Glycine receptors (GlyRs) are pentameric, ‘Cys-loop’ receptors that form chloride-permeable channels and mediate fast inhibitory signalling throughout the central nervous system1,2. In the spinal cord and brainstem, GlyRs regulate locomotion and cause movement disorders when mutated2,3. However, the stoichiometry of native GlyRs and the mechanism by which they are assembled remain unclear, despite extensive investigation4,5,6,7,8. Here we report cryo-electron microscopy structures of native GlyRs from pig spinal cord and brainstem, revealing structural insights into heteromeric receptors and their predominant subunit stoichiometry of 4α:1β. Within the heteromeric pentamer, the β(+)–α(−) interface adopts a structure that is distinct from the α(+)–α(−) and α(+)–β(−) interfaces. Furthermore, the β-subunit contains a unique phenylalanine residue that resides within the pore and disrupts the canonical picrotoxin site. These results explain why inclusion of the β-subunit breaks receptor symmetry and alters ion channel pharmacology. We also find incomplete receptor complexes and, by elucidating their structures, reveal the architectures of partially assembled α-trimers and α-tetramers.
This is a preview of subscription content
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Legendre, P. The glycinergic inhibitory synapse. Cell. Mol. Life Sci. 58, 760–793 (2001).
Lynch, J. W. Molecular structure and function of the glycine receptor chloride channel. Physiol. Rev. 84, 1051–1095 (2004).
Shiang, R. et al. Mutations in the alpha 1 subunit of the inhibitory glycine receptor cause the dominant neurologic disorder, hyperekplexia. Nat. Genet. 5, 351–358 (1993).
Grudzinska, J. et al. The β subunit determines the ligand binding properties of synaptic glycine receptors. Neuron 45, 727–739 (2005).
Langosch, D., Thomas, L. & Betz, H. Conserved quaternary structure of ligand-gated ion channels: the postsynaptic glycine receptor is a pentamer. Proc. Natl Acad. Sci. USA 85, 7394–7398 (1988).
Yang, Z., Taran, E., Webb, T. I. & Lynch, J. W. Stoichiometry and subunit arrangement of alpha1beta glycine receptors as determined by atomic force microscopy. Biochemistry 51, 5229–5231 (2012).
Burzomato, V., Groot-Kormelink, P. J., Sivilotti, L. G. & Beato, M. Stoichiometry of recombinant heteromeric glycine receptors revealed by a pore-lining region point mutation. Recept. Channels 9, 353-361 (2003).
Durisic, N. et al. Stoichiometry of the human glycine receptor revealed by direct subunit counting. J. Neurosci. 32, 12915–12920 (2012).
Shan, Q., Han, L. & Lynch, J. W. Distinct properties of glycine receptor β+/α− interface: unambiguously characterizing heteromeric interface reconstituted in homomeric protein. J. Biol. Chem. 287, 21244–21252 (2012).
Shan, Q., Haddrill, J. L. & Lynch, J. W. A single β subunit M2 domain residue controls the picrotoxin sensitivity of αβ heteromeric glycine receptor chloride channels. J. Neurochem. 76 1109–1120 (2001).
Meyer, G., Kirsch, J., Betz, H. & Langosch, D. Identification of a gephyrin binding motif on the glycine receptor beta subunit. Neuron 15, 563–572 (1995).
Graham, D., Pfeiffer, F., Simler, R. & Betz, H. Purification and characterization of the glycine receptor of pig spinal cord. Biochemistry 24, 990–994 (1985).
Dutertre, S., Becker, C. M. & Betz, H. Inhibitory glycine receptors: an update. J. Biol. Chem. 287, 40216–40223 (2012).
Toyoshima, C. & Unwin, N. Three-dimensional structure of the acetylcholine receptor by cryoelectron microscopy and helical image reconstruction. J. Cell Biol. 111, 2623–2635 (1990).
Yu, H., Bai, X.-c. & Wang, W. Characterization of the subunit composition and structure of adult glycine receptors. Neuron 109, 2707–2716.e6 (2021).
Kuhse, J., Laube, B., Magalei, D. & Betz, H. Assembly of the inhibitory glycine receptor: identification of amino acid sequence motifs governing subunit stoichiometry. Neuron 11, 1049–1056 (1993).
Yu, J. et al. Mechanism of gating and partial agonist action in the glycine receptor. Cell 184, 957–968.e21 (2021).
Hille, B. Ion Channels of Excitable Membranes 3rd edn (Sinauer, 2001).
Kumar, A. et al. Mechanisms of activation and desensitization of full-length glycine receptor in lipid nanodiscs. Nat. Commun. 11, 3752 (2020).
Hibbs, R. E. & Gouaux, E. Principles of activation and permeation in an anion-selective Cys-loop receptor. Nature 474, 54–60 (2011).
Masiulis, S. et al. GABAA receptor signalling mechanisms revealed by structural pharmacology. Nature 565, 454–459 (2019).
Todorovic, J., Welsh, B. T., Bertaccini, E. J., Trudell, J. R. & Mihic, S. J. Disruption of an intersubunit electrostatic bond is a critical step in glycine receptor activation. Proc. Natl Acad. Sci. USA 107, 7987–7992 (2010).
Phulera, S. et al. Cryo-EM structure of the benzodiazepine-sensitive α1β1γ2S tri-heteromeric GABAA receptor in complex with GABA. eLife 7, e39383 (2018).
Kloda, J. H. & Czajkowski, C. Agonist-, antagonist-, and benzodiazepine-induced structural changes in the α1 Met113–Leu132 region of the GABAA receptor. Mol. Pharmacol. 71, 483–493 (2007).
Liu, S. et al. Cryo-EM structure of the human α5β3 GABAA receptor. Cell Res. 28, 958–961 (2018).
Walsh, R. M., Jr. et al. Structural principles of distinct assemblies of the human α4β2 nicotinic receptor. Nature 557, 261–265 (2018).
Green, W. N. & Claudio, T. Acetylcholine receptor assembly: subunit folding and oligomerization occur sequentially. Cell 74, 57–69 (1993).
Klausberger, T. et al. Detection and binding properties of GABAA receptor assembly intermediates. J. Biol. Chem. 276, 16024–16032 (2001).
Blount, P. & Merlie, J. P. Molecular basis of the two nonequivalent ligand binding sites of the muscle nicotinic acetylcholine receptor. Neuron 3, 349–357 (1989).
Hattori, M., Hibbs, R. E. & Gouaux, E. A fluorescence-detection size-exclusion chromatography-based thermostability assay for membrane protein precrystallization screening. Structure 20, 1293–1299 (2012).
Du, J., Lu, W., Wu, S., Cheng, Y. & Gouaux, E. Glycine receptor mechanism elucidated by electron cryo-microscopy. Nature 526, 224–229 (2015).
Kawate, T. & Gouaux, E. Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14, 673–681 (2006).
Goehring, A. et al. Screening and large-scale expression of membrane proteins in mammalian cells for structural studies. Nat. Protoc. 9, 2574–2585 (2014).
Cook, N., Harris, A., Hopkins, A. & Hughes, K. Scintillation proximity assay (SPA) technology to study biomolecular interactions. Curr. Protoc. Protein Sci. Ch. 19, Unit 19.8 (2002).
Cheng, Y. & Prusoff, W. H. Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 22, 3099–3108 (1973).
Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).
Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).
Zhang, K. Gctf: Real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).
Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).
Henderson, R. Avoiding the pitfalls of single particle cryo-electron microscopy: Einstein from noise. Proc. Natl Acad. Sci. USA 110, 18037–18041 (2013).
Jakobi, A. J., Wilmanns, M. & Sachse, C. Model-based local density sharpening of cryo-EM maps. eLife 6, e27131 (2017).
Arnold, K., Bordoli, L., Kopp, J. & Schwede, T. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22, 195–201 (2006).
Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).
Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D 74, 531–544 (2018).
We thank L. Sivilotti, F. Jalali-Yazdi, C. Sun and R. Hallford for discussions and suggestions; L. Vaskalis for assistance with figures; J. Guidry for assisting with the proteomics work; and D. Claxton and D. Cawley for the monoclonal antibody. The LSUHSC Proteomics Core is supported by the Louisiana State University School of Medicine Office of the Dean. A portion of this research was supported by NIH grant U24GM129547 and performed at the PNCC at OHSU and accessed through EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research. This work was supported by NIH grant 5R01 GM100400 to E.G. E.G. is an investigator of the Howard Hughes Medical Institute.
The authors declare no competing interests.
Peer review information Nature thanks Nathan Absalom and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, Flow chart for native GlyR purification. b, Representative SEC profile for native GlyR in complex with the 3D1 Fab. Inset shows a typical silver staining of sodium dodecyl sulphate-polyacrylamide gel electrophoresis of native GlyR sample for cryo-EM grid preparation. c, Results from mass spectrometry (See Methods for more details). The table shows the identified peptides within the sample and the corresponding proteins with their gene accession numbers. d, Western blot analysis of isolated native GlyR eluted from strychnine column using antibodies against α1, α2, and α3. Positive control is the membrane extracts from rat brain. The experiments were repeated two times with similar results. e, FSEC profiles for mixing of different concentration of recombinant homomeric α pentamer with 3D1 Fab. f, g, FSEC profiles for mixing of YFP-tagged homomeric α1 GlyR (f) and CFP-tagged β GlyR (g), respectively. h–j, Saturation binding of 3H strychnine to native GlyRs with 3D1 Fab (h), recombinant expressed pig heteromeric GlyRs with (i) and without 3D1 Fab (j), respectively. Results are the average of three replicates and the error bars represent standard error of the mean (SEM) (n = 3). k–m, The competitive binding of glycine to native GlyRs with 3D1 Fab (k), recombinant expressed pig heteromeric GlyR with (l) and without 3D1 Fab (m), respectively. Results are the average of three replicates and the error bars represent SEM (n = 3). The hot ligand used here is 3H strychnine.
a, A typical cryo-EM micrograph for native GlyRs. The experiments were repeated three times with similar results. b, Selected 2D class averages for native GlyR-Fab complex. c, Flow chart for cryo-EM data analysis of native GlyRs. d, f, h, j, Local resolution maps for unsharpened heteromeric pentamer (d), homomeric α tetramer (f), locally refined ECD (h) and TMD map (j) of homomeric pentamer. e, g, i, k, FSC curves for heteromeric pentamer (e), homomeric α tetramer (g), locally refined ECD (i) and TMD map (k) of homomeric α pentamer.
a, b, EM density segments for α.A (a) and β (b) subunits, respectively. The model is shown in carton representation. The density is shown in surface representation. c, d, Representative densities for light (c) and heavy chain (d) of 3D1 Fab. Regions are numbered. e, f, Representative densities for glycosylation on α.A (e) and β (f) subunits. g–i, Representative densities of the binding pockets formed by β(+)/α.A(−) (g), α.D(+)/β(−) (h) and α.B(+)/α.C(−) (i), respectively. The related key amino acids are labeled. j, k, Representative densities for transmembrane helices including M1, M2, M3 and M4 from β (j) and α.A (k), respectively. All of the isolated densities are contoured at 8σ.
a, b, Comparison of isolated representative densities for α1 and β subunits contoured at 8σ. Two pairs of representative residues have been selected. These key amino acids are labeled. Black stars highlight the mismatched residues. c, Isolated densities with different amino acids between the α1 and α2 subunit from native heteromeric and homomeric pentamer maps contoured at 8σ, respectively. d, f, ECD (d) and TMD (f) of heteromeric pentamer shown in cartoon representation. The α subunits are colored in blue and β subunit is in salmon. The centers of mass for ECD and TMD are shown in green and orange, respectively. e, g, Schematic diagram illustrating the neighboring distances of centers of mass of heteromeric ECD (e) and TMD (g), respectively. h, i, Schematic diagram illustrating the neighboring distances of centers of mass of homomeric α1 pentamer ECD (h) and TMD (i), respectively. j, Top-down view of heteromeric GlyR-Fab complex. GlyRs are in cartoon representation, with N-glycans and lipids in sphere representation. 3D1 Fabs, α, β, N-glycans, ligands glycine and lipids are colored in green, blue, salmon, orange, purple and yellow, respectively. All of the distances are denoted in Å.
a, d, Side views of 3D1 Fab bound to the isolated α.A (a) and α.B subunit (d) in carton representation, respectively. b, c, e, Close-up view of the binding site of the region indicated in panel (a) and (d) viewed approximately parallel to the plane of the membrane. The key amino acids involved in interactions are shown in ball-stick representation. The potential hydrogen bonds, cation-π and π-π interactions are indicated in dashed lines. f, Side view of 3D1 Fab bound to the isolated β subunit in carton representation. g, Close-up view of the binding site of the region indicated in panel (f) viewed approximately parallel to the plane of the membrane.
a, Sequence alignment of M2 helices among GABAAR, GlyR and GluCl. Higher prime numbers approach ECD, lower prime numbers approach intracellular domain. The −2’ position is the first amino acid of M2 helix. Sequence alignment was performed by PROMALS3D. b–d, Isolated M2 helices bound with picrotoxin from GABAAR (b; PDB ID: 6HUG), GluCl (c; PDB ID: 3RI5) and homomeric GlyR (d; PDB ID: 6UD3). The important amino acids 6’T or 2’T interacting with picrotoxin are labeled. The M2 helices and picrotoxin are shown in cartoon and stick representation, respectively. e, f, Isolated M2 helices from native homomeric GlyR (e) and heteromeric GlyR (f), respectively. The 6’T and 6’F are shown in stick representation. The M2 helices are shown in cartoon representation. All distances are denoted in Å.
a–c, View of the interface interactions of native homomeric α1 pentamer (see Fig. 2c–e). d, The summary of the buried areas for heteromeric pentamer, homomeric α tetramer and homomeric α1 pentamer. The areas are given in Å2. e, Top down view of heteromeric GlyR in surface and ribbon representation. The glycine molecules are shown in sphere representation. The α.A and α.D are in blue. The β subunit and α.C subunits are colored in salmon and lime, respectively. The boxed areas are enlarged in panels (f) to (h). f–h, Views of the binding pockets at α.D(+)/β(−) (f), α.B(+)/α.C(−) (g) and β(+)/α.A(−) (h) interfaces, respectively. The glycine molecules are shown in ball-stick representations with oxygen in red, nitrogen in blue and carbon in green. The possible hydrogen bonds and cation-pi interactions are shown as dashed lines. i, Superposition of the orthostatic binding sites. The binding sites are overlapped by the ECD of the principle side subunits. Orange arrows indicate the movement of loop C. j, Schematic diagram illustrating the relative positions of the amino acids in the binding pockets. The blue, pink, green and red polygon are created by the connection of the Cα atoms of these crucial amino acids at the β(+)/α.A(−), α.D(+)/β(−), α.B(+)/α.C(−) and native homomeric α(+)/α(−) interfaces, respectively. k–n, Schematic diagram illustrating the distances and angles related with the interfaces of Cys-loop family members including GABAAR (k, PDB ID: 6A96; l, PDB ID: 6DW1) and nAChR (m, PDB ID: 6CNJ; n, PDB ID: 6CNK; see Fig. 2f). The black star indicates the binding pocket bound with ligand. All distances are given in Å and the angles are given in degree.
a, b, Representative FSEC profiles for recombinantly expressed homomeric GlyR tagged with YFP (a) and heteromeric GlyR tagged with CFP on β subunit (b), respectively. Melting temperatures (Tm) were determined by fitting the curves to a sigmoidal dose-response equation. c, A typical cryo-EM micrograph for recombinant GlyRs. The experiments were repeated three times with similar results. d, 2D class averages for recombinant GlyRs bound with 3D1 Fabs. e, f, Top down and side views for the recombinant heteromeric GlyR map, respectively. g, h, Top down and side views for the recombinant homomeric GlyR map, respectively. i, Side view of isolated α.B-α.C dimer from tetramer. Subunits are shown in cartoon representation. α.B and α.C are colored in blue and lime, respectively. The boxed areas are enlarged in panel (j) and (l). j, l, Superposition of the interfaces in the upper ECD (j) and the region near loop C (l) of α.B(+)/α.C(-) interface from homomeric α tetramer, heteromeric pentamer and homomeric pentamer. Orange arrows indicate the displacements of the Cα atoms. k, m, Schematic diagram illustrating the relative positions of the amino acids of the homomeric pentamer and tetramer. All distances are given in Å.
This file contains Supplementary Figs 1, 2. Supplementary Fig. 1 shows the original gel and original results for the western blots and Supplementary Fig. 2 displays amino acid sequence alignment, secondary structure and posttranslational modifications for pig GlyR α1 (GlyRA1) and β (GlyRB) subunits.
About this article
Cite this article
Zhu, H., Gouaux, E. Architecture and assembly mechanism of native glycine receptors. Nature 599, 513–517 (2021). https://doi.org/10.1038/s41586-021-04022-z