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 mechanism2,3,4,5. Glutamate transporters also conduct chloride ions by means of a channel-like process that is thermodynamically uncoupled from transport6,7,8. 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.
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Mutation in glutamate transporter homologue GltTk provides insights into pathologic mechanism of episodic ataxia 6
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All relevant crystallography, cryo-EM and molecular dynamics data are available from the corresponding author upon request. The maps and the coordinates of the refined models have been deposited into the Protein Data Bank and the Electron Microscopy Data Bank (EMDB) under the following accession numbers: GltPh-XL1 (PDB: 6X01), GltPh-XL3 (PDB: 6WZB), GltPh-XL2 - iOFS (PDB: 6WYJ; EMDB: EMD-21966), GltPh-XL2 – ClCS (PDB: 6WYK; EMDB: EMD-21967), GltPh-XL2 trimer – iOFS (PDB: 6WYL; EMDB: EMD-21968).
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This work was supported by the Australian National Health and Medical Research Council Project Grant APP1164494 (to R.M.R., R.J.V. and J.F.) and Fellowship APP1159347 (to A.G.S.), by National Institutes of Health grants P41-GM104601 (to E.T.) and R01-GM067887 (to E.T.), a Research Training Program Scholarship (to I.C.) and a Beckman Institute Graduate Fellowship (to S.P.). Computational resources were provided by XSEDE (grant MCA06N060 to E.T.), NCSA Blue Waters (to E.T.) and Microsoft Azure (to E.T.). We acknowledge the use of the Victor Chang Cardiac Research Institute Innovation Centre, funded by the NSW Government; the Electron Microscope Unit at UNSW Sydney, funded in part by the NSW Government; the MX2 beamline at the Australian Synchrotron, part of ANSTO; and the Australian Cancer Research Foundation (ACRF) detector. We acknowledge the facilities and technical assistance of J. Bouwer and S. Brown from Cryo Electron Microscopy - Molecular Horizons, University of Wollongong; and W. Close from Microscopy Australia at the Australian Centre for Microscopy and Microanalysis, University of Sydney. We thank D. Chappell, X. Wang and O. Boudker for discussions; Z. Zhao for assistance with simulations; and C. Handford and those that support the X. laevis colony at the University of Sydney.
The authors declare no competing interests.
Peer review information Nature thanks Aravind Penmatsa and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, SDS–PAGE gel shift assay showing the extent of crosslinking in detergent-solubilized CLGltPh and the double-cysteine GltPh transporters under untreated conditions, upon mPEG5K-maleimide treatment, and after incubation with HgCl2; arrows indicate the positions of differentially crosslinked protomers and mPEG5K-bound proteins. Data are representative from one experiment that was replicated at least two times from two separate protein purifications. For gel source data, see Supplementary Fig. 1. b, Crystal structure of GltPh-XL1 (purple) and GltPh-XL3 (pink) superimposed on the OFS (PDB: 2NWX; left, grey) and the IFS (PDB: 3KBC; right, grey), respectively. c, SDS–PAGE analysis of purified GltPh-XL3 in nanodiscs. d, Cryo-EM structure of GltPh-XL2 (green) superimposed on the iOFS (PDB: 3V8G, chain C; grey).
a, Data processing flow chart for GltPh reconstituted into nanodiscs in the presence of NaCl and aspartate. b, Fourier shell correlation curves indicating the resolution at the 0.143 threshold of final masked (blue) and unmasked (red) maps for GltPh trimer iOFS (left), GltPh protomer ClCS (middle) and GltPh protomer iOFS (right). c, Final maps after Relion post-processing, coloured according to local resolution estimation using Relion for GltPh trimer iOFS (left, 3.9Å resolution, contour level 7.2σ), GltPh protomer ClCS (middle, 4.0Å resolution, contour level 12.0σ) and GltPh protomer iOFS (right, 3.7Å resolution, contour level 9.5σ). Contour levels were calculated using Chimera.
a, b, Front (a) and top (b) views of the cryo-EM map and atomic model of GltPh-XL2 in the iOFS (contour level 9.5σ) and ClCS (contour level 12.0σ). Density attributed to the scaffold domain, transport domain and HP2 are shown in salmon, blue and red, respectively. c, Conformational changes undertaken by a GltPh protomer during the substrate-transport cycle viewed from the side and top. HP2 is coloured for easier visualization of the rotational changes observed in the transport domain. d, e, Close-up views of the L152C–G351C crosslink fitted in the iOFS (d) (contour level 12.9σ) and ClCS (e) (contour level 10.3σ) cryo-EM maps. Contour levels were calculated using Chimera.
A close-up view of the three Na+ coordination sites on the ClCS protomer. Residues interacting with the Na+ ion (purple circle, modelled) are shown in stick representation. The scaffold and the transport domains are shown in salmon and blue, respectively, with the substrate in black sticks.
Extended Data Fig. 5 Nanodisc deformation supports transport-domain movement by the ClCS and putative lipid-binding sites.
a, Percentage of GltPh-XL2 trimers containing all three protomers in the iOFS, in the ClCS, or in a mixture of both states. Out of 220,938 trimers, 79,809 contained one or more protomers in the ClCS. Particle counting within symmetry-expanded data showed that 63,470 trimers contained one protomer in the ClCS, 14,394 trimers contained two protomers in the ClCS and 1,945 trimers contained all three protomers in the ClCS. b, Density map of GltPh-XL2 trimer (unfocused refinement) containing one ClCS protomer and embedded in nanodiscs (viewed from the membrane plane). The two iOFS protomers are shown in orange and the ClCS in blue. The nanodisc is shown in yellow. c, Putative lipid-binding sites in the GltPh-XL2 trimer, in which the transport domain is shown in blue and the scaffold domain in salmon. Identical lipid densities (green) were observed between protomers (contour level 7.2σ). Transmembrane helices located within 5 Å of the putative lipid densities are labelled. Contour levels were calculated using Chimera.
Extended Data Fig. 6 Water conduction through GltPh-ClCS and setup of umbrella sampling simulations and convergence to capture Cl− movement through GltPh-ClCS.
a, The GltPh-ClCS structure was embedded into a lipid bilayer containing POPE, POPG and POPC lipids, mimicking experimental conditions. After an initial equilibration of 100 ns, the entire system was subjected to an external electric field of 800 mV, which resulted in a continuous water pathway through the interface of the scaffold and the transport domains. The GltPh-ClCS protomer is shown in cartoon representation, with the transport domain in blue and the scaffold domain in salmon. XL-2 residues L152 and G351 are shown in red and blue spheres, respectively. b, Residues lining the Cl− pathway have a higher solvent accessible surface area (SASA) in the GltPh-ClCS than in the OFS (calculated using the crystal structure of OFS PDB: 2NWX). c, Overlap between the corresponding windows used in umbrella sampling simulations. d, No substantial changes between the free-energy profile obtained at 10 ns (red), 15 ns (blue) and 20 ns (green) were observed, highlighting the convergence of umbrella sampling simulations.
a, l-[3H]glutamate uptake into oocytes expressing cysteine-less EAAT1 and double cysteine transporter mutants in control conditions (grey), and after pre-incubation with DTT (cyan) or copper phenanthroline (orange). Number of cells (n) used for each condition is indicated in each graph and all measurements presented were taken across at least two batches of oocytes. b–e, l-Glutamate elicited current–voltage relationships for cysteine-less E1 (b), E1-XL1 (c), E1-XL2 (d) and E1-XL3 (e) monitored under the same conditions as a. f, g, To confirm that crosslinks E1-XL1, E1-XL2 and E1-XL3 were occurring within an individual protomer, rather than between protomers of the trimeric complex, oocytes expressing single cysteine residues that make up E1-XL1 (K300C and W473C), E1-XL2 (L244C and G439C), and E1-XL3 (K300C and A470C) either alone or co-injected into an individual oocyte were also examined using the same approaches as in a, b–e. Data are mean ± s.e.m. h, EAAT1 (PBD: 5LLU) highlighting residues forming the extracellular and intracellular hydrophobic gates. The scaffold domain is shown in grey and the transport domain in gold. The Cα atoms of the two introduced cysteine residues are shown as spheres (L244 in red and G439 in blue). i, Membrane reversal potentials (Erev) measured in oocytes expressing wild-type (n = 6) and mutant (n = 5) EAAT1 transporters. Each data point (white circle) represents a response from a single cell. The black bar represents mean ± s.e.m. Significance was determined using one-way ANOVA with Bonferroni post hoc analysis for multiple comparisons performed using GraphPad Prism 8; exact P values are provided. j, Schematic of the substrate-transport cycle. A single protomer is shown with the scaffold domain in salmon, the transport domain in blue and the substrate in black.
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Chen, I., Pant, S., Wu, Q. et al. Glutamate transporters have a chloride channel with two hydrophobic gates. Nature 591, 327–331 (2021). https://doi.org/10.1038/s41586-021-03240-9
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