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The open gate of the AMPA receptor forms a Ca2+ binding site critical in regulating ion transport

Nature Structural & Molecular Biology volume 31, pages 688–700 (2024)Cite this article

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Abstract

Alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid receptors (AMPARs) are cation-selective ion channels that mediate most fast excitatory neurotransmission in the brain. Although their gating mechanism has been studied extensively, understanding how cations traverse the pore has remained elusive. Here we investigated putative ion and water densities in the open pore of Ca2+-permeable AMPARs (rat GRIA2 flip-Q isoform) at 2.3–2.6 Å resolution. We show that the ion permeation pathway attains an extracellular Ca2+ binding site (site-G) when the channel gate moves into the open configuration. Site-G is highly selective for Ca2+ over Na+, favoring the movement of Ca2+ into the selectivity filter of the pore. Seizure-related N619K mutation, adjacent to site-G, promotes channel opening but attenuates Ca2+ binding and thus diminishes Ca2+ permeability. Our work identifies the importance of site-G, which coordinates with the Q/R site of the selectivity filter to ensure the preferential transport of Ca2+ through the channel pore.

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Fig. 1: The ion permeation path of A2iQ/γ2(KKEE).
Fig. 2: Site-G as a Ca2+ binding site at the gate.
Fig. 3: External Ca2+ block of GluA2/γ2 and its voltage dependence.
Fig. 4: N619K mutation reduces Ca2+ binding at site-G, external Ca2+ block and Ca2+ permeability.
Fig. 5: N619K mutation promotes opening and alters kinetic properties of the ion channel.
Fig. 6: Calcium block and permeation are affected by N619K mutation.
Fig. 7: Schematic model for the function of site-G.

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Data availability

The structural data in this work have been deposited in the Protein Data Bank (PDB) and the Electron Microscopy Data Bank (EMDB) under the accession numbers PDB 8FQF (EMD-29386), PDB 8FP9 (EMD-29360), PDB 8FQ1 (EMD-29737), PDB 8FP4 (EMD-29359), PDB 8FQB (EMD-29382), PDB 8FQ5 (EMD-29378), PDB 8FPS (EMD-29369), PDB 8FPG (EMD-29363), PDB 8FQG (EMD-29387), PDB 8FQH (EMD-29388), PDB 8FQ0 (EMD-29394), PDB 8FPC (EMD-29361), PDB 8FPH (EMD-29364), PDB 8FQ6 (EMD-29379), PDB 8FPV (EMD-29370), PDB 8FPY (EMD-29371), PDB 8FPZ (EMD-29372), PDB 8FQD (EMD-29384), PDB 8FQE (EMD-29385), PDB 8FQ8 (EMD-29380), PDB 8FQA (EMD-29381), PDB 8FQ2 (EMD-29375), PDB 8FQ3 (EMD-29376), PDB 8FPK (EMD-29367) and PDB 8FPL (EMD-29368). The C1 maps are associated with each entry. Request for materials (plasmids and cell lines) will be fulfilled for reasonable inquiries and should be addressed to the corresponding author. Source data are provided with this paper.

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Acknowledgements

We acknowledge the use of the cryo-EM facility at the Center for Structural Biology and DORS Data Storage Core at Vanderbilt University. M. Chambers, S. Collier and M. Haider maintained the cryo-EM facility and facilitated data collection. We thank K. Kim and P. Christov at Vanderbilt Chemical Synthesis Core for synthesizing chemicals. Software was provided by SBGrid. The work was supported by funding from a National Institutes of Health grant R56/R01MH123474 (to T.N.), S10OD030292-01 (to T.N), Stanley Cohen Innovation Fund (to T.N.) and the Canadian Institutes of Health Research (FRN 163317, D.B.). X.-t.W. was funded by a Max Binz fellowship from McGill University’s Faculty of Medicine.

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Authors and Affiliations

  1. Department of Molecular Physiology and Biophysics, Vanderbilt University, School of Medicine, Nashville, TN, USA

    Terunaga Nakagawa

  2. Center for Structural Biology, Vanderbilt University, School of Medicine, Nashville, TN, USA

    Terunaga Nakagawa

  3. Vanderbilt Brain Institute, Vanderbilt University, School of Medicine, Nashville, TN, USA

    Terunaga Nakagawa

  4. Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada

    Xin-tong Wang, Federico J. Miguez-Cabello & Derek Bowie

  5. Integrated Program in Neuroscience, McGill University, Montreal, Quebec, Canada

    Xin-tong Wang

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  1. Terunaga Nakagawa
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Contributions

T.N. conceived the project, conducted cryo-EM experiments and pilot electrophysiology experiments, analyzed data and wrote the paper with inputs from other authors. D.B. provided insights into the divalent cation block and supervised electrophysiology experiments. X.-t.W. and F.J.M.-C. conducted electrophysiology experiments and analyzed data. T.N. and D.B. provided funding.

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Correspondence to Terunaga Nakagawa.

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Nature Structural & Molecular Biology thanks Janesh Kumar and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Sara Osman, in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 Expression, purification, and cryo-EM data processing.

A. DOX inducible expression scheme used in stable HEK cell line. In clone #53, GluA2(flip-Q isoform) and TARPγ-2(KKEE) were stably co-expressed, independently without tether. Clone #8 expresses the GluA2(flip-Q isoform, N619K mutant) and TARPγ2(KKEE). TARPγ2 is abbreviated as γ-2. B. Purified complex resolved in SDS-PAGE. C. Superdex200 (Sdx200) size exclusion chromatograph in the final step of purification. The peak with asterisk contains the complex formed of GluA2(flip-Q) and TARPγ2(KKEE). The N619K mutant complex exhibit identical chromatography profile and not shown. D. A representative motion corrected cryo-EM image of purified GluA2/TARPγ2(KKEE) complex of Open-Na260. The displayed image is representative among a total of ~16,000 micrographs. E. A set of representative 2D class averages obtained during the image processing of Open-Na260. The class averages displayed are from 1/4 of the total dataset. F. Image processing of Open-Na260. Other structures were obtained using similar procedures and described in the methods. G-H. The atomic model of Open-Na260. GluA2=blue, TARPγ2(KKEE)=yellow. TARPγ2(KKEE) in the A’/C’ positions (G) and B’/D’ positions (H) are shown. The zoomed views (left panels) correspond to the red square in the right panels. In all the structures determined in this work (see Table1), the regions containing the KKEE mutations were unresolved due to structural flexibility of the extracellular loop.

Source data

Extended Data Fig. 2 Fourier Shell Correlation (FSC) curves.

The FSC curves of the GluA2/TARP complexes presented in this work. The membrane embedded portion of the structures were refined using the TMD-STG mask, with (C2) and without (C1) imposing symmetry (A, C, E, G, I, K, and M). The LBDs were refined using the LBD mask with C2 symmetry imposed (B, D, F, H, J, L, and N). A-B. Open-CaNaMg. C-D. Open-Ca10. E-F. Open-Ca150. G-H. Open-Na610. I-J. Open-Na110. K-L. Open-CaNaMg/N619K. M-N. Closed-CaNaMg. The FSCs and final resolutions were calculated using Relion. FSC = 0.143 is indicated by dashed line. Resolution is indicated by the arrows and numbers. Note: FSC curves of Open-Na260 are provided in Extended Data Fig. 1F2 and F4.

Extended Data Fig. 3 Local resolution and representative fit between map and model.

A-G. Local resolution was estimated by ResMap59, using as input the half-maps generated by 3D refinement in Relion. The heatmap for resolution is shown on the bottom right. Side view of each indicated structure. H. Overlay of map and model in a subregion of M1-3 helices. With an optimal threshold, the holes in the aromatic sidechains are discernable. I-N. Cross sections of local resolution heatmaps of the open pores. The arrows in I-K are the Ca2+ density at site−G. Note higher local resolution of the density in 150 mM Ca2+ (Open-Ca150) compared to 10 mM Ca2+ (Open-Ca10 and Open-CaNaMg), consistent with more binding at higher concentration. Note, the maps displayed are half-maps that are unfiltered/unsharpened, and thus the resolution appear lower than the full-maps displayed in other figures. O. Representative overlay of map and model in the TMD. Open-Na260 is shown. Others were at similar quality. Also see Extended Data Figs. 6–8.

Extended Data Fig. 4 Consistency of pore densities between the C1 and C2 maps.

A-B. Cross sections of the pores of Open-Na260. (A) C1 maps. (B) C2 maps. All maps were filtered according to the local resolution calculated by Relion. The cryo-EM density map (mesh) and the atomic model are superimposed. The atomic models were built based on the C2 map and superimposed into the C1 map in A. Putative water and calcium ion are in red mesh. The atomic models of the polypeptides of M2, SF, and M3 are shown in yellow, orange, and sky blue, where elements are colored according to oxygen=red, nitrogen=blue, and sulfate=yellow. Resides that define the gate in the M3 of each structure are colored magenta. C-E. Overlay of the model and map at site−G are shown. C. Open-Ca10. D. Open-Ca150. E. Open-CaNaMg. Top: C1 map. Bottom: C2 map. The C1 map was filtered using the local resolution function in Relion. T617 and A618 are in magenta. The Ca2+ is green.

Extended Data Fig. 5 Global structures of the A2iQ/γ2(KKEE).

A-E. Conformational heterogeneity of the LBD gating ring in Open-Na260. A. LBD-TMD sectors were reconstructed from particles that produced LBD Conf1, Conf2, and Conf3. The LBD-TMD were aligned and superimposed using the cytoplasmic half of the SF. The TMD and γ2(KKEE) are nearly perfectly aligned as expected; there was no obvious global conformational heterogeneity of the TMD and γ2(KKEE). Blue=Conf1, Yellow=Conf2, and Red=Conf3. The rectangle indicates the regions that are highlighted in panels C-E. B. The LBD gating ring of the aligned and superimposed structure shown in panel A is viewed from above. The helices of the LBDs do not superimpose. C. Conformational transition from Conf1 to Conf2. The Conf1 conformation is shown with the mode vectors (yellow arrows) that schematically represent the displacements of the Cα during the conformational transition from Conf1 to Conf2 (panel C1). The lengths of the vectors are increased by 50% than actual displacements for clarity. The Conf1 and Conf2 conformations are superimposed in the same view as in panel C1. D and E. The transition from Conf1 to Conf3 (panels D1 and D2) and from Conf2 to Conf3 (panels E1 and E2) are shown as in panel C. F-N. Global architecture of A2iQ/γ-2(KKEE). F and G. The atomic model of Open-CaNaMg at 2.4 Å resolution, where γ2(KKEE) in teal and GluA2 in orange, is superimposed to the A2iQ/γ2 wild-type complex in yellow (PDB:6dlz, 3.9 Å resolution). The root mean square deviation (RMSD) of the Cα is 1.909 Å. Top view (A) and cross section side view (B). H. The pore (that is, M2, SF, and M3) of Open-Na260 at 2.3 Å resolution is superimposed to the A2iQ/γ2 wild-type complex in yellow (PDB:5vot, 4.9 Å resolution). The RMSD of Cα of the M3 in B/D subunit pairs is 0.642 Å. I. Conformational difference between Closed-CaNaMg and Open-CaNaMg. The two structures were aligned at the cytoplasmic half of M3 (residue 597-610) at RMSD = 0.3 Å. GluA2 are colored in ogrange and light orange in Open-CaNaMg and Closed-CaNaMg, respectively. The four γ2(KKEE)s of Open-CaNaMg are colored in teal, and in Closed-CaNaMg they are in light green. The position of the γ2(KKEE)s relative to the GluA2 are indicated by the A’-D’ labels, as defined in Fig. 1B. The γ2(KKEE)s undergo counter rotations of 2-3o within the A’/B’ and C’/D’ pairs (curved arrows). The eye and arrow indicate the viewpoint of the structures in J-K. J. Closed-CaNaMg without the mode vector arrows. K. Closed-CaNaMg with the mode vector arrows that describe the directions and magnitudes of displacement of the Cα between Closed-CaNaMg and Open-CaNaMg. Arrows are shown for all motion greater than 0.5 Å. The counter rotation of the γ2(KKEE)s in the A’/B’ pairs are shown. L. Superposition of the Open-CaNaMg and Closed-CaNaMg shows the ~2 Å outward motion of the extracellular domain of the TARPs. M and N. The activation is also accompanied by a 2-3o tilt of the TARPs, which brings close the cytoplasmic extension of TM4 of γ2(KKEE) to the base of M1-M2 loop of GluA2. In the magnified view (I), the difference in the tilt angle in TM1 and TM4 of γ2(KKEE) is shown.

Extended Data Fig. 6 Comparison of cryo-EM maps and models in the pores in the cross-section containing the A/C subunits.

Cross sections containing A/C subunits of the pores of Open-CaNaMg (A), Open-Ca10 (B), Open-Ca150 (C), Open-Na110 (D), Open-Na260 (E), Open-Na610 (F), Open-CaNaMg/N619K (G), and Closed-CaNaMg (H) are displayed. The cryo-EM density map (mesh) and the atomic model are superimposed. The cryo-EM maps are displayed using the indicated sigma values. Putative water and calcium ion are in red and green spheres, respectively. The atomic models of the polypeptides of M2, SF, and M3 are shown in yellow, orange, and sky blue, where elements are colored according to oxygen=red, nitrogen=blue, and sulfate=yellow. Resides that define the gate in the M3 of each structure are colored magenta. The left bottom inset shows the locations of the key residues in the wild type open pore. C2 maps are displayed (also see Table 1).

Extended Data Fig. 7 Comparison of cryo-EM maps and models in the pores in the cross-section containing the B/D subunits.

Cross sections containing B/D subunits of the pores of Open-CaNaMg (A), Open-Ca10 (B), Open-Ca150 (C), Open-Na110 (D), Open-Na260 (E), Open-Na610 (F), Open-CaNaMg/N619K (G), and Closed-CaNaMg (H) are displayed. The cryo-EM density map (mesh) and the atomic model are superimposed. The cryo-EM maps are displayed using the indicated sigma values. Putative water and calcium ion are in red and green spheres, respectively. The atomic models of the polypeptides of M2, SF, and M3 are shown in yellow, orange, and sky blue, where elements are colored according to oxygen=red, nitrogen=blue, and sulfate=yellow. Resides that define the gate in the M3 of each structure are colored magenta. The left bottom inset shows the locations of the key residues in the wild type open pore. Each view is the orthogonal to the corresponding structure shown in the previous figure. C2 maps are displayed (also see Table 1).

Extended Data Fig. 8 Putative water in the vestibules that are common in Open-Na260 and Open-Na110.

The horizontal sections through the upper (A) and lateral vestibule (B-D). The sections were made at different levels that contain residue S614 (B), G588 (B), C589 (C), and D590 (D) of the SF. In each row, Open-Na260 (left), Open-Na110 (middle), and superimposed models of two structures are displayed. Blue and red mesh are density map of polypeptides and putative water, respectively. The M3 (blue model), M2 with M2-M3 linker (yellow model), and SF (orange model) are shown. Cyan and red spheres are putative water in Open-Na260 and Open-Na110, respectively. The location of putative water is nearly identical in both structures, as indicated by dotted ovals surrounding the cyan and red water pairs in the right panels. The water surrounded by the red dashed oval are the ones in the lateral vestibules. Cyan-red sphere pair distances were below 1 Å in most cases. In A (right) the actual distances are provided next to the oval for subset of cyan-red sphere pairs. The densities in the lateral vestibule interpreted as water, described above, are unlikely to be cation because these is always a nearby -NH group within 3 Å distance.

Extended Data Fig. 9 Solvent accessible pore radius.

The pore radius accessible to solvent was estimated using CHAP software64. A. (Left) The surface representation of the accessible pore is shown with the ribbon diagram of Open-Na260. GluA2: light gray. TARPγ-2: dark gray. Pore facing and pore lining residues are in yellow and orange, respectively. The darkness of orange surface corelates with narrower radius of the pore. The value of s is the coordinate along the pore pathway, whose origin is set at the center of mass of the pore forming residues. The relations between the key pore residues and the value of s (in the unit of nm) are shown. (right) Radius vs. s plot. The location of the site-G plus N619 (or N619K) and SF are in blue and green, respectively. The dashed line indicates 1.4 Å. The radius of closed pore (Closed-CaNaMg) plotted in B crosses below the 1.4 Å radius but the open pores don’t. Radius vs. s plots are shown for Open-Na110 (C), Open-Na610 (D), Open-CaNaMg/N619K (E), Open-CaNaMg (F), Open-Ca10 (G), and Open-Ca150 (H).

Source data

Extended Data Fig. 10 Characterization of A2iQ(N619K)/γ2 complex.

A-C. Averaged ramp currents (I-V) recorded in excised patches expressing wild-type A2iQ/γ2 prior to agonist stimulation (A, black), N619K/γ2 leak (B, blue) and agonist-evoked (C, dark cyan) responses using an internal patch solution containing 30 µM spermine. Dim colours show the SEM. The I-V plots both show bi-rectifying I-V relationships demonstrating that cytoplasmic spermine blocks the leak (B) and agonist-gated state (C) of the mutant A2iQ(N619K)/γ2 receptors. Data are presented as mean values ± SEM. (also see Supplementary Table 4). D-F. Conductance-voltage (G-V) plots converted from A-C for wild-type leak (D, black), N619K/γ2 leak (E, blue) and agonist-evoked (F, dark cyan) responses. In dim colours it is shown the SEM of the averaged conductance curves, respectively. Red lines in E and F are the fit for the G-V relationships for leak and agonist-evoked responses of N619K/γ2 receptors, respectively, using the single permeant blocker model (Eq. 1). Data are presented as mean values ± SEM. (Also see Supplementary Table 7) G and H. Fast jumps experiments comparing the ability of the AMPAR negative allosteric modulator, GYKI 52466 (100 µM) (G, Patch # 221129p3) or the competitive antagonist, NBQX (100 µM) (H, Patch # 221129p3) to block the leak currents mediated by N619K/γ2 receptors. The onset and off kinetics of GYKI (G) were estimated as τon = 12.7 ± 0.9 ms (n = 10) and τoff = 37.1 ± 3.9 ms (n = 10), respectively (also see Supplementary Table 5 and 6). I. I-V plot of the leak current of N619K/γ2 receptors with an internal patch solution containing 30 µM spermine in the absence of agonist. J. Typical I-V plots of leak currents of N619K/γ2 receptors before (grey) and after (pink) applying 100 µM GYKI (Patch # 221201p4), in the absence of agonist and containing 30 µM spermine in the internal solution. K and L. Conductance-voltage (G-V) plots of the leak N619K/γ2 receptors, converted from J (Patch # 221201p4), before (K, black) and after (L, pink) applying 100 µM GYKI. The G-V curves are fit (red in K and L) using a single permeant ion blocker model (Eq. 1). L. (inset) Normalized G-V plots before (black) and after (pink) the application of GYKI are superimposed.

Source data

Supplementary information

Source data

Source Data Fig. 3

Source data of electrophysiology in Fig. 3.

Source Data Fig. 5

Source data of electrophysiology in Fig. 5.

Source Data Fig. 6

Source data of electrophysiology in Fig. 6.

Source Data Extended Data Fig. 1

Chromatograph of Extended Data Fig. 1c.

Source Data Extended Data Fig. 1

Uncropped gel image of Extended Data Fig. 1b.

Source Data Extended Data Fig. 9

Source data of pore diameter plot in Extended Data Fig. 9a–h.

Source Data Extended Data Fig. 10

Source data of electrophysiology in Extended Data Fig. 10.

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Nakagawa, T., Wang, Xt., Miguez-Cabello, F.J. et al. The open gate of the AMPA receptor forms a Ca2+ binding site critical in regulating ion transport. Nat Struct Mol Biol 31, 688–700 (2024). https://doi.org/10.1038/s41594-024-01228-3

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