Article | Published:

Structure and allosteric inhibition of excitatory amino acid transporter 1

Nature volume 544, pages 446451 (27 April 2017) | Download Citation

Abstract

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    , & Structural features of the glutamate transporter family. Microbiol. Mol. Biol. Rev. 63, 293–307 (1999)

  2. 2.

    & The glutamate and neutral amino acid transporter family: physiological and pharmacological implications. Eur. J. Pharmacol. 479, 237–247 (2003)

  3. 3.

    Glutamate uptake. Prog. Neurobiol. 65, 1–105 (2001)

  4. 4.

    & The number of glutamate transporter subtype molecules at glutamatergic synapses: chemical and stereological quantification in young adult rat brain. J. Neurosci. 18, 8751–8757 (1998)

  5. 5.

    & Flux coupling in a neuronal glutamate transporter. Nature 383, 634–637 (1996)

  6. 6.

    , , , & Selective loss of glial glutamate transporter GLT-1 in amyotrophic lateral sclerosis. Ann. Neurol. 38, 73–84 (1995)

  7. 7.

    , & A point mutation associated with episodic ataxia 6 increases glutamate transporter anion currents. Brain 135, 3416–3425 (2012)

  8. 8.

    et al. Late-onset episodic ataxia associated with SLC1A3 mutation. J. Hum. Genet. 62, 443–446 (2016)

  9. 9.

    , & The role of excitatory amino acid transporters in cerebral ischemia. Neurochem. Res. 35, 1224–1230 (2010)

  10. 10.

    , & Glutamate-based antidepressants: preclinical psychopharmacology. Biol. Psychiatry 73, 1125–1132 (2013)

  11. 11.

    & Glutamate transporters in the biology of malignant gliomas. Cell. Mol. Life Sci. 71, 1839–1854 (2014)

  12. 12.

    & ASCT-1 is a neutral amino acid exchanger with chloride channel activity. J. Biol. Chem. 271, 27991–27994 (1996)

  13. 13.

    et al. Targeting glutamine transport to suppress melanoma cell growth. Int. J. Cancer 135, 1060–1071 (2014)

  14. 14.

    et al. ASC amino-acid transporter 2 (ASCT2) as a novel prognostic marker in non-small cell lung cancer. Br. J. Cancer 110, 2030–2039 (2014)

  15. 15.

    et al. Targeting ASCT2-mediated glutamine uptake blocks prostate cancer growth and tumour development. J. Pathol. 236, 278–289 (2015)

  16. 16.

    et al. ASCT2/SLC1A5 controls glutamine uptake and tumour growth in triple-negative basal-like breast cancer. Oncogene 35, 3201–3208 (2016)

  17. 17.

    Glutamate transporter blockers for elucidation of the function of excitatory neurotransmission systems. Chem. Rec. 8, 182–199 (2008)

  18. 18.

    & New inhibitors for the neutral amino acid transporter ASCT2 reveal its Na+-dependent anion leak. J. Physiol. (Lond.) 557, 747–759 (2004)

  19. 19.

    et al. Discovery of the first selective inhibitor of excitatory amino acid transporter subtype 1. J. Med. Chem. 52, 912–915 (2009)

  20. 20.

    et al. Allosteric modulation of an excitatory amino acid transporter: the subtype-selective inhibitor UCPH-101 exerts sustained inhibition of EAAT1 through an intramonomeric site in the trimerization domain. J. Neurosci. 33, 1068–1087 (2013)

  21. 21.

    , , & Structure of a glutamate transporter homologue from Pyrococcus horikoshii. Nature 431, 811–818 (2004)

  22. 22.

    , & Transport mechanism of a bacterial homologue of glutamate transporters. Nature 462, 880–885 (2009)

  23. 23.

    et al. Transport domain unlocking sets the uptake rate of an aspartate transporter. Nature 518, 68–73 (2015)

  24. 24.

    , , , & Coupling substrate and ion binding to extracellular gate of a sodium-dependent aspartate transporter. Nature 445, 387–393 (2007)

  25. 25.

    , , , & Molecular determinant of ion selectivity of a (Na+ + K+)-coupled rat brain glutamate transporter. Proc. Natl Acad. Sci. USA 95, 751–755 (1998)

  26. 26.

    & A reentrant loop domain in the glutamate carrier EAAT1 participates in substrate binding and translocation. Neuron 21, 1487–1498 (1998)

  27. 27.

    et al. Mechanism of cation binding to the glutamate transporter EAAC1 probed with mutation of the conserved amino acid residue Thr101. J. Biol. Chem. 285, 17725–17733 (2010)

  28. 28.

    et al. Evidence for a third sodium-binding site in glutamate transporters suggests an ion/substrate coupling model. Proc. Natl Acad. Sci. USA 107, 13912–13917 (2010)

  29. 29.

    , , , & Coupled binding mechanism of three sodium ions and aspartate in the glutamate transporter homologue GltTk. Nat. Commun. 7, 13420 (2016)

  30. 30.

    , , & Inward-facing conformation of glutamate transporters as revealed by their inverted-topology structural repeats. Proc. Natl Acad. Sci. USA 106, 20752–20757 (2009)

  31. 31.

    , & Opposite movement of the external gate of a glutamate transporter homolog upon binding cotransported sodium compared with substrate. J. Neurosci. 31, 6255–6262 (2011)

  32. 32.

    , , & Proximity of two oppositely oriented reentrant loops in the glutamate transporter GLT-1 identified by paired cysteine mutagenesis. J. Biol. Chem. 277, 3985–3992 (2002)

  33. 33.

    & Substrates and non-transportable analogues induce structural rearrangements at the extracellular entrance of the glial glutamate transporter GLT-1/EAAT2. J. Biol. Chem. 283, 26391–26400 (2008)

  34. 34.

    , & A model for the topology of excitatory amino acid transporters determined by the extracellular accessibility of substituted cysteines. Neuron 25, 695–706 (2000)

  35. 35.

    , & Biotinylation of single cysteine mutants of the glutamate transporter GLT-1 from rat brain reveals its unusual topology. Neuron 21, 623–632 (1998)

  36. 36.

    , , & The position of an arginine residue influences substrate affinity and K+ coupling in the human glutamate transporter, EAAT1. J. Neurochem. 114, 565–575 (2010)

  37. 37.

    & Arginine 445 controls the coupling between glutamate and cations in the neuronal transporter EAAC-1. J. Biol. Chem. 279, 2513–2519 (2004)

  38. 38.

    et al. Loss-of-function mutations in the glutamate transporter SLC1A1 cause human dicarboxylic aminoaciduria. J. Clin. Invest. 121, 446–453 (2011)

  39. 39.

    , , & Regulation of glial glutamate transporters by C-terminal domains. J. Biol. Chem. 286, 1927–1937 (2011)

  40. 40.

    & Cholesterol is required for the reconstruction of the sodium- and chloride-coupled, gamma-aminobutyric acid transporter from rat brain. J. Biol. Chem. 265, 6002–6008 (1990)

  41. 41.

    , , & Association of excitatory amino acid transporters, especially EAAT2, with cholesterol-rich lipid raft microdomains: importance for excitatory amino acid transporter localization and function. J. Biol. Chem. 279, 34388–34396 (2004)

  42. 42.

    , & Transport rates of a glutamate transporter homologue are influenced by the lipid bilayer. J. Biol. Chem. 290, 9780–9788 (2015)

  43. 43.

    , , & Arachidonic acid elicits a substrate-gated proton current associated with the glutamate transporter EAAT4. Nat. Neurosci. 1, 105–113 (1998)

  44. 44.

    et al. Heterologously expressed GLT-1 associates in approximately 200-nm protein-lipid islands. Biophys. J. 91, 3718–3726 (2006)

  45. 45.

    & Hydrogen exchange mass spectrometry for the analysis of protein dynamics. Mass Spectrom. Rev. 25, 158–170 (2006)

  46. 46.

    , & Hydrogen exchange mass spectrometry for studying protein structure and dynamics. Chem. Soc. Rev. 40, 1224–1234 (2011)

  47. 47.

    et al. Characterization of novel l-threo-β-benzyloxyaspartate derivatives, potent blockers of the glutamate transporters. Mol. Pharmacol. 65, 1008–1015 (2004)

  48. 48.

    & The anion conductance of the glutamate transporter EAAC1 depends on the direction of glutamate transport. FEBS Lett. 503, 121–125 (2001)

  49. 49.

    , & Binding thermodynamics of a glutamate transporter homolog. Nat. Struct. Mol. Biol. 20, 634–640 (2013)

  50. 50.

    & Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14, 673–681 (2006)

  51. 51.

    , , & Sequence statistics reliably predict stabilizing mutations in a protein domain. J. Mol. Biol. 240, 188–192 (1994)

  52. 52.

    Xds. Acta Crystallogr. D 66, 125–132 (2010)

  53. 53.

    & How good are my data and what is the resolution? Acta Crystallogr. D 69, 1204–1214 (2013)

  54. 54.

    et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

  55. 55.

    , , & Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

  56. 56.

    et al. Refinement of severely incomplete structures with maximum likelihood in BUSTER-TNT. Acta Crystallogr. D 60, 2210–2221 (2004)

  57. 57.

    et al. Structural models of intrinsically disordered and calcium-bound folded states of a protein adapted for secretion. Sci. Rep. 5, 14223 (2015)

  58. 58.

    et al. MEMHDX: an interactive tool to expedite the statistical validation and visualization of large HDX-MS datasets. Bioinformatics 32, 3413–3419 (2016)

  59. 59.

    , , , & Jalview Version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189–1191 (2009)

Download references

Acknowledgements

We thank O. Boudker for comments on the manuscript and discussion on consensus mutagenesis; P. V. Krasteva for comments on the manuscript; A. Haouz and the staff at the crystallogenesis core facility of the Institut Pasteur for assistance with crystallization screens; Staff at Synchrotron SOLEIL and the European Synchrotron Radiation Facility for assistance with data collection; D. O’Brien for discussion of HDX results. The work was funded by the ERC Starting grant 309657 (N.R.). Further support from G5 Institut Pasteur funds (N.R.), CACSICE grant (ANR-11-EQPX-008), and CNRS UMR3528 (N.R., J.C.-R.) is acknowledged.

Author information

Author notes

    • Juan C. Canul-Tec
    •  & Reda Assal

    These authors contributed equally to this work.

Affiliations

  1. Molecular Mechanisms of Membrane Transport Laboratory, Institut Pasteur, 25–28 rue du Docteur Roux, 75015 Paris, France

    • Juan C. Canul-Tec
    • , Reda Assal
    • , Erica Cirri
    •  & Nicolas Reyes
  2. UMR 3528, CNRS, Institut Pasteur, 25–28 rue du Docteur Roux, 75015 Paris, France

    • Juan C. Canul-Tec
    • , Reda Assal
    • , Erica Cirri
    • , Sébastien Brier
    • , Julia Chamot-Rooke
    •  & Nicolas Reyes
  3. Synchrotron SOLEIL, L’Orme des Merisiers, 91192 Gif-sur-Yvette, France

    • Pierre Legrand
  4. Structural Mass Spectrometry and Proteomics Unit, Institut Pasteur, 25–28 rue du Docteur Roux, 75015 Paris, France

    • Sébastien Brier
    •  & Julia Chamot-Rooke

Authors

  1. Search for Juan C. Canul-Tec in:

  2. Search for Reda Assal in:

  3. Search for Erica Cirri in:

  4. Search for Pierre Legrand in:

  5. Search for Sébastien Brier in:

  6. Search for Julia Chamot-Rooke in:

  7. Search for Nicolas Reyes in:

Contributions

J.C.C.-T. and R.A. optimized and performed protein expression, purification and crystallization, and R.A. performed molecular biology; J.C.C.-T., R.A. and N.R. collected crystallographic data, and J.C.C.-T., P.L. and N.R. analysed diffraction data and structures; E.C. and R.A performed and analysed uptake experiments; E.C. prepared protein samples for HDX-MS; S.B. collected and analysed HDX-MS data with help from E.C.; All authors contributed to the experimental design of the project and manuscript preparation. N.R. conceived and supervised the project.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Nicolas Reyes.

Reviewer Information Nature thanks C. Miller 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

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature22064

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.