distinct stoichiometries. A bacterial D-galactonate transporter (DgoT) from E. coli shows high sequence similarity with mammalian VGLUTs and sialin. We used a combination of experimental and computational approaches to explore DgoT transport mechanism. In solid-supported membrane (SSM)-based electrophysiology, rapid galactonate application triggers a transient current generated by substrate binding and subsequent conformational changes, followed by a slower electrogenic transport. Transport current at different pH values demonstrates inhibition of H+ release and binding with apparent pK values of 6.7 and 9.2, respectively. We demonstrated that DgoT functions as galactonate/H+ symporter with 1:2 stoichiometry. There are four charged residues present in the membrane spanning region of DgoT, of which three (R47, R126 and E133) are conserved among SLC17 family. Mutations D46N, E133Q, R47Q and R126Q lead to loss of transport activity, while selective binding of galactonate remains intact. Analysis of pre-steady currents indicates that D46 and E133 are involved in proton translocation, while R47 and R126 are necessary for conformational changes. In all-atom molecular dynamics simulations, protonation of D46 and E133 leads to rearrangement of transmembrane (TM) helices TM1 and TM7 that gate access to the substrate-binding site from the extracellular side. Substrate binding then induces the closure of the periplasmic gate and transition to the inward-open conformation, where substrate is released, in agreement with the alternating access mechanism used by other MFS transporters. Based on our experimental and computational results, we propose a detailed transport model for galactonate/H+ cotransport in DgoT.

Distinct roles of the Na+ binding sites in the allosteric coupling mechanism of the glutamate transporter homolog, GltPh

Glutamate transporters carry out the concentrative uptake of glutamate by harnessing the ionic gradients present across cellular membranes. A central step in the transport mechanism is the coupled binding of Na+ and substrate. The sodium coupled Asp transporter, GltPh is an archaeal homolog of glutamate transporters that has been extensively used to probe the transport mechanism. Previous studies have shown that hairpin-2 (HP2) functions as the extracellular gate for the aspartate binding site and plays a key role in the coupled binding of sodium and aspartate to GltPh. The binding sites for three Na+ ions (Na1-3) have been identified in GltPh, but the specific roles of the individual Na+ sites in the binding process have not been elucidated. In this study, we developed assays to probe Na+ binding to the Na1 and Na3 sites and to monitor the conformational switch in the NMDGT motif. We used these assays along with a fluorescence assay to monitor HP2 movement and EPR spectroscopy to show that Na+ binding to the Na3 site is required for the NMDGT conformational switch while Na+ binding to the Na1 site is responsible for the partial opening of HP2. Complete opening of HP2 requires the conformational switch of the NMDGT motif and therefore Na+ binding to both the Na1 and the Na3 sites. Based on our studies, we also propose an alternate pathway for the coupled binding of Na+ and Asp.

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, GltPh, 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 GltPh, which exclusively populates the outward-facing state, at least two substates coexist in slow equilibrium, binding the substrate with different apparent affinities. Wild type GltPh 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.

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, therefore its precise control is vital for maintaining normal brain function and preventing excitotoxicity1. 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 via a channel-like process that is thermodynamically uncoupled from transport6–8. However, the molecular mechanisms that allow 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, revealing an aqueous cavity that is formed during the transport cycle. By studying functional properties combined with molecular dynamics simulations, we show that this cavity is an aqueous-accessible chloride permeation pathway 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 a crucial piece of information to aid mapping of the complete transport cycle shared by the SLC1A transporter family.

Glutamate Transporters (EAATS) Contain a Conserved Chloride Channel with Two Hydrophobic Gates

Glutamate is the most abundant excitatory neurotransmitter in the central nervous system, therefore its precise control is vital for maintaining normal brain function and preventing excitotoxicity. Removal of extracellular glutamate is achieved by plasma membrane-bound transporters known as Excitatory Amino Acid Transporters (EAATs) which belong to the solute carrier 1A (SLC1A) family. The EAATs couple glutamate transport to sodium, potassium and pH gradients using an elevator transport mechanism. Glutamate transporters also conduct chloride ions via a channel-like process that is thermodynamically uncoupled from transport. However, the molecular mechanisms that allow 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 (GltPh) in an open-channel state, revealing an aqueous cavity that is formed at the interface of the transport and scaffold domains during the transport cycle. Using functional studies and molecular dynamics simulations, we show that this cavity is an aqueous-accessible chloride permeation pathway gated by two hydrophobic regions and is conserved across mammalian and archaeal glutamate transporters. Our findings reveal the mechanism by which glutamate transporters support their dual functions and provide a crucial piece of information to aid mapping of the complete transport cycle shared by the SLC1A transporter family.

Structural Aspects of Photopharmacology: Insight into the Binding of Photoswitchable and Photocaged Inhibitors to the Glutamate Transporter Homologue.

Photopharmacology addresses the challenge of drug selectivity and side effects through creation of photoresponsive molecules activated with light with high spatiotemporal precision. This is achieved through incorporation of molecular photoswitches and photocages into the pharmacophore. However, the structural basis for the light-induced modulation of inhibitory potency in general is still missing, which poses a major design challenge for this emerging field of research. Here we solved crystal structures of the glutamate transporter homologue GltTk in complex with photoresponsive transport inhibitors—azobenzene derivative of TBOA (both in trans and cis configuration) and with the photocaged compound ONB-hydroxyaspartate. The essential role of glutamate transporters in the functioning of the central nervous system renders them potential therapeutic targets in the treatment of neurodegenerative diseases. The obtained structures provide a clear structural insight into the origins of photocontrol in photopharmacology and lay the foundation for application of photocontrolled ligands to study the transporter dynamics by using time-resolved X-ray crystallography.

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 GltPh 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 GltPh that limits the rate of the substrate translocation process. Using bioinformatics, we identified GltPh 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.

Use of paramagnetic 19F NMR to monitor domain movement in a glutamate transporter homolog.

In proteins where conformational changes are functionally important, the number of accessible states and their dynamics are often difficult to establish. Here we describe a novel 19F-NMR spectroscopy approach to probe dynamics of large membrane proteins. We labeled a glutamate transporter homologue with a 19F probe via cysteine chemistry and with a Ni2+ ion via chelation by a di-histidine motif. We used distance-dependent enhancement of the longitudinal relaxation of 19F nuclei by the paramagnetic metal to assign the observed resonances. We identified two outward- and one inward-facing states of the transporter, in which the substrate-binding site is near the extracellular and intracellular solutions, respectively. We then resolved the structure of the unanticipated second outward-facing state by Cryo-EM. Finally, we showed that the rates of the conformational exchange are accessible from measurements of the metal-enhanced longitudinal relaxation of 19F nuclei.

Single-molecule transport kinetics of a glutamate transporter homolog shows static disorder.

Kinetic properties of membrane transporters are typically poorly defined because high-resolution functional assays analogous to single-channel recordings are lacking. Here, we measure single-molecule transport kinetics of a glutamate transporter homolog from Pyrococcus horikoshii, GltPh, using fluorescently labeled periplasmic amino acid binding protein as a fluorescence resonance energy transfer-based sensor. We show that individual transporters can function at rates varying by at least two orders of magnitude that persist for multiple turnovers. A gain-of-function mutant shows increased population of the fast-acting transporters, leading to a 10-fold increase in the mean transport rate. These findings, which are broadly consistent with earlier single-molecule measurements of GltPh conformational dynamics, suggest that GltPh transport is defined by kinetically distinct populations that exhibit long-lasting "molecular memory."

Structural ensemble of a glutamate transporter homologue in lipid nanodisc environment

Glutamate transporters are cation-coupled secondary active membrane transporters that clear the neurotransmitter L-glutamate from the synaptic cleft. These transporters are homotrimers, with each protomer functioning independently by an elevator-type mechanism, in which a mobile transport domain alternates between inward- and outward-oriented states. Using single-particle cryo-EM we have determined five structures of the glutamate transporter homologue GltTk, a Na+- L-aspartate symporter, embedded in lipid nanodiscs. Dependent on the substrate concentrations used, the protomers of the trimer adopt a variety of asymmetrical conformations, consistent with the independent movement. Six of the 15 resolved protomers are in a hitherto elusive state of the transport cycle in which the inward-facing transporters are loaded with Na+ ions. These structures explain how substrate-leakage is prevented – a strict requirement for coupled transport. The belt protein of the lipid nanodiscs bends around the inward oriented protomers, suggesting that membrane deformations occur during transport.

The 4b-4c loop of excitatory amino acid transporter 1 containing four critical residues essential for substrate transport

In the mammalians, the 4b-4c loop of excitatory amino acid transporters (EAATs) spans more than 50 amino-acid residues that are absent in glutamate transporter homologue of Pyrococcus horikoshii (GltPh). This part of insertion is unique for metazoans and indispensable to the localization of EAATs. The excitatory amino acid transporter (EAAT) 1 is one of the two glial glutamate transporters, which are responsible for efficiently clearing glutamate from the synaptic cleft to prevent neurotoxicity and cell death. Although the crystal structure of EAAT1cryst (a human thermostable EAAT1) was resolved in 2017, the structure-function relationship of the 4b-4c loop has not been elucidated in EAAT1cryst. To investigate the role of the 4b-4c loop, we performed alanine-scanning mutagenesis in the mutants and observed dramatically decreased transport activities in T192A, Y194A, N242A, and G245A mutants. The surface expression of T192A and Y194A mutants even decreased by more than 80%, and most of them were detained in the cytoplasm. However, when T192 and Y194 were substituted with conservative residues, the transport activities and the surface expressions of T192S and Y194F were largely recovered, and their kinetic parameters (Km values) were comparable to the wild-type EAAT1 as well. In contrast, N242 and G245 substituted with conservative residues could not rescue the uptake function, suggesting that N242 and G245 may play irreplaceable roles in the glutamate uptake process. These results indicate that the 4b-4c loop of EAAT1 may not only affect the glutamate uptake activity, but also influence the surface localization of EAAT1 by T192 and Y194.Communicated by Ramaswamy H. Sarma

Investigation of the allosteric coupling mechanism in a glutamate transporter homolog via unnatural amino acid mutagenesis

Glutamate transporters harness the ionic gradients across cell membranes for the concentrative uptake of glutamate. The sodium-coupled Asp symporter, GltPh is an archaeal homolog of glutamate transporters and has been extensively used to understand the transport mechanism. A critical aspect of the transport cycle in GltPh is the coupled binding of sodium and aspartate. Previous studies have suggested a major role for hairpin-2 (HP2), which functions as the extracellular gate for the aspartate binding site, in the coupled binding of sodium and aspartate to GltPh In this study, we develop a fluorescence assay for monitoring HP2 movement by incorporating tryptophan and the unnatural amino acid, p-cyanophenylalanine into GltPh We use the HP2 assays to show that HP2 opening with Na+ follows an induced-fit mechanism. We also determine how residues in the substrate binding site affect the opening and closing of HP2. Our data, combined with previous studies, provide the molecular sequence of events in the coupled binding of sodium and aspartate to GltPh.

Binding and transport of D-aspartate by the glutamate transporter homolog GltTk.

Mammalian glutamate transporters are crucial players in neuronal communication as they perform neurotransmitter reuptake from the synaptic cleft. Besides L-glutamate and L-aspartate, they also recognize D-aspartate, which might participate in mammalian neurotransmission and/or neuromodulation. Much of the mechanistic insight in glutamate transport comes from studies of the archeal homologs GltPh from Pyrococcus horikoshii and GltTk from Thermococcus kodakarensis. Here, we show that GltTk transports D-aspartate with identical Na+: substrate coupling stoichiometry as L-aspartate, and that the affinities (Kd and Km) for the two substrates are similar. We determined a crystal structure of GltTk with bound D-aspartate at 2.8 Å resolution. Comparison of the L- and D-aspartate bound GltTk structures revealed that D-aspartate is accommodated with only minor rearrangements in the structure of the binding site. The structure explains how the geometrically different molecules L- and D-aspartate are recognized and transported by the protein in the same way.

Investigation of Extracellular Gate Movement in a Glutamate Homologue

Glutamate transporters harness the ionic gradients across cell membranes to carry out the concentrative uptake of glutamate. The sodium coupled Asp symporter, GltPh is an archaeal homologue of glutamate transporters which has been extensively used to understand the transport mechanism. A critical aspect of the transport mechanism is the coupled binding of sodium and aspartate. The crystal structures of GltPh shows that the Na+ ions do not form direct interactions with the substrate, suggesting that the binding of Na+ and substrate is coupled through an allosteric mechanism. Previous studies have suggested a critical role for hairpin-2 (HP2), the extracellular gate for the substrate binding site, in coupling the binding of sodium and asparate to GltPh. We have developed a fluorescence assay for monitoring HP2 movement by incorporating tryptophan and the unnatural amino acid, p-cyanophenylalanine into GltPh. We use the HP2 fluorescence assay in concert with traditional Na+ and Asp binding assays to investigate how key residues in the substrate binding sites modulate HP2 movement. Our studies reveal the intricate choreography of side chain conformational changes linked to HP2 opening and closing that is the basis for the coupled binding of Na+ and Asp to GltPh.

Millisecond Time Resolution by HS-AFM Line Scanning of Fast GltPh Dynamics

Mammalian glutamate transporter proteins play the crucial role in removing excitatory neurotransmitters from the synaptic cleft. Malfunction of Glutamate transporters is identified in various neurological disorders such as cerebral stroke, epilepsy, Alzheimer's disease, dementia, Huntington's disease, amyotrophic lateral sclerosis (ALS) and malignant glioma. During the past decade, our understanding of the structure and function of these proteins have evolved profoundly from studying a prokaryotic glutamate transporter homolog (GltPh). Here, we report the development and use of novel high-speed atomic force microscopy (HS-AFM) based techniques that reach millisecond time resolution, and explored previously undetectable short-lived transport states in unlabeled membrane reconstituted GltPh. We find that GltPh exhibits much faster dynamics than previously thought.

A facile approach for the in vitro assembly of multimeric membrane transport proteins

Membrane proteins such as ion channels and transporters are frequently homomeric. The homomeric nature raises important questions regarding coupling between subunits and complicates the application of techniques such as FRET or DEER spectroscopy. These challenges can be overcome if the subunits of a homomeric protein can be independently modified for functional or spectroscopic studies. Here, we describe a general approach for in vitro assembly that can be used for the generation of heteromeric variants of homomeric membrane proteins. We establish the approach using GltPh, a glutamate transporter homolog that is trimeric in the native state. We use heteromeric GltPh transporters to directly demonstrate the lack of coupling in substrate binding and demonstrate how heteromeric transporters considerably simplify the application of DEER spectroscopy. Further, we demonstrate the general applicability of this approach by carrying out the in vitro assembly of VcINDY, a Na+-coupled succinate transporter and CLC-ec1, a Cl-/H+ antiporter.

Sucrose and the Lipid Environment Modulate Conformational Heterogeneity in the Glutamate Transporter Homologue Gltph

Gltph is a sodium dependent aspartate transporter that is structurally homologous to glutamate transporters; as a result, it provides a model for excitatory amino acid transporters (EAATs) that facilitate glutamate reuptake. Crystal structures suggest a model for transport but do not provide information on biologically relevant intermediate states or effects of the lipid bilayer on the transport cycle. In the present work, distance distributions were measured for Gltph in three different bilayer systems though site directed spin labelling (SDSL) and double electron-electron resonance (DEER), and membrane depths were measured through SDSL and power saturation to determine if the crystal structures are representative of biologically relevant conformations. Transport domain distances were measured across the subunits of the trimer, and the protective osmolyte sucrose was used to modulate conformational populations. Distance distributions for T375R1 revealed populations with average distances that were close to crystal structure predictions. However, narrowing of the distance distributions and a shift to shorter distances in the presence of substrate suggest that there are biologically relevant states that are not seen in the crystal structures. Sucrose mediated stabilization of the inward facing state suggests increased lipid contacts, and membrane depth measurements showed that T375R1 comes into contact with the lipid environment in the presence of substrate, which suggests a currently unknown role for lipids during the transport cycle. This work was supported by NIGMS, GM035215.