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Channel opening and gating mechanism in AMPA-subtype glutamate receptors

Abstract

AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-subtype ionotropic glutamate receptors mediate fast excitatory neurotransmission throughout the central nervous system. Gated by the neurotransmitter glutamate, AMPA receptors are critical for synaptic strength, and dysregulation of AMPA receptor-mediated signalling is linked to numerous neurological diseases. Here we use cryo-electron microscopy to solve the structures of AMPA receptor–auxiliary subunit complexes in the apo, antagonist- and agonist-bound states and determine the iris-like mechanism of ion channel opening. The ion channel selectivity filter is formed by the extended portions of the re-entrant M2 loops, while the helical portions of M2 contribute to extensive hydrophobic interfaces between AMPA receptor subunits in the ion channel. We show how the permeation pathway changes upon channel opening and identify conformational changes throughout the entire AMPA receptor that accompany activation and desensitization. Our findings provide a framework for understanding gating across the family of ionotropic glutamate receptors and the role of AMPA receptors in excitatory neurotransmission.

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Figure 1: GluA2–GSG1L and ion channel structure.
Figure 2: Cryo-EM of an activated GluA2–STZ complex.
Figure 3: Structure of the GluA2–STZ complex.
Figure 4: Ion channel pore in open, closed and desensitized states.
Figure 5: Structural rearrangements during gating.

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Acknowledgements

We thank H. Kao for computational support, A. des Georges, I. S. Fernandez, M. Fislage and A. K. Singh for processing advice. E.C.T. is supported by National Institutes of Health (NIH) F31 NS093838. A.I.S. is supported by the NIH (R01 NS083660, R01 CA206573), the Pew Scholar Award in Biomedical Sciences, and the Irma T. Hirschl Career Scientist Award. J.F. is supported by the Howard Hughes Medical Institute and the NIH (R01 GM029169). Cryo-EM data were collected at the Columbia University Medical Center cryo-EM facility and at the Simons Electron Microscopy Center and National Resource for Automated Molecular Microscopy located at the New York Structural Biology Center, supported by grants from the Simons Foundation (349247), NYSTAR, and the NIH National Institute of General Medical Sciences (GM103310).

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Authors

Contributions

E.C.T. designed the constructs, prepared the protein samples, carried out cryo-EM data collection and processing, built models, analysed data and wrote the manuscript. M.V.Y. carried out electrophysiology experiments, assisted in protein production and edited the manuscript. R.A.G. assisted in cryo-EM data collection. J.F. advised on the cryo-EM workflow and provided funding. A.I.S. supervised the project, built models, analysed data, wrote the manuscript and provided funding. E.C.T. and A.I.S. designed the project.

Corresponding author

Correspondence to Alexander I. Sobolevsky.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks M. Mayer, S. Traynelis and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 FSC curves for cryo-EM reconstructions.

FSC curves calculated between half-maps for GluA2–GSG1LZK-1, GluA2–GSG1LZK-2, GluA2–GSG1Lapo-1, GluA2–GSG1Lapo-2 and GluA2–STZGlu+CTZ cryo-EM reconstructions, as well as for the GluA2–STZGlu+CTZ TMD reconstruction from directed refinement. The dashed line indicates FSC = 0.143.

Extended Data Figure 2 Local resolution and fitting of cryo-EM maps.

ap, Local resolution calculated using Resmap and two unfiltered halves of the reconstruction for GluA2–GSG1LZK-1, GluA2–GSG1LZK-2, GluA2–GSG1Lapo-1, GluA2–GSG1Lapo-2 and GluA2–STZGlu+CTZ structures viewed parallel to the membrane as a surface (a, d, h, k, n) and slice through the centre of the receptor (b, e, i, l, o), with the cross-validation FSC curves for the refined model versus unfiltered half maps (one used in the refinement, work, and another one not, free) and the unfiltered summed maps shown on the right (c, f, j, m, p).

Extended Data Figure 3 Closed state 1 cryo-EM density and comparison of ZK200775-bound and apo states.

ad, Fragments of GluA2–GSG1LZK-1 and GluA2–GSG1Lapo-1 with the corresponding cryo-EM density. a, d, ATD and LBD of subunit A in GluA2–GSG1LZK-1 (a) and GluA2–GSG1Lapo-1 (d) with density for ZK200775 indicated in the GluA2–2×GSG1LZK-1 structure. b, c, M2 helix (b) and selectivity filter (c) with the Q/R-site Gln586 side chains pointing towards the centre of the pore in GluA2–GSG1LZK-1. e, Superposition of GluA2–GSG1LZK-1 (blue) and GluA2–GSG1Lapo-1 (red) viewed parallel to the membrane. Note that the structures are almost indistinguishable (r.m.s.d. = 0.526 Å). Densities are shown at 6σ.

Extended Data Figure 4 Closed state 2 structure and digitonin-binding pocket.

a, b, Structures of GluA2–GSG1LZK-1 (a) and GluA2–GSG1LZK-2 (b) viewed parallel to the membrane. The GluA2 subunits A and C are coloured purple, B and D in green and GSG1L in red. The competitive antagonist ZK200775 and digitonin are shown as space-filling models. In b, inset shows expanded view of the boxed region, demonstrating cryo-EM density for digitonin (blue mesh, 4σ). Digitonin and the surrounding residues in the inset are shown in stick representation. ch, Top down views along the axis of the overall two-fold rotational symmetry on the ATD (c, d), LBD (e, f) and TMD (g, h) layers. Rigid-body rotation of the ATD tetramer in d and rotation of LBD dimers in f are indicated by red arrows. i, j, Superposition of GluA2–GSG1LZK-2 (blue) and GluA2–GSG1Lapo-2 (red) viewed parallel to the membrane. Note that the structures are almost indistinguishable (r.m.s.d. = 0.701 Å).

Extended Data Figure 5 Cryo-EM density for the open state.

ae, Fragments of GluA2–STZGlu+CTZ with the corresponding cryo-EM density. a, Zoomed view of the glutamate-binding pocket. b, c, The ion channel pore with a central density at the selectivity filter, probably for a sodium ion that is hydrated based on the pore diameter, viewed from the top of the selectivity filter looking down into the cytoplasm (b) or parallel to the membrane with two (front and back) GluA2 subunits removed (c). d, Density for CTZ. e, Transmembrane domain segments for GluA2 (top row) and STZ (bottom row).

Extended Data Figure 6 Overview of single-particle cryo-EM and stoichiometry for GluA2–STZ and GluA2–GSG1L solubilized in digitonin.

a, b, Two-dimensional class averages for GluA2–STZGlu+CTZ (a) and GluA2nGSG1LZK (b) indicating three-layer architecture of the particles. c, d, Final densities for GluA2–STZGlu+CTZ (c) and GluA2–GSG1LZK-1 (d) with the GluA2 subunits A and C coloured purple, B and D in green, STZ in cyan and GSG1L in red. Insets show 2D slices made parallel to the membrane through the refined, nonfiltered map. Note, although four STZ molecules bind one receptor, only two copies of GSG1L can bind per GluA2 tetramer.

Extended Data Figure 7 Conformational differences between the closed, open and desensitized states.

ac, Structures of GluA2–GSG1LZK-1 in the closed state (a), GluA2–STZGlu+CTZ in the open state (b) and GluA2–2×GSG1LQuis in the desensitized state (c), viewed parallel to the membrane. The GluA2 subunits A and C are coloured purple, B and D in green, GSG1L in red and STZ in cyan. The competitive antagonist ZK200775, agonists Glu and Quis and positive allosteric modulator CTZ are shown as space-filling models. dl, Top down views along the axis of the overall two-fold rotational symmetry on the layers of ATD (df), LBD (gi) and TMD (jl). Rigid-body rotation of the ATD tetramers in e and f, broadening of LBD layer in h and rotation of subunit A/C LBDs in i are indicated by red arrows. Note the dramatic opening in the middle of the LBD layer (h) and pore dilation (k) in the open state.

Extended Data Figure 8 iGluR gating mechanism.

Two out of four iGluR subunits are shown with the ATDs omitted. Four basic states of iGluR gating are illustrated: resting, represented by apo (GluA2–GSG1Lapo-1) or antagonist-bound closed state (GluA2–GSG1LZK-1) structures; closed, agonist-bound (pre-active state crystal structures10,11); open (GluA2–STZGlu+CTZ); and desensitized (GluA2–2×GSG1LQuis complex16). Transitions between the states are indicated by black arrows, conformational rearrangements by blue arrows, and ionic current through the open channel by an orange arrow. Upper and lower gates are indicated by one and two red asterisks, respectively, with red sticks at the upper gate representing channel occluding residues at the bundle crossing and the Q/R site at the lower gate. Glutamate molecules are illustrated by orange wedges. The receptor sits in a resting, closed state, with its LBD clamshells in the maximally open conformations, unoccupied by the neurotransmitter glutamate. Upon glutamate binding, the LBD clamshells close, as described in the pre-activated crystal structures, to an intermediate state that does not put enough strain on the LBD–TMD linkers to open the channel. The LBDs then transition to their maximally closed state, which strains the LBD–TMD linkers, causing the channel pore to open and conduct ions. Most AMPARs, however, quickly desensitize, transitioning to the desensitized state from the open state via the agonist-bound, closed state. Desensitization is accompanied by the rupture of the upper LBD interfaces, with the LBDs adapting their maximally closed clamshell conformations, as described in the desensitized-state GluA2–GSG1L complex.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics

Supplementary information

Reporting Summary (PDF 67 kb)

Conformational changes between GluA2 closed, open and desensitized states

A morph between GluA2 closed, open and desensitized stated represented by the GluA2-GSG1LZK-1, GluA2-STZGlu-CTZ and GluA2-2xGSG1LQuis structures, respectively. The GluA2 subunits A and C are colored purple and B and D green. The agonists Glu are represented by space-filling models. Note, the pre-M4 helices in subunits A and C unfold during channel opening, while the entire TMD becomes wider. The receptor undergoes a corkscrew twist and becomes shorter while progressing from the closed to open to desensitized states. (MP4 9845 kb)

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Twomey, E., Yelshanskaya, M., Grassucci, R. et al. Channel opening and gating mechanism in AMPA-subtype glutamate receptors. Nature 549, 60–65 (2017). https://doi.org/10.1038/nature23479

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