Structural basis of kainate subtype glutamate receptor desensitization

Abstract

Glutamate receptors are ligand-gated tetrameric ion channels that mediate synaptic transmission in the central nervous system. They are instrumental in vertebrate cognition and their dysfunction underlies diverse diseases1,2. In both the resting and desensitized states of AMPA and kainate receptor subtypes, the ion channels are closed, whereas the ligand-binding domains, which are physically coupled to the channels, adopt markedly different conformations3,4,5,6. Without an atomic model for the desensitized state, it is not possible to address a central problem in receptor gating: how the resting and desensitized receptor states both display closed ion channels, although they have major differences in the quaternary structure of the ligand-binding domain. Here, by determining the structure of the kainate receptor GluK2 subtype in its desensitized state by cryo-electron microscopy (cryo-EM) at 3.8 Å resolution, we show that desensitization is characterized by the establishment of a ring-like structure in the ligand-binding domain layer of the receptor. Formation of this ‘desensitization ring’ is mediated by staggered helix contacts between adjacent subunits, which leads to a pseudo-four-fold symmetric arrangement of the ligand-binding domains, illustrating subtle changes in symmetry that are important for the gating mechanism. Disruption of the desensitization ring is probably the key switch that enables restoration of the receptor to its resting state, thereby completing the gating cycle.

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Figure 1: Desensitized kainate receptor at 3.8 Å resolution.
Figure 2: Desensitization ring.
Figure 3: Desensitized state ion channel.
Figure 4: LBD–TMD linkers mediate channel closing and LBD reorganization.

Accession codes

Data deposits

Cryo-EM density maps for GluK2EM with 2s,4R-4-methylglutamate, and GluK2EM with LY466195, have been deposited in the Electron Microscopy Data Bank under accession codes EMD- 8289 and EMD- 8290, respectively. Atomic coordinates for GluK2EM with 2s,4R-4-methylglutamate, GluK2EM with LY466195, GluK2EM LBD with 2s,4R-4-methylglutamate, and GluK2EM LBD with LY466195, have been deposited in the Protein Data Bank under accession codes, 5KUF, 5KUH, 5CMM and 5CMK, respectively.

Change history

  • 21 September 2016

    Missing labels were added to Fig. 4a, and minor orientation changes were made to Fig. 4e.

References

  1. 1

    Burnashev, N. & Szepetowski, P. NMDA receptor subunit mutations in neurodevelopmental disorders. Curr. Opin. Pharmacol. 20, 73–82 (2015)

    CAS  Article  Google Scholar 

  2. 2

    Lesca, G. et al. GRIN2A mutations in acquired epileptic aphasia and related childhood focal epilepsies and encephalopathies with speech and language dysfunction. Nat. Genet. 45, 1061–1066 (2013)

    CAS  Article  Google Scholar 

  3. 3

    Dürr, K. L. et al. Structure and dynamics of AMPA receptor GluA2 in resting, pre-open, and desensitized states. Cell 158, 778–792 (2014)

    Article  Google Scholar 

  4. 4

    Meyerson, J. R. et al. Structural mechanism of glutamate receptor activation and desensitization. Nature 514, 328–334 (2014)

    CAS  ADS  Article  Google Scholar 

  5. 5

    Schauder, D. M. et al. Glutamate receptor desensitization is mediated by changes in quaternary structure of the ligand binding domain. Proc. Natl Acad. Sci. USA 110, 5921–5926 (2013)

    CAS  ADS  Article  Google Scholar 

  6. 6

    Sobolevsky, A. I., Rosconi, M. P. & Gouaux, E. X-ray structure, symmetry and mechanism of an AMPA-subtype glutamate receptor. Nature 462, 745–756 (2009)

    CAS  ADS  Article  Google Scholar 

  7. 7

    Traynelis, S. F. et al. Glutamate receptor ion channels: structure, regulation, and function. Pharmacol. Rev. 62, 405–496 (2010)

    CAS  Article  Google Scholar 

  8. 8

    Mayer, M. L. Glutamate receptors at atomic resolution. Nature 440, 456–462 (2006)

    CAS  ADS  Article  Google Scholar 

  9. 9

    Watkins, J. C. & Evans, R. H. Excitatory amino acid transmitters. Annu. Rev. Pharmacol. Toxicol. 21, 165–204 (1981)

    CAS  Article  Google Scholar 

  10. 10

    Chen, L., Dürr, K. L. & Gouaux, E. X-ray structures of AMPA receptor-cone snail toxin complexes illuminate activation mechanism. Science 345, 1021–1026 (2014)

    CAS  ADS  Article  Google Scholar 

  11. 11

    Herguedas, B. et al. Structure and organization of heteromeric AMPA-type glutamate receptors. Science 352, aad3873 (2016)

    Article  Google Scholar 

  12. 12

    Nakagawa, T., Cheng, Y., Ramm, E., Sheng, M. & Walz, T. Structure and different conformational states of native AMPA receptor complexes. Nature 433, 545–549 (2005)

    CAS  ADS  Article  Google Scholar 

  13. 13

    Twomey, E. C., Yelshanskaya, M. V., Grassucci, R. A., Frank, J. & Sobolevsky, A. I. Elucidation of AMPA receptor-stargazin complexes by cryo-electron microscopy. Science 353, 83–86 (2016)

    CAS  ADS  Article  Google Scholar 

  14. 14

    Yelshanskaya, M. V., Li, M. & Sobolevsky, A. I. Structure of an agonist-bound ionotropic glutamate receptor. Science 345, 1070–1074 (2014)

    CAS  ADS  Article  Google Scholar 

  15. 15

    Zhao, Y., Chen, S., Yoshioka, C., Baconguis, I. & Gouaux, E. Architecture of fully occupied GluA2 AMPA receptor–TARP complex elucidated by cryo-EM. Nature 536, 108–111 (2016)

    CAS  ADS  Article  Google Scholar 

  16. 16

    Alushin, G. M., Jane, D. & Mayer, M. L. Binding site and ligand flexibility revealed by high resolution crystal structures of GluK1 competitive antagonists. Neuropharmacology 60, 126–134 (2011)

    CAS  Article  Google Scholar 

  17. 17

    Jones, K. A., Wilding, T. J., Huettner, J. E. & Costa, A. M. Desensitization of kainate receptors by kainate, glutamate and diastereomers of 4-methylglutamate. Neuropharmacology 36, 853–863 (1997)

    CAS  Article  Google Scholar 

  18. 18

    Möykkynen, T., Coleman, S. K., Semenov, A. & Keinänen, K. The N-terminal domain modulates α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor desensitization. J. Biol. Chem. 289, 13197–13205 (2014)

    Article  Google Scholar 

  19. 19

    Dutta, A. et al. Cooperative dynamics of intact AMPA and NMDA glutamate receptors: similarities and subfamily-specific differences. Structure 23, 1692–1704 (2015)

    CAS  Article  Google Scholar 

  20. 20

    Carbone, A. L. & Plested, A. J. Coupled control of desensitization and gating by the ligand binding domain of glutamate receptors. Neuron 74, 845–857 (2012)

    CAS  Article  Google Scholar 

  21. 21

    Fleck, M. W., Cornell, E. & Mah, S. J. Amino-acid residues involved in glutamate receptor 6 kainate receptor gating and desensitization. J. Neurosci. 23, 1219–1227 (2003)

    CAS  Article  Google Scholar 

  22. 22

    Chaudhry, C., Plested, A. J., Schuck, P. & Mayer, M. L. Energetics of glutamate receptor ligand binding domain dimer assembly are modulated by allosteric ions. Proc. Natl Acad. Sci. USA 106, 12329–12334 (2009)

    CAS  ADS  Article  Google Scholar 

  23. 23

    Dawe, G. B. et al. Distinct structural pathways coordinate the activation of AMPA peceptor-auxiliary subunit complexes. Neuron 89, 1264–1276 (2016)

    CAS  Article  Google Scholar 

  24. 24

    Plested, A. J., Vijayan, R., Biggin, P. C. & Mayer, M. L. Molecular basis of kainate receptor modulation by sodium. Neuron 58, 720–735 (2008)

    CAS  Article  Google Scholar 

  25. 25

    Patneau, D. K., Mayer, M. L., Jane, D. E. & Watkins, J. C. Activation and desensitization of AMPA/kainate receptors by novel derivatives of willardiine. J. Neurosci. 12, 595–606 (1992)

    CAS  Article  Google Scholar 

  26. 26

    Mayer, M. L. Crystal structures of the GluR5 and GluR6 ligand binding cores: molecular mechanisms underlying kainate receptor selectivity. Neuron 45, 539–552 (2005)

    CAS  Article  Google Scholar 

  27. 27

    Meyerson, J. R. et al. Self-assembled monolayers improve protein distribution on holey carbon cryo-EM supports. Sci. Rep. 4, 7084 (2014)

    Article  Google Scholar 

  28. 28

    Li, X. et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat. Methods 10, 584–590 (2013)

    CAS  Article  Google Scholar 

  29. 29

    Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007)

    CAS  Article  Google Scholar 

  30. 30

    Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012)

    CAS  Article  Google Scholar 

  31. 31

    Mindell, J. A. & Grigorieff, N. Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334–347 (2003)

    Article  Google Scholar 

  32. 32

    Scheres, S. H. Beam-induced motion correction for sub-megadalton cryo-EM particles. eLife 3, e03665 (2014)

    Article  Google Scholar 

  33. 33

    Pettersen, E. F. et al. UCSF Chimera--a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    The PyMOL Molecular Graphics System. Version 1.8 Schrödinger, LLC. (DeLano Scientific, 2002)

  35. 35

    Smart, O. S., Goodfellow, J. M. & Wallace, B. A. The pore dimensions of gramicidin A. Biophys. J. 65, 2455–2460 (1993)

    CAS  Article  Google Scholar 

  36. 36

    Heymann, J. B. Bsoft: image and molecular processing in electron microscopy. J. Struct. Biol. 133, 156–169 (2001)

    CAS  Article  Google Scholar 

  37. 37

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010)

    CAS  Article  Google Scholar 

  38. 38

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010)

    CAS  Article  Google Scholar 

  39. 39

    Wang, R. Y. et al. De novo protein structure determination from near-atomic-resolution cryo-EM maps. Nat. Methods 12, 335–338 (2015)

    CAS  Article  Google Scholar 

  40. 40

    Horning, M. S. & Mayer, M. L. Regulation of AMPA receptor gating by ligand binding core dimers. Neuron 41, 379–388 (2004)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This research was supported by the intramural programs of the NCI, and NICHD, NIH, the IATAP program at NIH, and the NIH-FEI Living Laboratory for Structural Biology. Synchrotron diffraction data was collected at Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline at the Advanced Photon Source (APS), Argonne National Laboratory. Use of APS was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract number W-31-109-Eng-38. We thank D. Bleakman for the gift of LY466195, C. Glasser for preparing DNA constructs, and F. DiMaio and colleagues for assistance with use of the program Rosetta. We also thank V. Falconieri for preparing the schematic illustration panel. This study used the capabilities of the HPC Biowulf cluster at NIH, Bethesda, MD (http://hpc.nih.gov).

Author information

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Authors

Contributions

J.R.M., M.L.M. and S.S. were involved in all stages of design of experiments and interpretation of results; S.C. carried out protein expression and purification; S.C. and M.L.M. carried out X-ray crystallography; T.H., E.S. and M.L.M. performed electrophysiological experiments; J.R.M., A.M., P.R. and S.C. carried out cryo-EM data collection; J.R.M. carried out cryo-EM image processing; J.R.M. and M.L.M. carried out structural analysis; J.R.M., M.L.M. and S.S. integrated all of the data, analysis of the implications and mechanism, and wrote the manuscript, with help from S.C.

Corresponding authors

Correspondence to Joel R. Meyerson or Mark L. Mayer or Sriram Subramaniam.

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Competing interests

The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks L. Wollmuth and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Desensitized GluK2 imaging and structure determination.

a, b, Representative cryo-EM image of GluK2EM solubilized in DDM–CHS and bound by 2S,4R-4-methylglutamate (a), with the corresponding image power spectrum and CTF estimate showing signal beyond 3 Å resolution (b; solid and dotted lines, respectively). The defocus value for the image is 1.5 μm. Particles in a are highlighted with circles, and image binning at 4× and uniform level adjustments were used to make particles apparent. Scale bar, 500 Å. c, Subset of selected two-dimensional class averages. d, FSC curve with reported resolution of 3.8 Å at the 0.143 crossing. e, Structure of agonist-bound GluK2EM, coloured according to local resolution, shown at three progressively increasing contours.

Extended Data Figure 2 Desensitized GluK2 transmembrane and glycosylation features.

a, b, Cryo-EM density for resolved S1–M1 (a) and M3–S2 (b) linkers and transmembrane helices, displayed with Cα trace of the atomic model. c, Representative sites with densities for complex glycans at Asn244, Asn347 and Asn399.

Extended Data Figure 3 Reconstruction of desensitized GluK2 without computational symmetry.

a, Cryo-EM density map for the reconstruction of agonist-bound GluK2 without imposition of computational symmetry, and coloured according to local resolution. b, FSC curve with reported resolution of 4.4 Å at the 0.143 crossing. c, Segmentation of individual GluK2 chains of the asymmetric reconstruction. The density map segmentation is shown fitted with a trace representation of the model displayed in Fig. 1b.

Extended Data Figure 4 Inhibition of GluK2EM by LY466195 and LBD crystal structures for agonist and antagonist complexes.

a, Crystal structure for the GluK2EM-isolated LBD dimer assembly complex with 2S,4R-4-methylglutamate. The upper and lower lobes for the two subunits are coloured orange and pale yellow, and teal and pale cyan, respectively; the dashed line indicates the separation of the lower lobes measured as the distance between the Cα positions of Ile637. b, Crystal structure for the GluK2EM-isolated LBD dimer assembly complex with LY466195 illustrating the large decrease in separation of the lower lobes compared to the agonist complex. Colouring is the same as in a. c, Responses to 100 μM glutamate recorded under two electrode voltage clamp for GluK2EM (top) and wild-type GluK2 (bottom); the initial response to glutamate recorded after prior application of 300 nM LY466195 showed nearly complete block for GluK2EM, with no change in amplitude for wild type. d, Concentration dependence for inhibition of GluK2EM by LY466195 yielded an IC50 of 30 nM; data points show mean ± s.e.m. of 4–7 observations per concentration.

Extended Data Figure 5 Imaging and structure of GluK2EM bound by antagonist LY466195.

a, b, Representative cryo-EM image of GluK2EM solubilized in DDM-CHS and bound by LY466195 (a), with the corresponding image power spectrum and CTF estimate showing signal beyond 6 Å resolution (b, solid and dotted lines, respectively). The defocus value for the image is 2.7 μm. Particles in a are highlighted with circles, and image binning at 4× and uniform level adjustments were used to make particles apparent. Scale bar, 500 Å. c, Subset of selected two-dimensional class averages. d, Cryo-EM density map for GluA2 bound to ZK200775 (ref. 4) (left), density map for GluK2EM bound to LY466195 (middle) and its corresponding molecular model built from ATD and LBD dimers (right). e, FSC curve with reported resolution of 11.6 Å at 0.143 crossing.

Extended Data Figure 6 ATD–LBD interface of desensitized GluK2.

a, Desensitized GluK2 shown in surface representation with ATD–LBD interfaces highlighted. b, Top-down view of LBD layer shown with perspective indicated by eye icon in a. c, Underside of the ATD layer as viewed after peeling away from LBD layer. Dashed lines in a highlight where the layers are separated. In all panels, interfaces on chain A and C are in green and blue, respectively. d, Table with residues that mediate ATD–LBD interaction. e, f, Cartoon representation of LBD and ATD layers from same views as in b and c, with interface residues coloured to correspond with table in d.

Extended Data Figure 7 Desensitization ring residues that influence recovery kinetics.

ad, Rate of recovery from desensitization measured using twin pulse applications of 10 mM glutamate (data points show mean ± s.d.; fits are shown in red). a, Wild-type GluK2 fit with a single exponential. b, D672R fit with the sum of two exponentials with the response for wild type shown as a black line; c, S669R D672R double mutant fit with the sum of two exponentials with the response for wild type shown as a black line. d, Time to 50% recovery in seconds. eh, Top views of the GluK2 desensitization ring with residues found to influence recovery kinetics when mutated. Each panel shows the wild-type residue Cα position as a sphere. Positions for the S669R and D672R mutations from the present study are shown in e and f, respectively. The A676T and S679R positions found in previous studies20,21 are in g and h, respectively.

Extended Data Table 1 Electrophysiological analysis
Extended Data Table 2 Cryo-EM data collection and structural analysis
Extended Data Table 3 Crystallographic data collection and refinement statistics

Supplementary information

Model for the kainate receptor state transitions

The transitions between resting, activated, and desensitized states are shown here. The resting and desensitized state models were built as described in the methods section. The activated state model was built using the GluA2EM activated state and GluA2cryst structures as guides to reveal conformational changes in the ATD and LBD layers, with the GluA2cryst ion channel used as a reference for positioning in the vertical plane. (MOV 13322 kb)

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Meyerson, J., Chittori, S., Merk, A. et al. Structural basis of kainate subtype glutamate receptor desensitization. Nature 537, 567–571 (2016). https://doi.org/10.1038/nature19352

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