Abstract
Fast excitatory neurotransmission in the mammalian central nervous system is largely carried out by AMPA-sensitive ionotropic glutamate receptors1. Localized within the postsynaptic density of glutamatergic spines, AMPA receptors are composed of heterotetrameric receptor assemblies associated with auxiliary subunits, the most common of which are transmembrane AMPA receptor regulatory proteins (TARPs). The association of TARPs with AMPA receptors modulates receptor trafficking and the kinetics of receptor gating and pharmacology2. Here we report the cryo-electron microscopy (cryo-EM) structure of the homomeric rat GluA2 AMPA receptor saturated with TARP γ2 subunits, which shows how the TARPs are arranged with four-fold symmetry around the ion channel domain and make extensive interactions with the M1, M2 and M4 transmembrane helices. Poised like partially opened ‘hands’ underneath the two-fold symmetric ligand-binding domain (LBD) ‘clamshells’, one pair of TARPs is juxtaposed near the LBD dimer interface, whereas the other pair is near the LBD dimer–dimer interface. The extracellular ‘domains’ of TARP are positioned to not only modulate LBD clamshell closure, but also affect conformational rearrangements of the LBD layer associated with receptor activation and desensitization, while the TARP transmembrane domains buttress the ion channel pore.
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References
Traynelis, S. F. et al. Glutamate receptor ion channels: structure, regulation, and function. Pharmacol. Rev. 62, 405–496 (2010)
Jackson, A. C. & Nicoll, R. A. The expanding social network of ionotropic glutamate receptors: TARPs and other transmembrane auxiliary subunits. Neuron 70, 178–199 (2011)
Chen, L. et al. Stargazin regulates synaptic targeting of AMPA receptors by two distinct mechanisms. Nature 408, 936–943 (2000)
Saitoh, Y. et al. Tight junctions. Structural insight into tight junction disassembly by Clostridium perfringens enterotoxin. Science 347, 775–778 (2015)
Milstein, A. D. & Nicoll, R. A. Regulation of AMPA receptor gating and pharmacology by TARP auxiliary subunits. Trends Pharmacol. Sci. 29, 333–339 (2008)
Shi, Y., Lu, W., Milstein, A. D. & Nicoll, R. A. The stoichiometry of AMPA receptors and TARPs varies by neuronal cell type. Neuron 62, 633–640 (2009)
Tomita, S. et al. Stargazin modulates AMPA receptor gating and trafficking by distinct domains. Nature 435, 1052–1058 (2005)
Milstein, A. D., Zhou, W., Karimzadegan, S., Bredt, D. S. & Nicoll, R. A. TARP subtypes differentially and dose-dependently control synaptic AMPA receptor gating. Neuron 55, 905–918 (2007)
Soto, D., Coombs, I. D., Kelly, L., Farrant, M. & Cull-Candy, S. G. Stargazin attenuates intracellular polyamine block of calcium-permeable AMPA receptors. Nat. Neurosci. 10, 1260–1267 (2007)
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)
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)
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)
Dürr, K. L. et al. Structure and dynamics of AMPA receptor GluA2 in resting, pre-open, and desensitized states. Cell 158, 778–792 (2014)
Meyerson, J. R. et al. Structural mechanism of glutamate receptor activation and desensitization. Nature 514, 328–334 (2014)
Herguedas, B. et al. Structure and organization of heteromeric AMPA-type glutamate receptors. Science 352, aad3873 (2016)
Kuusinen, A., Abele, R., Madden, D. R. & Keinänen, K. Oligomerization and ligand-binding properties of the ectodomain of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunit GluRD. J. Biol. Chem. 274, 28937–28943 (1999)
Sun, Y. et al. Mechanism of glutamate receptor desensitization. Nature 417, 245–253 (2002)
Armstrong, N., Sun, Y., Chen, G.-Q. & Gouaux, E. Structure of a glutamate-receptor ligand-binding core in complex with kainate. Nature 395, 913–917 (1998)
Armstrong, N. & Gouaux, E. Mechanisms for activation and antagonism of an AMPA-sensitive glutamate receptor: crystal structures of the GluR2 ligand binding core. Neuron 28, 165–181 (2000)
Armstrong, N., Jasti, J., Beich-Frandsen, M. & Gouaux, E. Measurement of conformational changes accompanying desensitization in an ionotropic glutamate receptor. Cell 127, 85–97 (2006)
Keinänen, K. et al. A family of AMPA-selective glutamate receptors. Science 249, 556–560 (1990)
Sommer, B., Köhler, M., Sprengel, R. & Seeburg, P. H. RNA editing in brain controls a determinant of ion flow in glutamate-gated channels. Cell 67, 11–19 (1991)
Sommer, B. et al. Flip and flop: a cell-specific functional switch in glutamate-operated channels of the CNS. Science 249, 1580–1585 (1990)
Letts, V. A. et al. The mouse stargazer gene encodes a neuronal Ca2+-channel gamma subunit. Nat. Genet. 19, 340–347 (1998)
Shanks, N. F., Maruo, T., Farina, A. N., Ellisman, M. H. & Nakagawa, T. Contribution of the global subunit structure and stargazin on the maturation of AMPA receptors. J. Neurosci. 30, 2728–2740 (2010)
Turetsky, D., Garringer, E. & Patneau, D. K. Stargazin modulates native AMPA receptor functional properties by two distinct mechanisms. J. Neurosci. 25, 7438–7448 (2005)
Kawate, T. & Gouaux, E. Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14, 673–681 (2006)
Turski, L. et al. ZK200775: a phosphonate quinoxalinedione AMPA antagonist for neuroprotection in stroke and trauma. Proc. Natl Acad. Sci. USA 95, 10960–10965 (1998)
Kazi, R., Dai, J., Sweeney, C., Zhou, H. X. & Wollmuth, L. P. Mechanical coupling maintains the fidelity of NMDA receptor-mediated currents. Nat. Neurosci. 17, 914–922 (2014)
Dawe, G. B. et al. Distinct structural pathways coordinate the activation of AMPA receptor-auxiliary subunit complexes. Neuron 89, 1264–1276 (2016)
Kaae, B. H. et al. Structural proof of a dimeric positive modulator bridging two identical AMPA receptor-binding sites. Chem. Biol. 14, 1294–1303 (2007)
Grant, T. & Grigorieff, N. Measuring the optimal exposure for single particle cryo-EM using a 2.6 A reconstruction of rotovirus VP6. eLife 4, e06980 (2015)
Mindell, J. A. & Grigorieff, N. Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334–347 (2003)
Voss, N. R., Yoshioka, C. K., Radermacher, M. & Potter, C. S. & Carragher, B. DoG Picker and TiltPicker: software tools to facilitate particle selection in single particle electron microscopy. J. Struct. Biol. 166, 205–213 (2009)
Penczek, P. A., Frank, J. & Spahn, C. M. A method of focused classification, based on the bootstrap 3D variance analysis, and its application to EF-G-dependent translocation. J. Struct. Biol. 154, 184–194 (2006)
Zhu, S. et al. Mechanism of NMDA receptor inhibition and activation. Cell 165, 704–714 (2016)
Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012)
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)
Sievers, F. et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539 (2011)
Stothard, P. The sequence manipulation suite: JavaScript programs for analyzing and formatting protein and DNA sequences. Biotechniques 28, 1102, 1104 (2000)
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)
Drozdetskiy, A., Cole, C., Procter, J. & Barton, G. J. JPred4: a protein secondary structure prediction server. Nucleic Acids Res. 43 (W1), W389–W394 (2015)
Baker, M. L., Ju, T. & Chiu, W. Identification of secondary structure elements in intermediate-resolution density maps. Structure 15, 7–19 (2007)
Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)
Pintilie, G. D., Zhang, J., Goddard, T. D., Chiu, W. & Gossard, D. C. Quantitative analysis of cryo-EM density map segmentation by watershed and scale-space filtering, and fitting of structures by alignment to regions. J. Struct. Biol. 170, 427–438 (2010)
Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002)
The PyMOL Molecular Graphics System. (DeLano Scientific, San Carlos, California, USA, 2002)
Kucukelbir, A., Sigworth, F. J. & Tagare, H. D. Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2014)
Acknowledgements
We thank T. Nakagawa for providing the clone 10 cell line, Z. H. Yu, R. Huang and C. Hong (Janelia Campus) for assistance with microscope operation and data collection and the Advanced Computing Center (OHSU) for computational support. We are grateful to A. Goehring for help with cell culture, the Multiscale Microscopy Core (OHSU) for support with microscopy, L. Vaskalis for assistance with figures, H. Owen for help with proofreading and other Gouaux laboratory members for helpful discussions. S.C. is supported by an American Heart Association postdoctoral fellowship (16POST27790099). This work was supported by the NIH (E.G., NS038631). E.G. is an investigator with the Howard Hughes Medical Institute.
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Y.Z., S.C. and E.G designed the project. Y.Z. and S.C. performed sample preparation and cryo-EM data collection. Y.Z. and C.Y. analysed the data. I.B. performed electrophysiology experiments. Y.Z., S.C., C.Y. and E.G. wrote the manuscript with input from I.B.
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Extended data figures and tables
Extended Data Figure 1 Digitonin is a suitable detergent for the purification of GluA2 receptor–TARP γ2 complex.
a, The receptor–TARP complex in digitonin disassociates when diluted into DDM. A complex composed of the GluA2 receptor and GFP-tagged TARP γ2 was diluted in digitonin (green) or DDM (red) before being subjected to GFP-tuned FSEC analysis. b, Coomassie-blue-stained SDS–PAGE gel of the purified complex. c, Tryptophan-tuned FSEC profile of the purified complex was comprised of a major peak containing the tetrameric complex and a minor shoulder, the latter suggestive of either incompletely assembled or partially dissociated complexes. Only the full-size tetrameric species was used for single-particle cryo-EM analysis. d, A representative, motion-corrected micrograph of the GluA2 receptor–TARP γ2 complex is shown. A few distinct complexes with the characteristic capital Y shape of the non-desensitized state of the AMPA receptor are circled. e, Representative 2D class averages showing a range of projections of the receptor–TARP γ2 complex.
Extended Data Figure 2 The work-flow of cryo-EM data processing.
The raw data set used in this study was composed of 2,675 micrographs. Particles (257,378) were picked from motion-corrected and contrast transfer function (CTF)-estimated micrographs for subsequent classifications. After multiple rounds of 2D classification, the remaining 61,539 particles were subjected to several rounds of 3D classification. Initial 3D classification yielded four major classes, where the most populated one contained 49% of total particles. An initial 3D reconstruction without imposed symmetry resulted in a moderate resolution at 9.6 Å. With C2 symmetry imposed, subsequent 3D refinement focused on the LBD and TMD layer improved the density map, allowing a reconstruction at 7.6 Å resolution. An additional two iterations of 3D classification and refinement using updated map as the reference further improved the reconstruction. The resolution of the final cryo-EM density map was estimated to be 7.3 Å.
Extended Data Figure 3 Statistics for the cryo-EM reconstruction.
a, Euler angular distribution of all particles included in the final 3D reconstruction. The number of particles viewed from each specific orientation was indicated by the size of the corresponding sphere. b, Gold-standard Fourier shell correlation (FSC) curves calculated between two independently refined half-maps before (red) and after (blue) post-processing, overlaid with FSC curve calculated between cryo-EM density map and structural model. c, RESMAP48 analysis of the unfiltered and unsharpened EM density map indicating the range of local resolution by colour code.
Extended Data Figure 4 Structures of the TARP γ2 subunits in the context of the respective cryo-EM density map.
a, Sequence alignment between TARP γ2 and claudin-19 calculated using Clustal omega. Also shown above the alignments are the secondary structure elements of TARP γ2 based on the model reported here, and below the aligned sequences are the secondary structure elements derived from the claudin-19 crystal structure. The ECD region rich in negative charges is conserved throughout the TARP family and highlighted in red. b, EM density for B' TARP and pseudo-atoms placed by SSEhunter, each coloured according to a calculated secondary structure score. Positive and negative scores indicate α-helix and β-sheet propensity, respectively. Dashed-line circles a map region where high scores were found, suggesting the presence of helical structure. A scale bar ranging from a maximum positive value (α-helix) to the minimum negative score (β-strand) is shown. c, The A' TARP of the A'–C' pair. d, The B' TARP of the B'–D' pair. The first and last visible residues Arg6 and Thr215 were labelled. Secondary structure elements were colour-coded as in a.
Extended Data Figure 5 Structural comparison between TARP γ2 and claudin-19.
A superimposition of the TARP γ2 structure (in blue) derived from this study and claudin-19 (in grey) is consistent in the conserved overall fold, with the exception that there is a short α1 helix present only in TARP γ2.
Extended Data Figure 6 Cryo-EM density map for the pore-helix of the GluA2 receptor.
Clear density (blue mesh) is present for the pore-lining M2 helices, secondary structure elements that are weak or absent in all previous crystal structures. The N terminus of each pore helix is involved in extensive interactions with TM4 from TARP subunits and we suggest that interactions of receptor TM helices that include M2, with TARP TM helices, stabilize the ion channel pore.
Extended Data Figure 7 Possible interaction between receptor LBD and TARP in an active state.
Shown on the left is a ‘top-down view’ of MPQX-bound receptor–TARP complex structure (in colour) superimposed with the crystal structure of an active state GluA2 receptor (in grey) in complex with a partial agonist, fluorowillardiine (FW) and a positive allosteric modulator, (R, R)-2b, using the central M3 helices as a reference. ATDs and LBDs were omitted for clarity. The modelled pseudo-complex consisting of TARPs and the active state receptor illustrates a possible mechanism for how TARP interacts with receptor LBD during activation. Enlarged views of the MPQX-bound complex structure and FW–(R, R)-2b-bound complex model were shown side by side at both D' and A' TARP positions. LBD helices and a S2–M4 linker were labelled according to convention.
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Zhao, Y., Chen, S., Yoshioka, C. et al. Architecture of fully occupied GluA2 AMPA receptor–TARP complex elucidated by cryo-EM. Nature 536, 108–111 (2016). https://doi.org/10.1038/nature18961
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DOI: https://doi.org/10.1038/nature18961
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