N-Methyl-d-aspartate receptors (NMDARs) play a crucial role in excitatory neurotransmission, with numerous pathogenic variants identified in the GluN subunits, including their ligand-binding domains (LBDs). The prevailing hypothesis postulates that the endoplasmic reticulum (ER) quality control machinery verifies the agonist occupancy of NMDARs, but this was tested in a limited number of studies. Using microscopy and electrophysiology in the human embryonic kidney 293 (HEK293) cells, we found that surface expression of GluN1/GluN2A receptors containing a set of alanine substitutions within the LBDs correlated with the measured EC50 values for glycine (GluN1 subunit mutations) while not correlating with the measured EC50 values for l-glutamate (GluN2A subunit mutations). The mutant cycle of GluN1-S688 residue, including the pathogenic GluN1-S688Y and GluN1-S688P variants, showed a correlation between relative surface expression of the GluN1/GluN2A receptors and the measured EC50 values for glycine, as well as with the calculated ΔG binding values for glycine obtained from molecular dynamics simulations. In contrast, the mutant cycle of GluN2A-S511 residue did not show any correlation between the relative surface expression of the GluN1/GluN2A receptors and the measured EC50 values for l-glutamate or calculated ΔG binding values for l-glutamate. Coexpression of both mutated GluN1 and GluN2A subunits led to additive or synergistic alterations in the surface number of GluN1/GluN2A receptors. The synchronized ER release by ARIAD technology confirmed the altered early trafficking of GluN1/GluN2A receptors containing the mutated LBDs. The microscopical analysis from embryonal rat hippocampal neurons (both sexes) corroborated our conclusions from the HEK293 cells.

Biomedical Research Center University Hospital Hradec Kralove Sokolska 581 Hradec Kralove 500 05 Czech Republic

Department of Neurochemistry Institute of Experimental Medicine of the Czech Academy of Sciences Prague 14220 Czech Republic

Department of Physiology Faculty of Science Charles University Prague Prague 12843 Czech Republic

IT4Innovations VSB Technical University of Ostrava Ostrava Poruba 708 00 Czech Republic

Regional Center of Advanced Technologies and Materials Czech Advanced Technology and Research Institute Palacky University Olomouc Olomouc 779 00 Czech Republic

Zobrazit více v PubMed

Anandakrishnan R, Aguilar B, Onufriev AV (2012) H++ 3.0: automating pK prediction and the preparation of biomolecular structures for atomistic molecular modeling and simulations. Nucleic Acids Res 40:W537–W541. 10.1093/nar/gks375 PubMed DOI PMC

Anson LC, Chen PE, Wyllie DJA, Colquhoun D, Schoepfer R (1998) Identification of amino acid residues of the NR2A subunit that control glutamate potency in recombinant NR1/NR2A NMDA receptors. J Neurosci 18:581–589. 10.1523/JNEUROSCI.18-02-00581.1998 PubMed DOI PMC

Berendsen HJC, Postma JPM, van Gunsteren WF, DiNola A, Haak JR (1984) Molecular dynamics with coupling to an external bath. J Chem Phys 81:3684–3690.. 10.1063/1.448118 DOI

Blanke ML, VanDongen AMJ (2008) Constitutive activation of the N-methyl-d-aspartate receptor via cleft-spanning disulfide bonds. J Biol Chem 283:21519–21529. 10.1074/jbc.M709190200 PubMed DOI PMC

Case DA, et al. (2024) Amber 2024, University of California, San Francisco.

Chen JZ, Church WB, Bastard K, Duff AP, Balle T (2023) Binding and dynamics demonstrate the destabilization of ligand binding for the S688Y mutation in the NMDA receptor GluN1 subunit. Molecules 28:4108. 10.3390/molecules28104108 PubMed DOI PMC

Chen PE, Geballe MT, Stansfeld PJ, Johnston AR, Yuan H, Jacob AL, Snyder JP, Traynelis SF, Wyllie DJA (2005) Structural features of the glutamate binding site in recombinant NR1/NR2A N-methyl-d-aspartate receptors determined by site-directed mutagenesis and molecular modeling. Mol Pharmacol 67:1470–1484. 10.1124/mol.104.008185 PubMed DOI

Chen PE, Wyllie DJA (2006) Pharmacological insights obtained from structure–function studies of ionotropic glutamate receptors. Br J Pharmacol 147:839–853. 10.1038/sj.bjp.0706689 PubMed DOI PMC

Coleman SK, Möykkynen T, Jouppila A, Koskelainen S, Rivera C, Korpi ER, Keinänen K (2009) Agonist occupancy is essential for forward trafficking of AMPA receptors. J Neurosci 29:303–312. 10.1523/JNEUROSCI.3953-08.2009 PubMed DOI PMC

Coleman SK, Möykkynen T, Hinkkuri S, Vaahtera L, Korpi ER, Pentikäinen OT, Keinänen K (2010) Ligand-binding domain determines endoplasmic reticulum exit of AMPA receptors. J Biol Chem 285:36032–36039. 10.1074/jbc.M110.156943 PubMed DOI PMC

Collett VJ, Collingridge GL (2004) Interactions between NMDA receptors and mGlu5 receptors expressed in HEK293 cells. Br J Pharmacol 142:991–1001. 10.1038/sj.bjp.0705861 PubMed DOI PMC

Conroy J, et al. (2014) Towards the identification of a genetic basis for Landau-Kleffner syndrome. Epilepsia 55:858–865. 10.1111/epi.12645 PubMed DOI

Dai J, Zhou H-X (2016) Semiclosed conformations of the ligand-binding domains of NMDA receptors during stationary gating. Biophys J 111:1418–1428. 10.1016/j.bpj.2016.08.010 PubMed DOI PMC

Darden T, York D, Pedersen L (1993) Particle mesh Ewald: an N⋅log(N) method for Ewald sums in large systems. J Chem Phys 98:10089–10092. 10.1063/1.464397 DOI

Erreger K, et al. (2007) Subunit-specific agonist activity at NR2A-, NR2B-, NR2C-, and NR2D-containing N-methyl-d-aspartate glutamate receptors. Mol Pharmacol 72:907–920. 10.1124/mol.107.037333 PubMed DOI

Fernández-Marmiesse A, et al. (2019) Rare variants in 48 genes account for 42% of cases of epilepsy with or without neurodevelopmental delay in 246 pediatric patients. Front Neurosci 13:1135. 10.3389/fnins.2019.01135 PubMed DOI PMC

Frisch MJ, et al. (2016) Gaussian 16, Revision C.01; Gaussian, Inc., Wallingford CT.

Furukawa H, Singh SK, Mancusso R, Gouaux E (2005) Subunit arrangement and function in NMDA receptors. Nature 438:185–192. 10.1038/nature04089 PubMed DOI

Furukawa H, Gouaux E (2003) Mechanisms of activation, inhibition and specificity: crystal structures of the NMDA receptor NR1 ligand-binding core. EMBO J 22:2873–2885. 10.1093/emboj/cdg303 PubMed DOI PMC

Hackos DH, et al. (2016) Positive allosteric modulators of GluN2A-containing NMDARs with distinct modes of action and impacts on circuit function. Neuron 89:983–999. 10.1016/j.neuron.2016.01.016 PubMed DOI

Hangen E, Cordelières FP, Petersen JD, Choquet D, Coussen F (2018) Neuronal activity and intracellular calcium levels regulate intracellular transport of newly synthesized AMPAR. Cell Rep 24:1001–1012.e3. 10.1016/j.celrep.2018.06.095 PubMed DOI PMC

Hansen KB, et al. (2021) Structure, function, and pharmacology of glutamate receptor ion channels. Pharmacol Rev 73:298–487. 10.1124/pharmrev.120.000131 PubMed DOI PMC

Hanus C, Ehlers MD (2016) Specialization of biosynthetic membrane trafficking for neuronal form and function. Curr Opin Neurobiol 39:8–16. 10.1016/j.conb.2016.03.004 PubMed DOI

Hawkins LM, Prybylowski K, Chang K, Moussan C, Stephenson FA, Wenthold RJ (2004) Export from the endoplasmic reticulum of assembled N-methyl-D-aspartic acid receptors is controlled by a motif in the C terminus of the NR2 subunit*. J Biol Chem 279:28903–28910. 10.1074/jbc.M402599200 PubMed DOI

Hayashi T, Thomas GM, Huganir RL (2009) Dual palmitoylation of NR2 subunits regulates NMDA receptor trafficking. Neuron 64:213–226. 10.1016/j.neuron.2009.08.017 PubMed DOI PMC

Horak M, Vlcek K, Petrovic M, Chodounska H, Vyklicky L (2004) Molecular mechanism of pregnenolone sulfate action at NR1/NR2B receptors. J Neurosci 24:10318–10325. 10.1523/JNEUROSCI.2099-04.2004 PubMed DOI PMC

Horak M, Al-Hallaq RA, Chang K, Wenthold RJ (2008) Role of the fourth membrane domain of the NR2B subunit in the assembly of the NMDA receptor. Channels 2:159. 10.4161/chan.2.3.6188 PubMed DOI PMC

Horak M, Petralia RS, Kaniakova M, Sans N (2014) ER to synapse trafficking of NMDA receptors. Front Cell Neurosci 8:394. 10.3389/fncel.2014.00394 PubMed DOI PMC

Horak M, Barackova P, Langore E, Netolicky J, Rivas-Ramirez P, Rehakova K (2021) The extracellular domains of GluN subunits play an essential role in processing NMDA receptors in the ER. Front Neurosci 15:603715. 10.3389/fnins.2021.603715 PubMed DOI PMC

Horak M, Wenthold RJ (2009) Different roles of C-terminal cassettes in the trafficking of full-length NR1 subunits to the cell surface. J Biol Chem 284:9683–9691. 10.1074/jbc.M807050200 PubMed DOI PMC

Huh KH, Wenthold RJ (1999) Turnover analysis of glutamate receptors identifies a rapidly degraded pool of the N-methyl-D-aspartate receptor subunit, NR1, in cultured cerebellar granule cells. J Biol Chem 274:151–157. 10.1074/jbc.274.1.151 PubMed DOI

Inanobe A, Furukawa H, Gouaux E (2005) Mechanism of partial agonist action at the NR1 subunit of NMDA receptors. Neuron 47:71–84. 10.1016/j.neuron.2005.05.022 PubMed DOI

Izadi S, Anandakrishnan R, Onufriev AV (2014) Building water models: a different approach. J Phys Chem Lett 5:3863–3871. 10.1021/jz501780a PubMed DOI PMC

Jespersen A, Tajima N, Fernandez-Cuervo G, Garnier-Amblard EC, Furukawa H (2014) Structural insights into competitive antagonism in NMDA receptors. Neuron 81:366–378. 10.1016/j.neuron.2013.11.033 PubMed DOI PMC

Jeyifous O, et al. (2009) SAP97 and CASK mediate sorting of N-methyl-D-aspartate receptors through a novel secretory pathway. Nat Neurosci 12:1011–1019. 10.1038/nn.2362 PubMed DOI PMC

Kalbaugh TL, VanDongen HMA, VanDongen AMJ (2004) Ligand-binding residues integrate affinity and efficacy in the NMDA receptor. Mol Pharmacol 66:209–219. 10.1124/mol.66.2.209 PubMed DOI

Kenny AV, Cousins SL, Pinho L, Stephenson FA (2009) The integrity of the glycine co-agonist binding site of N-methyl-d-aspartate receptors is a functional quality control checkpoint for cell surface delivery*. J Biol Chem 284:324–333. 10.1074/jbc.M804023200 PubMed DOI

Kinarsky L, Feng B, Skifter DA, Morley RM, Sherman S, Jane DE, Monaghan DT (2005) Identification of subunit- and antagonist-specific amino acid residues in the N-methyl-D-aspartate receptor glutamate-binding pocket. J Pharmacol Exp Ther 313:1066–1074. 10.1124/jpet.104.082990 PubMed DOI

Kolcheva M, et al. (2023) The pathogenic N650K variant in the GluN1 subunit regulates the trafficking, conductance, and pharmacological properties of NMDA receptors. Neuropharmacology 222:109297. 10.1016/j.neuropharm.2022.109297 PubMed DOI

Kussius CL, Popescu GK (2010) NMDA receptors with locked glutamate-binding clefts open with high efficacy. J Neurosci 30:12474–12479. 10.1523/JNEUROSCI.3337-10.2010 PubMed DOI PMC

Kvist T, Greenwood JR, Hansen KB, Traynelis SF, Bräuner-Osborne H (2013) Structure-based discovery of antagonists for GluN3-containing N-methyl-D-aspartate receptors. Neuropharmacology 75:324–336. 10.1016/j.neuropharm.2013.08.003 PubMed DOI PMC

Laube B, Schemm R, Betz H (2004) Molecular determinants of ligand discrimination in the glutamate-binding pocket of the NMDA receptor. Neuropharmacology 47:994–1007. 10.1016/j.neuropharm.2004.07.041 PubMed DOI

Lesca G, et al. (2013) GRIN2A mutations in acquired epileptic aphasia and related childhood focal epilepsies and encephalopathies with speech and language dysfunction. Nat Genet 45:1061–1066. 10.1038/ng.2726 PubMed DOI

Lichnerova K, Kaniakova M, Park SP, Skrenkova K, Wang Y-X, Petralia RS, Suh YH, Horak M (2015) Two N-glycosylation sites in the GluN1 subunit are essential for releasing N-methyl-d-aspartate (NMDA) receptors from the endoplasmic reticulum. J Biol Chem 290:18379–18390. 10.1074/jbc.M115.656546 PubMed DOI PMC

Maier W, Schemm R, Grewer C, Laube B (2007) Disruption of interdomain interactions in the glutamate binding pocket affects differentially agonist affinity and efficacy of N-methyl-D-aspartate receptor activation. J Biol Chem 282:1863–1872. 10.1074/jbc.M608156200 PubMed DOI

Meddows E, Le Bourdellès B, Grimwood S, Wafford K, Sandhu S, Whiting P, McIlhinney RAJ (2001) Identification of molecular determinants that are important in the assembly of N-methyl-d-aspartate receptors*. J Biol Chem 276:18795–18803. 10.1074/jbc.M101382200 PubMed DOI

Miller BRI, McGee TD Jr, Swails JM, Homeyer N, Gohlke H, Roitberg AE (2012) MMPBSA.py: an efficient program for end-state free energy calculations. J Chem Theory Comput 8:3314–3321. 10.1021/ct300418h PubMed DOI

Monyer H, Burnashev N, Laurie DJ, Sakmann B, Seeburg PH (1994) Developmental and regional expression in the rat brain and functional properties of four NMDA receptors. Neuron 12:529–540. 10.1016/0896-6273(94)90210-0 PubMed DOI

Nakagomi S, Barsoum MJ, Bossy-Wetzel E, Sütterlin C, Malhotra V, Lipton SA (2008) A Golgi fragmentation pathway in neurodegeneration. Neurobiol Dis 29:221–231. 10.1016/j.nbd.2007.08.015 PubMed DOI PMC

Nykamp K, et al. (2017) Sherloc: a comprehensive refinement of the ACMG–AMP variant classification criteria. Genet Med 19:1105–1117. 10.1038/gim.2017.37 PubMed DOI PMC

Okabe S, Miwa A, Okado H (1999) Alternative splicing of the C-terminal domain regulates cell surface expression of the NMDA receptor NR1 subunit. J Neurosci 19:7781–7792. 10.1523/JNEUROSCI.19-18-07781.1999 PubMed DOI PMC

Paoletti P, Bellone C, Zhou Q (2013) NMDA receptor subunit diversity: impact on receptor properties, synaptic plasticity and disease. Nat Rev Neurosci 14:383–400. 10.1038/nrn3504 PubMed DOI

Penn AC, Williams SR, Greger IH (2008) Gating motions underlie AMPA receptor secretion from the endoplasmic reticulum. EMBO J 27:3056–3068. 10.1038/emboj.2008.222 PubMed DOI PMC

Prybylowski K, Fu Z, Losi G, Hawkins LM, Luo J, Chang K, Wenthold RJ, Vicini S (2002) Relationship between availability of NMDA receptor subunits and their expression at the synapse. J Neurosci 22:8902–8910. 10.1523/JNEUROSCI.22-20-08902.2002 PubMed DOI PMC

Qiu S, Zhang X, Cao J, Yang W, Yan Y, Shan L, Zheng J, Luo J (2009) An endoplasmic reticulum retention signal located in the extracellular amino-terminal domain of the NR2A subunit of N-methyl-d-aspartate receptors. J Biol Chem 284:20285. 10.1074/jbc.M109.004960 PubMed DOI PMC

Roe DR, Cheatham TE (2013) PTRAJ and CPPTRAJ: software for processing and analysis of molecular dynamics trajectory data. J Chem Theory Comput 9:3084–3095. 10.1021/ct400341p PubMed DOI

Ryckaert JP, Ciccotti G, Berendsen HJC (1977) Numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes. J Comp Phys 23:327–341. 10.1016/0021-9991(77)90098-5 DOI

Santos-Gómez A, et al. (2021) Paradigmatic de novo GRIN1 variants recapitulate pathophysiological mechanisms underlying GRIN1-related disorder clinical spectrum. Int J Mol Sci 22:12656. 10.3390/ijms222312656 PubMed DOI PMC

Sanz-Clemente A, Nicoll RA, Roche KW (2012) Diversity in NMDA receptor composition: many regulators, many consequences. Neuroscientist 19:62–75. 10.1177/1073858411435129 PubMed DOI PMC

Scholefield CL, Atlason PT, Jane DE, Molnár E (2019) Assembly and trafficking of homomeric and heteromeric kainate receptors with impaired ligand binding sites. Neurochem Res 44:585–599. 10.1007/s11064-018-2654-0 PubMed DOI PMC

Schüler T, Mesic I, Madry C, Bartholomäus I, Laube B (2008) Formation of NR1/NR2 and NR1/NR3 heterodimers constitutes the initial step in N-methyl-D-aspartate receptor assembly*. J Biol Chem 283:37–46. 10.1074/jbc.M703539200 PubMed DOI

Scott DB, Blanpied TA, Swanson GT, Zhang C, Ehlers MD (2001) An NMDA receptor ER retention signal regulated by phosphorylation and alternative splicing. J Neurosci 21:3063–3072. 10.1523/JNEUROSCI.21-09-03063.2001 PubMed DOI PMC

Seljeset S, Sintsova O, Wang Y, Harb HY, Lynagh T (2024) Constitutive activity of ionotropic glutamate receptors via a hydrophobic plug in the ligand-binding domain. Structure 32:966–978. 10.1016/j.str.2024.04.001 PubMed DOI

Sengupta A, Li Z, Song LF, Li P, Merz KM Jr (2021) Parameterization of monovalent ions for the OPC3, OPC, TIP3P-FB, and TIP4P-FB water models. J Chem Inf Model 61:869–880. 10.1021/acs.jcim.0c01390 PubMed DOI PMC

She K, Ferreira JS, Carvalho AL, Craig AM (2012) Glutamate binding to the GluN2B subunit controls surface trafficking of N-methyl-d-aspartate (NMDA) receptors♦. J Biol Chem 287:27432–27445. 10.1074/jbc.M112.345108 PubMed DOI PMC

Sheng M, Cummings J, Roldan LA, Jan YN, Jan LY (1994) Changing subunit composition of heteromeric NMDA receptors during development of rat cortex. Nature 368:144–147. 10.1038/368144a0 PubMed DOI

Skrenkova K, Hemelikova K, Kolcheva M, Kortus S, Kaniakova M, Krausova B, Horak M (2019) Structural features in the glycine-binding sites of the GluN1 and GluN3A subunits regulate the surface delivery of NMDA receptors. Sci Rep 9:12303. 10.1038/s41598-019-48845-3 PubMed DOI PMC

Skrenkova K, et al. (2020) The pathogenic S688Y mutation in the ligand-binding domain of the GluN1 subunit regulates the properties of NMDA receptors. Sci Rep 10:18576. 10.1038/s41598-020-75646-w PubMed DOI PMC

Soliman K, Grimm F, Wurm CA, Egner A (2021) Predicting the membrane permeability of organic fluorescent probes by the deep neural network based lipophilicity descriptor DeepFl-LogP. Sci Rep 11:6991. 10.1038/s41598-021-86460-3 PubMed DOI PMC

Standley S, Roche KW, McCallum J, Sans N, Wenthold RJ (2000) PDZ domain suppression of an ER retention signal in NMDA receptor NR1 splice variants. Neuron 28:887–898. 10.1016/S0896-6273(00)00161-6 PubMed DOI

Strehlow V, et al. (2019) GRIN2A-related disorders: genotype and functional consequence predict phenotype. Brain 142:80–92. 10.1093/brain/awy304 PubMed DOI PMC

Stroebel D, Paoletti P (2021) Architecture and function of NMDA receptors: an evolutionary perspective. J Physiol 599:2615–2638. 10.1113/JP279028 PubMed DOI

Swanger SA, et al. (2016) Mechanistic insight into NMDA receptor dysregulation by rare variants in the GluN2A and GluN2B agonist binding domains. Am J Hum Genet 99:1261–1280. 10.1016/j.ajhg.2016.10.002 PubMed DOI PMC

Tian C, et al. (2020) ff19SB: amino-acid-specific protein backbone parameters trained against quantum mechanics energy surfaces in solution. J Chem Theory Comput 16:528–552. 10.1021/acs.jctc.9b00591 PubMed DOI

Traynelis SF, Wollmuth LP, McBain CJ, Menniti FS, Vance KM, Ogden KK, Hansen KB, Yuan H, Myers SJ, Dingledine R (2010) Glutamate receptor ion channels: structure, regulation, and function (Sibley D, ed). Pharmacol Rev 62:405–496. 10.1124/pr.109.002451 PubMed DOI PMC

Vieira M, Yong XLH, Roche KW, Anggono V (2020) Regulation of NMDA glutamate receptor functions by the GluN2 subunits. J Neurochem 154:121–143. 10.1111/jnc.14970 PubMed DOI PMC

Waterhouse A, , et al. (2018) Swiss-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res 46:W296–W303. 10.1093/nar/gky427 PubMed DOI PMC

Williams K, Chao J, Kashiwagi K, Masuko T, Igarashi K (1996) Activation of N-methyl-D-aspartate receptors by glycine: role of an aspartate residue in the M3-M4 loop of the NR1 subunit. Mol Pharmacol 50:701–708. 10.1016/S0026-895X(25)09369-1 PubMed DOI

Xia H, Hornby ZD, Malenka RC (2001) An ER retention signal explains differences in surface expression of NMDA and AMPA receptor subunits. Neuropharmacology 41:714–723. 10.1016/S0028-3908(01)00103-4 PubMed DOI

Zehavi Y, Mandel H, Zehavi A, Rashid MA, Straussberg R, Jabur B, Shaag A, Elpeleg O, Spiegel R (2017) De novo GRIN1 mutations: an emerging cause of severe early infantile encephalopathy. Eur J Med Genet 60:317–320. 10.1016/j.ejmg.2017.04.001 PubMed DOI

Zhou X, Hollern D, Liao J, Andrechek E, Wang H (2013) NMDA receptor-mediated excitotoxicity depends on the coactivation of synaptic and extrasynaptic receptors. Cell Death Dis 4:e560. 10.1038/cddis.2013.82 PubMed DOI PMC