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GluA2-containing AMPA receptors form a continuum of Ca2+-permeable channels

Nature volume 641, pages 537–544 (2025)Cite this article

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Abstract

Fast excitatory neurotransmission in the mammalian brain is mediated by cation-selective AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors (AMPARs)1. AMPARs are critical for the learning and memory mechanisms of Hebbian plasticity2 and glutamatergic synapse homeostasis3, with recent work establishing that AMPAR missense mutations can cause autism and intellectual disability4,5,6,7. AMPARs have been grouped into two functionally distinct tetrameric assemblies based on the inclusion or exclusion of the GluA2 subunit that determines Ca2+ permeability through RNA editing8,9. GluA2-containing AMPARs are the most abundant in the central nervous system and considered to be Ca2+ impermeable10. Here we show this is not the case. Contrary to conventional understanding, GluA2-containing AMPARs form a continuum of polyamine-insensitive ion channels with varying degrees of Ca2+ permeability. Their ability to transport Ca2+ is shaped by the subunit composition of AMPAR tetramers as well as the spatial orientation of transmembrane AMPAR regulatory proteins and cornichon auxiliary subunits. Ca2+ crosses the ion-conduction pathway by docking to an extracellular binding site that helps funnel divalent ions into the pore selectivity filter. The dynamic range in Ca2+ permeability, however, arises because auxiliary subunits primarily modify the selectivity filter. Taken together, our work proposes a broader role for AMPARs in Ca2+ signalling in the mammalian brain and offers mechanistic insight into the pathogenic nature of missense mutations.

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Fig. 1: TARP γ2 regulates Ca2+ permeability of GluA2-containing AMPARs.
Fig. 2: CNIHs further regulate Ca2+ permeability of GluA2-containing AMPARs.
Fig. 3: GluA2-containing AMPARs form a continuum of Ca2+ permeable channels.
Fig. 4: Block of GluA2-containing AMPARs by external Ca2+.
Fig. 5: Permeation and block of GluA2-containing AMPARs by external Ca2+.

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Data availability

Data supporting the findings of this study are available upon request. The referred protein structure has the Protein Data bank (PDB) accession code 7OCA. Supplementary Information is available for this paper. The data obtained to create Extended Data Fig. 1c have been taken from refs. 16,31,34. Source data are provided with this paper.

Code availability

Code used in the current study is publicly available on GitHub (https://github.com/niklasbrake/AMPAR_permeation_modelling).

References

  1. Hansen, K. B. et al. Structure, function, and pharmacology of glutamate receptor ion channels. Pharmacol. Rev. 73, 298–487 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  2. Nicoll, R. A. A brief history of long-term potentiation. Neuron 93, 281–290 (2017).

    Article  CAS  PubMed  Google Scholar 

  3. Turrigiano, G. G. The self-tuning neuron: synaptic scaling of excitatory synapses. Cell 135, 422–435 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Salpietro, V. et al. AMPA receptor GluA2 subunit defects are a cause of neurodevelopmental disorders. Nat. Commun. 10, 3094 (2019).

    Article  PubMed  PubMed Central  ADS  Google Scholar 

  5. Martin, S. et al. De novo variants in GRIA4 lead to intellectual disability with or without seizures and gait abnormalities. Am. J. Hum. Genet. 101, 1013–1020 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Wu, Y. et al. Mutations in ionotropic AMPA receptor 3 alter channel properties and are associated with moderate cognitive impairment in humans. Proc. Natl Acad. Sci. USA 104, 18163–18168 (2007).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  7. Peall, K. J., Owen, M. J. & Hall, J. Rare genetic brain disorders with overlapping neurological and psychiatric phenotypes. Nat. Rev. Neurol. 20, 7–21 (2024).

    Article  CAS  PubMed  Google Scholar 

  8. Sommer, B., Kohler, 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).

    Article  CAS  PubMed  Google Scholar 

  9. Burnashev, N., Monyer, H., Seeburg, P. H. & Sakmann, B. Divalent ion permeability of AMPA receptor channels is dominated by the edited form of a single subunit. Neuron 8, 189–198 (1992).

    Article  CAS  PubMed  Google Scholar 

  10. Bowie, D. Polyamine-mediated channel block of ionotropic glutamate receptors and its regulation by auxiliary proteins. J. Biol. Chem. 293, 18789–18802 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Herring, B. E. & Nicoll, R. A. Long-term potentiation: from CaMKII to AMPA receptor trafficking. Annu. Rev. Physiol. 78, 351–365 (2016).

    Article  CAS  PubMed  Google Scholar 

  12. Turrigiano, G. G. The dialectic of Hebb and homeostasis. Philos. Trans. R. Soc. Lond. B Biol. Sci. 372, 20160258 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Bowie, D. Redefining the classification of AMPA-selective ionotropic glutamate receptors. J. Physiol. 590, 49–61 (2012).

    Article  CAS  PubMed  Google Scholar 

  14. Jonas, P. The time course of signaling at central glutamatergic synapses. News Physiol. Sci. 15, 83–89 (2000).

    CAS  PubMed  ADS  Google Scholar 

  15. Jonas, P., Racca, C., Sakmann, B., Seeburg, P. H. & Monyer, H. Differences in Ca2+ permeability of AMPA-type glutamate receptor channels in neocortical neurons caused by differential GluR-B subunit expression. Neuron 12, 1281–1289 (1994).

    Article  CAS  PubMed  Google Scholar 

  16. Geiger, J. R. et al. Relative abundance of subunit mRNAs determines gating and Ca2+ permeability of AMPA receptors in principal neurons and interneurons in rat CNS. Neuron 15, 193–204 (1995).

    Article  CAS  PubMed  Google Scholar 

  17. Mosbacher, J. et al. A molecular determinant for submillisecond desensitization in glutamate receptors. Science 266, 1059–1062 (1994).

    Article  CAS  PubMed  ADS  Google Scholar 

  18. Burnashev, N. et al. Calcium-permeable AMPA-kainate receptors in fusiform cerebellar glial cells. Science 256, 1566–1570 (1992).

    Article  CAS  PubMed  ADS  Google Scholar 

  19. Blaschke, M. et al. A single amino acid determines the subunit-specific spider toxin block of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor channels. Proc. Natl Acad. Sci. USA 90, 6528–6532 (1993).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  20. Herlitze, S. et al. Argiotoxin detects molecular differences in AMPA receptor channels. Neuron 10, 1131–1140 (1993).

    Article  CAS  PubMed  Google Scholar 

  21. Mahanty, N. K. & Sah, P. Calcium-permeable AMPA receptors mediate long-term potentiation in interneurons in the amygdala. Nature 394, 683–687 (1998).

    Article  CAS  PubMed  ADS  Google Scholar 

  22. Liu, S. Q. & Cull-Candy, S. G. Synaptic activity at calcium-permeable AMPA receptors induces a switch in receptor subtype. Nature 405, 454–458 (2000).

    Article  CAS  PubMed  ADS  Google Scholar 

  23. Plant, K. et al. Transient incorporation of native GluR2-lacking AMPA receptors during hippocampal long-term potentiation. Nat. Neurosci. 9, 602–604 (2006).

    Article  CAS  PubMed  Google Scholar 

  24. Granzotto, A., Weiss, J. H. & Sensi, S. L. Editorial: excitotoxicity turns 50. The death that never dies. Front Neurosci 15, 831809 (2021).

    Article  PubMed  Google Scholar 

  25. Kwak, S. & Weiss, J. H. Calcium-permeable AMPA channels in neurodegenerative disease and ischemia. Curr. Opin. Neurobiol. 16, 281–287 (2006).

    Article  CAS  PubMed  Google Scholar 

  26. Weiss, J. H. & Sensi, S. L. Ca2+-Zn2+ permeable AMPA or kainate receptors: possible key factors in selective neurodegeneration. Trends Neurosci. 23, 365–371 (2000).

    Article  CAS  PubMed  Google Scholar 

  27. Cueva Vargas, J. L. et al. Soluble tumor necrosis factor alpha promotes retinal ganglion cell death in glaucoma via calcium-permeable AMPA receptor activation. J. Neurosci. 35, 12088–12102 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  28. Schwenk, J. et al. High-resolution proteomics unravel architecture and molecular diversity of native AMPA receptor complexes. Neuron 74, 621–633 (2012).

    Article  CAS  PubMed  Google Scholar 

  29. Schwenk, J. et al. Functional proteomics identify cornichon proteins as auxiliary subunits of AMPA receptors. Science 323, 1313–1319 (2009).

    Article  CAS  PubMed  ADS  Google Scholar 

  30. Perozzo, A. M., Brown, P. & Bowie, D. Alternative splicing of the flip/flop cassette and TARP auxiliary subunits engage in a privileged relationship that fine-tunes AMPA receptor gating. J. Neurosci. 43, 2837–2849 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Dawe, G. B. et al. Nanoscale mobility of the apo state and TARP stoichiometry dictate the gating behavior of alternatively spliced AMPA receptors. Neuron 102, 976–992 e975 (2019).

    Article  CAS  PubMed  Google Scholar 

  32. Zhao, Y., Chen, S., Swensen, A. C., Qian, W. J. & Gouaux, E. Architecture and subunit arrangement of native AMPA receptors elucidated by cryo-EM. Science 364, 355–362 (2019).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  33. Twomey, E. C., Yelshanskaya, M. V., Grassucci, R. A., Frank, J. & Sobolevsky, A. I. Elucidation of AMPA receptor-stargazin complexes by cryo-electron microscopy. Science 353, 83–86 (2016).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  34. Perozzo, A. M. et al. GSG1L-containing AMPA receptor complexes are defined by their spatiotemporal expression, native interactome and allosteric sites. Nat. Commun. 14, 6799 (2023).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  35. Twomey, E. C., Yelshanskaya, M. V., Grassucci, R. A., Frank, J. & Sobolevsky, A. I. Structural bases of desensitization in AMPA receptor-auxiliary subunit complexes. Neuron 94, 569–580 e565 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Zhang, D., Watson, J. F., Matthews, P. M., Cais, O. & Greger, I. H. Gating and modulation of a hetero-octameric AMPA glutamate receptor. Nature 594, 454–458 (2021).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  37. Nakagawa, T. Structures of the AMPA receptor in complex with its auxiliary subunit cornichon. Science 366, 1259–1263 (2019).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  38. Matt, L. et al. SynDIG4/Prrt1 is required for excitatory synapse development and plasticity underlying cognitive function. Cell Rep. 22, 2246–2253 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Diaz, E. Beyond the AMPA receptor: diverse roles of SynDIG/PRRT brain-specific transmembrane proteins at excitatory synapses. Curr. Opin. Pharmacol. 58, 76–82 (2021).

    Article  Google Scholar 

  40. Yu, J. et al. Hippocampal AMPA receptor assemblies and mechanism of allosteric inhibition. Nature 594, 448–453 (2021).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  41. Boudkkazi, S., Brechet, A., Schwenk, J. & Fakler, B. Cornichon2 dictates the time course of excitatory transmission at individual hippocampal synapses. Neuron 82, 848–858 (2014).

    Article  CAS  PubMed  Google Scholar 

  42. von Engelhardt, J. et al. CKAMP44: a brain-specific protein attenuating short-term synaptic plasticity in the dentate gyrus. Science 327, 1518–1522 (2010).

    Article  ADS  Google Scholar 

  43. Nakagawa, T., Wang, X. T., Miguez-Cabello, F. J. & Bowie, D. The open gate of the AMPA receptor forms a Ca(2+) binding site critical in regulating ion transport. Nat. Struct. Mol. Biol. 31, 688–700 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Tempia, F. et al. Fractional calcium current through neuronal AMPA-receptor channels with a low calcium permeability. J. Neurosci. 16, 456–466 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Gangwar, S. P. et al. Modulation of GluA2-gamma5 synaptic complex desensitization, polyamine block and antiepileptic perampanel inhibition by auxiliary subunit cornichon-2. Nat. Struct. Mol. Biol. 30, 1481–1494 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Hawken, N. M., Zaika, E. I. & Nakagawa, T. Engineering defined membrane-embedded elements of AMPA receptor induces opposing gating modulation by cornichon 3 and stargazin. J. Physiol. 595, 6517–6539 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Dingledine, R., Hume, R. I. & Heinemann, S. F. Structural determinants of barium permeation and rectification in non-NMDA glutamate receptor channels. J. Neurosci. 12, 4080–4087 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Brown, P., McGuire, H. & Bowie, D. Stargazin and cornichon-3 relieve polyamine block of AMPA receptors by enhancing blocker permeation. J. Gen. Physiol. 150, 67–82 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Ohkura, M. et al. Genetically encoded green fluorescent Ca2+ indicators with improved detectability for neuronal Ca2+ signals. PLoS ONE 7, e51286 (2012).

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  50. Bowie, D., Lange, G. D. & Mayer, M. L. Activity-dependent modulation of glutamate receptors by polyamines. J. Neurosci. 18, 8175–8185 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Isaac, J. T., Ashby, M. C. & McBain, C. J. The role of the GluR2 subunit in AMPA receptor function and synaptic plasticity. Neuron 54, 859–871 (2007).

    Article  CAS  PubMed  Google Scholar 

  52. Osswald, I. K., Galan, A. & Bowie, D. Light triggers expression of philanthotoxin-insensitive Ca2+-permeable AMPA receptors in the developing rat retina. J. Physiol. 582, 95–111 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Mattison, H. A. et al. Evidence of calcium-permeable AMPA receptors in dendritic spines of CA1 pyramidal neurons. J. Neurophysiol. 112, 263–275 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Schwenk, J. et al. Regional diversity and developmental dynamics of the AMPA-receptor proteome in the mammalian brain. Neuron 84, 41–54 (2014).

    Article  CAS  PubMed  Google Scholar 

  55. XiangWei, W. et al. Clinical and functional consequences of GRIA variants in patients with neurological diseases. Cell. Mol. Life Sci. 80, 345 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Brown, P. M., Aurousseau, M. R., Musgaard, M., Biggin, P. C. & Bowie, D. Kainate receptor pore-forming and auxiliary subunits regulate channel block by a novel mechanism. J. Physiol. 594, 1821–1840 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Bowie, D., Garcia, E. P., Marshall, J., Traynelis, S. F. & Lange, G. D. Allosteric regulation and spatial distribution of kainate receptors bound to ancillary proteins. J. Physiol. 547, 373–385 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Alexander, R. P. D. & Bowie, D. Intrinsic plasticity of cerebellar stellate cells is mediated by NMDA receptor regulation of voltage-gated Na(+) channels. J. Physiol. 599, 647–665 (2021).

    Article  CAS  PubMed  Google Scholar 

  59. Wong, A. Y., Fay, A. M. & Bowie, D. External ions are coactivators of kainate receptors. J. Neurosci. 26, 5750–5755 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Woodhull, A. M. Ionic blockage of sodium channels in nerve. J. Gen. Physiol. 61, 687–708 (1973).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Konishi, S. & Kitagawa, G. Information Criteria and Statistical Modeling (Springer, 2008).

  62. Efron, B. & Tibshirani, R. An Introduction to the Bootstrap (Chapman and Hall, 1993).

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Acknowledgements

We thank former Bowie laboratory members, D. MacLean (University of Rochester), P. Brown (University of Cologne), M. Aurousseau and H. McGuire for their preliminary experiments on this project and current laboratory members for their feedback on the manuscript, especially C. Koens. We also thank T. Nakagawa (Vanderbilt) for fruitful and interesting discussions about Ca2+ permeation and I. Greger (LMB) for providing the tethered TARP-γ8 complementary DNA. We thank S. Bosse (Delta Phototonics) for his technical support with the imaging system. The work was supported by finance from the Canadian Institutes of Health Research (grant on. FRN 163317, D.B.), and from the Natural Sciences and Engineering Research Council of Canada (Discovery Grant, A.K.). A.M.P and R.P.D.A. were supported by the Natural Sciences and Engineering Research Council of Canada. Y.Y. was supported by Fonds de recherche du Québec-Santé (FRQS). N.B. was supported by Fonds de recherche du Québec-Nature et technologies. X-T.W. was supported by a studentship from the Integrated Program in Neuroscience at McGill University.

Author information

Author notes

  1. These authors contributed equally: Federico Miguez-Cabello, Xin-tong Wang, Yuhao Yan

Authors and Affiliations

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

    Federico Miguez-Cabello, Xin-tong Wang, Yuhao Yan, Ryan P. D. Alexander & Amanda M. Perozzo

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

    Xin-tong Wang, Yuhao Yan, Ryan P. D. Alexander, Amanda M. Perozzo & Derek Bowie

  3. Department of Physiology, McGill University, Montreal, Quebec, Canada

    Niklas Brake & Anmar Khadra

  4. Quantitative Life Sciences PhD program, McGill University, Montreal, Quebec, Canada

    Niklas Brake

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  1. Federico Miguez-Cabello
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  4. Niklas Brake
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Contributions

F.M.-C., X.-t.W. and Y.Y. conducted recombinant electrophysiology experiments and analysed data and contributed equally. N.B. contributed to the computational modelling. R.P.D.A. conducted the native electrophysiology experiments and analysed data. A.M.P. generated figures, edited the manuscript and was involved in the discussions. A.K. supervised and provided insights into the computational modelling. D.B. conceived the project, supervised all the experiments, wrote the manuscript and provided financial support.

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Correspondence to Derek Bowie.

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Extended data figures and tables

Extended Data Fig. 1 AMPARs with tethered TARP auxiliary subunits faithfully reproduce the gating behaviour of native AMPARs.

a. (Upper) Example electrophysiological records of L-Glu responses (10 mM) of GluA1/A2 AMPAR heteromers co-expressed with (left) or tethered to (right) TARP γ2. (Lower) Statistical analyses of the degree of AMPAR desensitization (left, SS/Pk) or rate of onset of desensitization (right, τfast) reveals that the gating properties are indistinguishable when co-expressed with or tethered to TARP γ2. (Adapted from Perozzo et al., 2023). b. Plots comparing the gating properties of recombinant A1/A2 heteromers (orange symbol) and native (black symbol) AMPARs expressed by inhibitory stellate (upper) and Purkinje (lower) cells from the cerebellum. In both cases, recombinant AMPARs were expressed as fusion proteins with TARP γ2 tethered to either GluA1 or GluA2 subunit (upper) or to both (lower). Importantly, the desensitization properties of recombinant receptor exactly matched with functional behavior of stellate cells when only two TARP γ2 subunits were present (upper) whereas the Purkinje cell data was exactly matched by recombinant data where all four AMPAR subunits were tethered to TARP γ2. (Adapted from Perozzo et al., 2023). c. Desensitization rates (τdes) of native and recombinant AMPARs arranged in ascending order shows that they form a continuum of functional behavior. Values for native data were taken from Geiger et al.16 and Dawe et al.31 and the values for the recombinant were taken from this study. The black bars correspond to AMPAR heteromers that are tethered to at least two TARP subunits whereas the unfilled bar corresponds to GluA1/A2 heteromers lacking TARPs.

Source data

Extended Data Fig. 2 TARPs promote Ca2+ permeability of GluA2-containing AMPARs while remaining polyamine insensitive.

a. Different AMPAR-TARP complexes arranged according to the reversal potential observed in 108 mM external Ca2+ solution (A1/A2: ErevCa2+ = −44.8 ± 2.8 mV, n = 6; A1γ8/A2: ErevCa2+ = −29.7 ± 1.8 mV, n = 5; A1/A2γ8: ErevCa2+ = −25.6 ± 2.9 mV, n = 5; A2γ2/A3γ2: ErevCa2+ = −31.4 ± 2.3 mV, n = 11; A1γ2/A2: ErevCa2+ = −31.5 ± 1.2 mV, n = 5; A1/A2γ2: ErevCa2+ = −7.2 ± 1.8 mV, n = 16; A1γ2/A2γ2: ErevCa2+ = −27.0 ± 1.8 mV, n = 6). b. Pooled data of PCa/PNa of AMPAR-TARP complexes co-assembled with TARP γ2 or γ 8 (A1/A2 PCa/PNa = 0.06 ± 0.01, n = 6; A1γ8/A2 PCa/PNa = 0.12 ± 0.01, n = 5; A1/A2γ8 PCa/PNa = 0.18 ± 0.03, n = 5; A2γ2/A3γ2 PCa/PNa = 0.12 ± 0.01, n = 11; A1γ2/A2 PCa/PNa = 0.12 ± 0.01, n = 5; A1/A2γ2 PCa/PNa = 0.43 ± 0.04, n = 16; A1γ2/A2γ2 PCa/PNa = 0.14 ± 0.01, n = 6). Two-sided Kruskal-Wallis ANOVA followed by Mann-Whitney U tests with Bonferroni-Holmes correction. p-value * <0.05, ** <0.01, *** <0.001. c. IV plots of different AMPAR-TARP complexes in external 150 mM Na+ (open circles fitted by a 4th order polynomial function) solution. d. GV plots of AMPAR-TARP complexes obtained from IV curves above (c). Open circles show average normalized response and colored shading show s.e.m. (A1/A2: n = 6; A1γ2/A2: n = 5; A1/A2γ2: n = 16; A1γ2/A2γ2: n = 6).

Source data

Extended Data Fig. 3 Simulations of current-voltage (IV) and conductance-voltage (GV) plots establishes that data from edited GluA2-containing AMPARs is not impacted by unedited AMPARs.

a. GV plots of unedited GluA2(Q) AMPARs showing that tethering TARP γ2 significantly attenuates the degree of block by cytoplasmic spermine (30 μM). Adapted from the data shown in Supplementary Table 3. b. A series of GV plots where the contribution (%) of unedited AMPARs to the overall AMPAR response is reduced in a stepwise manner. Note that shape of the GV plot with only 1 % contribution of unedited AMPARs best fits the data we have observed with GluA1/A2 heteromers. c. A series of Ca2+ IV plots where the contribution (%) of unedited AMPARs to the overall AMPAR response was adjusted in an incremental manner. Note, that at least 50% of unedited AMPARs would be needed for the reversal potential to be in the positive voltage range. d. The relative Ca2+ permeability of AMPARs estimated from the simulated Ca2+ IV plot data shown in c. Note, that for the relative Ca2+ permeability to be close to 1 (dotted line), as described for GluA1/A2 heteromers with TARP γ2 and CNIH3, almost 75 % of the overall response would need to contain unedited AMPARs.

Source data

Extended Data Fig. 4 Current-voltage (IV) and conductance-voltage (GV) plots confirm that GluA2-containing AMPARs are polyamine insensitive and kinetic properties corroborate CNIH presence in the AMPAR complex.

a. IV plots of different AMPAR-TARP + CNIH complexes in external 150 mM Na+ (open circles fitted by a 4th order polynomial function) solution. b. GV plots of the AMPAR-TARP complexes obtained from IV curves above (a). Open circles show average normalized response and colored shading show se.m. (A1γ2/A2 + CNIH-2: n = 9; A1/A2γ2 + CNIH-2: n = 8; A1γ2/A2 + CNIH-3: n = 6; A1/A2γ2 + CNIH-3: n = 9). c. Example membrane currents evoked by 10 mM L-Glu (250 ms duration) on GluA1/A2 AMPARs co-assembled with either TARP γ2 and CNIH2 or TARP γ2 and CNH3 (c). The grey shadow shows the SEM of the response. d. Comparison of gating properties between A1/A2γ2 (in grey) vs A1/A2γ2 + CNIH-3 (black) receptors. e. CNIH-3 slows desensitization kinetics of A1/A2γ2 receptors (A1/A2γ2: τdes = 7.5 ± 0.6 ms, n = 16; A1/A2γ2 + CNIH-3: τdes = 22.5 ± 1.7 ms, n = 9). Equilibrium current was enhanced by CNIH-3 (A1/A2γ2: Equilibrium current = 2.9 ± 0.4 %, n = 16; A1/A2γ2 + CNIH-3: Equilibrium current = 14.7 ± 1.3 %, n = 9). CNIH-3 also slowed the off-kinetics of AMPARs (A1/A2γ2: τoff = 6.8 ± 0.5 ms, n = 19; A1/A2γ2 + CNIH-3: τoff = 23.5 ± 1.5 ms, n = 14) and the deactivation kinetics (A1/A2γ2: τdeact = 1.5 ± 0.1 ms, n = 6; A1/A2γ2 + CNIH-3: τdeact = 7.0 ± 0.8 ms, n = 9). Two-sided unpaired t-tests with Welch correction. p-value *** <0.001. f. AMPAR-TARP + CNIH complexes arranged by the reversal potential observed in 108 mM external Ca2+ solution (A1/A2: ErevCa2+ = −44.8 ± 2.8 mV, n = 6; A1γ2/A2 + CNIH-3: ErevCa2+ = −33.6 ± 1.7 mV, n = 6; A1γ2/A2 + CNIH-2: ErevCa2+ = −25.8 ± 2.2 mV, n = 9; A2γ2/A3 + CNIH-3: ErevCa2+ = −29.7 ± 2.2 mV, n = 5; A1γ8/A2 + CNIH-3: ErevCa2+ = −31.2 ± 2.7 mV, n = 5; A1/A2γ8 + CNIH-3: ErevCa2+ = −18.9 ± 3.4 mV, n = 6). Orange square shows the A1/A2γ2 AMPAR to highlight changes in the Ca2+ reversal potential induced by CNIH-2 and -3 auxiliary proteins (cyan squares, A1/A2γ2 + CNIH-2: ErevCa2+ = −1.8 ± 2.5 mV, n = 8; A1/A2γ2 + CNIH-3: ErevCa2+ = 7.5 ± 1.1 mV, n = 9). Sky-blue circle denotes A1/A2γ2 + CNIH-3 with the A789F mutation which left-shifts the Ca2+ reversal potential (A1AF/A2γ2 + CNIH-3: ErevCa2+ = −25.5 ± 3.1 mV, n = 8). g. Pooled data of PCa/PNa from the AMPAR complexes shown in (f) grouped by CNIH and TARP type (A1γ2/A2 + CNIH-2: PCa/PNa = 0.15 ± 0.02, n = 9; A1/A2γ2 + CNIH-2: PCa/PNa = 0.55 ± 0.11, n = 8; A1γ2/A2 + CNIH-3: PCa/PNa = 0.10 ± 0.01, n = 6; A1/A2γ2 + CNIH-3: PCa/PNa = 0.95 ± 0.05, n = 9; A1AF/A2γ2 + CNIH-3: PCa/PNa = 0.16 ± 0.02, n = 8; A1γ8/A2 + CNIH-3: PCa/PNa = 0.12 ± 0.02, n = 5; A1/A2γ8 + CNIH-3: PCa/PNa = 0.23 ± 0.04, n = 6; A2γ2/A3 + CNIH-3: PCa/PNa = 0.12 ± 0.02, n = 5). Two-sided Kruskal-Wallis ANOVA followed by Mann-Whitney U tests with Bonferroni-Holmes correction. p-value * <0.05, ** <0.01, *** <0.001.

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Extended Data Fig. 5 TARP γ2 increases the Ca2+ permeability of missense mutations.

a. The Ca2+ reversal potential shift with the GluA2 missense mutations (D611N in green, R607G in light blue and R607E in dark blue) in the absence and presence of TARP γ2. b. The Na+-relative Ca2+ permeability of GluA1+ GluA2 missense mutations (D611N in green, R607G in light blue and R607E in dark blue) in the absence and presence of TARP γ2. Two-sided two-way between-subject ANOVA was performed upon the dataset, decomposing with Tukey’s HSD post hoc analysis respectively. #: p < 0.05; ***: p < 0.001. c. IV plots of GluA2-containing AMPARs mutated at the pore region (D611N) and the pore selectivity filter (R607G and R607E). d. GV plots obtained from IV curves above (c). A1 + A2D611N channels exhibit a linear GV relationship while A1 + A2R607G and A1 + A2R607E GV curves show block by internal spermine (A1 + A2R607G Kd(0 mV) = 34 ± 6.5 μM n = 4 and A1 + A2R607E Kd(0 mV) = 4.4 ± 0.9 μM n = 5). Open circles show average normalized response, colored shading show se.m., and the solid red line corresponds to the fit of a single permeant blocker model. e. IV plots of different A1/A2γ2 AMPARs mutated at the +4 site (D611N) and selectivity filter (R607G and R607E). f. GV plots obtained from IV curves above (e). A1/A2γ2D611N channels show a linear GV relationship while A1/A2γ2R607G and A1/A2γ2R607E GV curves show sensitivity to block by internal spermine (A1/A2γ2R607G Kd(0 mV) = 38.2 ± 11.7 μM n = 5 and A1/A2γ2R607E Kd(0 mV) = 14.5 ± 3.4 μM n = 7). Open circles show average normalized response, colored shading show s.e.m., and the solid redline denotes fits using a single permeant blocker model.

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Extended Data Fig. 6 Current-voltage (I-V) plots from whole-cell recording and Linear regression relationships between Ca2+ induced fluorescent intensity (ΔFluorescence) and total charge transfer (Qtotal) of whole-cell current recording.

a-d show the I-V relationships recorded using a RAMP protocol, from −100mV to +60 mV, with whole-cell configuration for GluA1/A2 (black, n = 11), GluA1/A2γ2 (orange, n = 12), GluA1/A2γ2/CNIH-3 (cyan, n = 15) and GluA1/A2R607Eγ2 (dark blue, n = 12), respectively. The shades of each I-V plot indicates the SEM. Noted that GluA1/A2R607Eγ2 receptors are sensitive to internal polyamines with an affinity of Kd(0mV) value of 18.7 ± 3.0 μM. e-h show the linear regression relationships between Ca2+ induced fluorescent intensity (ΔFluorescence) and total charge transfer (Qtotal) of whole-cell current recording for GluA1/A2 (grey, n = 11), GluA1/A2γ2 (orange, n = 12), GluA1/A2γ2/CNIH-3 (cyan, n = 15) and GluA1/A2R607Eγ2 (dark blue, n = 12), respectively. Scatters indicate individual data points, while the solid lines are the linear regression for each group: GluA1/A2: y = 0.0173*x + 65.935, r = 0.56; GluA1/A2γ2: y = 4.8557*x-4068.4, r = 0.94; GluA1/A2γ2/CNIH-3: y = 3.4786*x-566.31, r = 0.87 and GluA1/A2R607Eγ2: y = 7.2528*x, r = 0.88.

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Extended Data Fig. 7 Ca2+ block of GluA2-containing AMPARs at two binding sites.

Fits of two binding site isotherm to Ca2+ inhibition data recorded at −100 mV of AMPARs composed of a. GluA1/A2 (n = 7), b. GluA1/A2-γ2 (n = 16), c. GluA1+CNIH−3/A2-γ2 (n = 9) and d. GluA1/A2-γ2 R607E (n = 8). The solid line corresponds to the sum of the two binding site isotherm and the dotted lines are the individual isotherm fits.

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Extended Data Fig. 8 A two-binding site permeation model best captures voltage-dependent calcium block of AMPARs.

a. Schematic illustrations of the five Ca2+ binding models studied. b. The black dots are the calcium-dependent Gnorm values at −60 mV for each AMPAR (top to bottom). The colored lines are the fits for the five models (left to right). Gnorm is modelled as one minus the probability of calcium binding to site B2 (except model 1, where block is modelled based on calcium binding to site B1). Notice that all the models either overestimate block at low calcium concentration or underestimate block at high calcium concentrations, except for Model 4. c. Bayesian information criterion (BIC) computed for each model and AMPAR. Model 4 exhibited the lowest BIC across all AMPARs, indicating that the model is the best trade-off between high accuracy and low complexity.

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Extended Data Fig. 9 Nested model comparison identifies similarity in voltage sensitivity among AMPARs (see Supplementary Information).

a. Illustration of Model 4.0. The voltage dependences of both sites B1 and B2 are allowed to vary among all the AMPAR complexes. Model 4.0 is identical to Model 4. b. Illustration of Model 4.1. The voltage dependence of site B2 is allowed to vary among all the AMPAR complexes, but the voltage dependence of site B1 (\({\delta }_{1}\)) is identical among all AMPARs. c. Illustration of Model 4.2. The voltage dependence of site B2 (\({\delta }_{3}\)) of A1 + A2R607Eγ2 is allowed to vary, but the other AMPARs have identical voltage dependence. d. Illustration of Model 4.3. The voltage dependence of all AMPARs are identical. e. The number of unique parameters and the residual squared error (RSS) after fitting each of the nested models. f. Results of F-tests for model comparison. Models 4.0, 4.1, and 4.2 all performed significantly better than Model 4.3. However, Models 4.0, 4.1, and 4.2 achieved statistically indistinguishable (p > 0.05) fits to the data when taking the number of model parameters into account.

Extended Data Fig. 10 Calcium block data of A1_A2, A1_A2γ2, and A1_A2γ2 + CIHN3 captured by permeation model (Model 4.2) with identical voltage dependences.

a. Black dots indicate normalized AMPAR conductance at various calcium concentrations for four different AMPAR complexes (top to bottom) and various voltages (left to right). The red lines show the fit of Model 4.2. Shading indicates 95% confidence interval of model fit (bootstrap, n = 100). b. Optimal parameter values for the voltage dependence of site B1 (\({\delta }_{1}\)) and site B2 (\({\delta }_{3}\)) after repeatedly fitting Model 4.2 to 100 bootstrap samples of the data. The voltage dependence of B2 was allowed to differ for GluA1+GluA2R607Eγ2. Notice the second peak in density for the voltage dependence of binding site B2, indicating that in the mutant receptor, binding site B2 sits more shallowly in the membrane electric field compared to the other AMPARs studied. c. Dissociation constants of each of the four transitions in Model 4.2 after fitting to each of the four AMPAR complexes. Vertical lines reflect 95% confidence interval (bootstrap, n = 100). d. Dissociation constants of calcium binding, same as panel c, now plotted against the calcium permeability of each AMPAR. The dissociation constants are positively associated with calcium permeability for CaO↔B1 (ρ = 0.95, spearman correlation, p < 10-9), CaO↔B2 (ρ = 0.96, p < 10-9), but not for Cai↔B2 (ρ = 0.08, p = 0.09).

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Miguez-Cabello, F., Wang, Xt., Yan, Y. et al. GluA2-containing AMPA receptors form a continuum of Ca2+-permeable channels. Nature 641, 537–544 (2025). https://doi.org/10.1038/s41586-025-08736-2

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