Article | Published:

Glycine receptor mechanism elucidated by electron cryo-microscopy

Nature volume 526, pages 224229 (08 October 2015) | Download Citation

Abstract

The strychnine-sensitive glycine receptor (GlyR) mediates inhibitory synaptic transmission in the spinal cord and brainstem and is linked to neurological disorders, including autism and hyperekplexia. Understanding of molecular mechanisms and pharmacology of glycine receptors has been hindered by a lack of high-resolution structures. Here we report electron cryo-microscopy structures of the zebrafish α1 GlyR with strychnine, glycine, or glycine and ivermectin (glycine/ivermectin). Strychnine arrests the receptor in an antagonist-bound closed ion channel state, glycine stabilizes the receptor in an agonist-bound open channel state, and the glycine/ivermectin complex adopts a potentially desensitized or partially open state. Relative to the glycine-bound state, strychnine expands the agonist-binding pocket via outward movement of the C loop, promotes rearrangement of the extracellular and transmembrane domain ‘wrist’ interface, and leads to rotation of the transmembrane domain towards the pore axis, occluding the ion conduction pathway. These structures illuminate the GlyR mechanism and define a rubric to interpret structures of Cys-loop receptors.

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Accessions

Primary accessions

Electron Microscopy Data Bank

Data deposits

Three three-dimensional cryo-EM density maps and coordinates of α1 glycine receptors in strychnine-bound, glycine-bound and glycine/ivermectin-bound forms have been deposited in the Electron Microscopy Data Bank under the accession numbers EMD-6344, EMD-6345, and EMD-6346 and deposited in the RCSB Protein Data Bank under the accession codes 3JAD, 3JAE, and 3JAF.

References

  1. 1.

    & A study of the ‘desensitization’ produced by acetylcholine at the motor end-plate. J. Physiol. (Lond.) 138, 63–80 (1957)

  2. 2.

    & The distribution of glycine in cat spinal cord and roots. Life Sci. 4, 2075–2083 (1965)

  3. 3.

    , & Inhibition of spinal neurons by glycine. Nature 215, 1502–1503 (1967)

  4. 4.

    , & Inhibition of motoneurones by iontophoresis of glycine. Nature 214, 681–683 (1967)

  5. 5.

    , , & The hyperpolarization of spinal motoneurones by glycine and related amino acids. Exp. Brain Res. 5, 235–258 (1968)

  6. 6.

    Molecular structure and function of the glycine receptor chloride channel. Physiol. Rev. 84, 1051–1095 (2004)

  7. 7.

    , & The multiple phenotypes of allosteric receptor mutants. Proc. Natl Acad. Sci. USA 93, 1853–1858 (1996)

  8. 8.

    The glycinergic inhibitory synapse. Cell. Mol. Life Sci. 58, 760–793 (2001)

  9. 9.

    & Glycine receptors: a new therapeutic target in pain pathways. Curr. Opin. Investig. Drugs 7, 48–53 (2006)

  10. 10.

    & The impact of human hyperekplexia mutations on glycine receptor structure and function. Mol. Brain 7, 2 (2014)

  11. 11.

    & Strychnine binding associated with glycine receptors of the central nervous system. Proc. Natl Acad. Sci. USA 70, 2832–2836 (1973)

  12. 12.

    , & Purification by affinity chromatography of the glycine receptor of rat spinal cord. J. Biol. Chem. 257, 9389–9393 (1982)

  13. 13.

    , & Photoaffinity-labelling of the glycine receptor of rat spinal cord. Eur. J. Biochem. 131, 519–525 (1983)

  14. 14.

    , & Mechanism of anion permeation through channels gated by glycine and gamma-aminobutyric acid in mouse cultured spinal neurones. J. Physiol. 385, 243–286 (1987)

  15. 15.

    & Anion permeation in GABA- and glycine-gated channels of mammalian cultured hippocampal neurons. Proc. R. Soc. Lond. B 253, 69–75 (1993)

  16. 16.

    , & Ivermectin, an unconventional agonist of the glycine receptor chloride channel. J. Biol. Chem. 276, 12556–12564 (2001)

  17. 17.

    & Allosteric receptors after 30 years. Neuron 21, 959–980 (1998)

  18. 18.

    & Binding, activation and modulation of Cys-loop receptors. Trends Pharmacol. Sci. 31, 161–174 (2010)

  19. 19.

    et al. X-ray structure of a pentameric ligand-gated ion channel in an apparently open conformation. Nature 457, 111–114 (2009)

  20. 20.

    & Structure of a potentially open state of a proton-activated pentameric ligand-gated ion channel. Nature 457, 115–118 (2009)

  21. 21.

    et al. Pentameric ligand-gated ion channel ELIC is activated by GABA and modulated by benzodiazepines. Proc. Natl Acad. Sci. USA 109, E3028–E3034 (2012)

  22. 22.

    et al. Crystal structures of a pentameric ligand-gated ion channel provide a mechanism for activation. Proc. Natl Acad. Sci. USA 111, 966–971 (2014)

  23. 23.

    et al. Allosteric and hyperekplexic mutant phenotypes investigated on an α1 glycine receptor transmembrane structure. Proc. Natl Acad. Sci. USA 112, 2865–2870 (2015)

  24. 24.

    , & Structure and gating mechanism of the acetylcholine receptor pore. Nature 423, 949–955 (2003)

  25. 25.

    Refined structure of the nicotinic acetylcholine receptor. J. Mol. Biol. 346, 967–989 (2005)

  26. 26.

    & Principles of activation and permeation in an anion-selective Cys-loop receptor. Nature 474, 54–60 (2011)

  27. 27.

    , , & X-ray structures of GluCl in apo states reveal a gating mechanism of Cys-loop receptors. Nature 512, 333–337 (2014)

  28. 28.

    et al. X-ray structure of the mouse serotonin 5–HT3 receptor. Nature 512, 276–281 (2014)

  29. 29.

    & Crystal structure of a human GABAA receptor. Nature 512, 270–275 (2014)

  30. 30.

    & Gating of pentameric ligand-gated ion channels: structural insights and ambiguities. Structure 21, 1271–1283 (2013)

  31. 31.

    & The nicotinic acetylcholine receptor and its prokaryotic homologues: structure, conformational transitions & allosteric modulation. Neuropharmacology 96, 137–149 (2015)

  32. 32.

    Ion Channels of Excitable Membranes (Sinauer Associates, 2001)

  33. 33.

    et al. Conformational changes underlying desensitization of the pentameric ligand-gated ion channel ELIC. Structure 23, 995–1004 (2015)

  34. 34.

    , & The desensitization gate of inhibitory Cys-loop receptors. Nature Commun. 6, 6829 (2015)

  35. 35.

    Using molecular dynamics to elucidate the structural basis for function in pLGICs. Proc. Natl Acad. Sci. USA 110, 16700–16701 (2013)

  36. 36.

    et al. A structural and mutagenic blueprint for molecular recognition of strychnine and d-tubocurarine by different cys-loop receptors. PLoS Biol. 9, e1001034 (2011)

  37. 37.

    , & Distinct agonist- and antagonist-binding sites on the glycine receptor. Neuron 9, 491–496 (1992)

  38. 38.

    et al. The glycine receptor: pharmacological studies and mathematical modeling of the allosteric interaction between the glycine- and strychnine-binding sites. Mol. Pharmacol. 30, 590–597 (1986)

  39. 39.

    , , , & Localization of the strychnine binding site on the 48-kilodalton subunit of the glycine receptor. Biochemistry 29, 7033–7040 (1990)

  40. 40.

    & Molecular mechanisms of inherited startle syndromes. Trends Neurosci. 18, 80–82 (1995)

  41. 41.

    et al. The unique extracellular disulfide loop of the glycine receptor is a principal ligand binding element. EMBO J. 14, 2987–2998 (1995)

  42. 42.

    et al. Agonist and antagonist binding in human glycine receptors. Biochemistry 53, 6041–6051 (2014)

  43. 43.

    et al. The β subunit determines the ligand binding properties of synaptic glycine receptors. Neuron 45, 727–739 (2005)

  44. 44.

    , & Initial coupling of binding to gating mediated by conserved residues in the muscle nicotinic receptor. J. Gen. Physiol. 126, 23–39 (2005)

  45. 45.

    et al. Structures of Aplysia AChBP complexes with nicotinic agonists and antagonists reveal distinctive binding interfaces and conformations. EMBO J. 24, 3635–3646 (2005)

  46. 46.

    & Loop C and the mechanism of acetylcholine receptor-channel gating. J. Gen. Physiol. 141, 467–478 (2013)

  47. 47.

    et al. Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures. Neuron 41, 907–914 (2004)

  48. 48.

    et al. Complex between α-bungarotoxin and an α7 nicotinic receptor ligand-binding domain chimaera. Biochem. J. 454, 303–310 (2013)

  49. 49.

    & An improved ivermectin-activated chloride channel receptor for inhibiting electrical activity in defined neuronal populations. J. Biol. Chem. 285, 14890–14897 (2010)

  50. 50.

    , , & Functional anatomy of an allosteric protein. Nature Commun. 4, 2984 (2013)

  51. 51.

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

  52. 52.

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

  53. 53.

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

  54. 54.

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

  55. 55.

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

  56. 56.

    & Prevention of overfitting in cryo-EM structure determination. Nature Methods 9, 853–854 (2012)

  57. 57.

    , , & The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22, 195–201 (2006)

  58. 58.

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

  59. 59.

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

  60. 60.

    et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011)

  61. 61.

    et al. Structure of the yeast mitochondrial large ribosomal subunit. Science 343, 1485–1489 (2014)

  62. 62.

    et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010)

  63. 63.

    The PyMOL Molecular Graphics System. (DeLano Scientific, San Carlos, USA, 2002)

  64. 64.

    , , , & HOLE: a program for the analysis of the pore dimensions of ion channel structural models. J. Mol. Graph. 14, 354–360 (1996)

  65. 65.

    , & Quantifying the local resolution of cryo-EM density maps. Nature Methods 11, 63–65 (2014)

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Acknowledgements

We are grateful to Z. H. Yu, N. Grigorieff, J. Cruz and C. Hong (Janelia Campus), C. Arthur (FEI) and M. Braunfeld (UCSF) for assistance with microscope operation, data collection and for comments, and to R. Stites, M. Hakanson and A. Trzynka (OHSU) for computational support. We acknowledge the support of R. Goodman and J. Gray. Microscopy at Oregon Health & Science University (OHSU) was performed at the Multiscale Microscopy Core (MMC) with technical support from the OHSU-FEI Living Lab, Intel and the OHSU Center for Spatial Systems Biomedicine (OCSSB). We thank L. Vaskalis for help with illustrations and H. Owen for proofreading. R. Hibbs is gratefully acknowledged for pre-screening the GlyR constructs and D. P. Claxton for optimizing the constructs. We thank Gouaux and Baconguis laboratory members for discussions. This work was supported by the National Institute of Health (E.G). E.G. is an investigator with the Howard Hughes Medical Institute.

Author information

Author notes

    • Juan Du
    •  & Wei Lü

    These authors contributed equally to this work.

Affiliations

  1. Vollum Institute, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97239, USA

    • Juan Du
    • , Wei Lü
    •  & Eric Gouaux
  2. Department of Biochemistry and Biophysics, University of California San Francisco, 600 16th Street, San Francisco, California 94158, USA

    • Shenping Wu
    •  & Yifan Cheng
  3. Howard Hughes Medical Institute, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97239, USA

    • Eric Gouaux

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Contributions

J.D., W.L. and E.G. designed the project, J.D. and W.L. performed sample preparation, cryo-EM data collection and data analysis, J.D., W.L. and E.G. wrote the manuscript, S.W. and Y.C. assisted in cryo-EM experiments at UCSF and participated in discussion and editing of the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Eric Gouaux.

Extended data

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    Supplementary Figures

    This file contains Supplementary Figure 1.

Videos

  1. 1.

    Ligand-induced conformational transitions of GlyR

    This video shows a morph of the GlyR from the strychnine-bound to the glycine/ivermectin-bound state, via the glycine-bound state. Shown on the left side of the video is the view from the cytoplasmic side of the membrane and on the right side is the view parallel to the membrane. One subunit is highlighted.

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DOI

https://doi.org/10.1038/nature14853

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