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.

• Keywords
• Golgi apparatus, endoplasmic reticulum, glutamate receptor, hippocampal neuron, ion channel, pathogenic variant,
• MeSH
• Glycine metabolism   MeSH
• HEK293 Cells MeSH
• Hippocampus cytology   metabolism   MeSH
• Rats MeSH
• Glutamic Acid metabolism   MeSH
• Humans MeSH
• Ligands MeSH
• Mutation genetics   MeSH
• Protein Domains MeSH
• Receptors, N-Methyl-D-Aspartate * metabolism   genetics   chemistry   MeSH
• Protein Transport physiology   genetics   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Glycine MeSH
• GRIN1 protein, human MeSH Browser
• Glutamic Acid MeSH
• Ligands MeSH
• N-methyl D-aspartate receptor subtype 2A MeSH Browser
• Nerve Tissue Proteins MeSH
• Receptors, N-Methyl-D-Aspartate * MeSH

The expression and activity of ionotropic glutamate receptors control signal transduction at the excitatory synapses in the CNS. The NMDAR comprises two obligatory GluN1 subunits and two GluN2 or GluN3 subunits in different combinations. Each GluN subunit consists of four domains: the extracellular amino-terminal and agonist-binding domains, the transmembrane domain, and the intracellular C-terminal domain (CTD). The CTD interaction with various classes of intracellular proteins is critical for trafficking and synaptic localization of NMDARs. Amino acid mutations or the inclusion of premature stop codons in the CTD could contribute to the emergence of neurodevelopmental and neuropsychiatric disorders. Here, we describe the method of preparing primary hippocampal neurons and lentiviral particles expressing GluN subunits that can be used as a model to study cell surface expression and synaptic localization of NMDARs. We also show a simple method of fluorescence immunostaining of eGFP-tagged GluN2 subunits and subsequent microscopy technique and image analysis to study the effects of disease-associated mutations in the CTDs of GluN2A and GluN2B subunits.

• Keywords
• Colocalization, Fluorescence immunostaining, Fluorescence microscopy, Glutamate receptor, ImageJ analysis, Lentivirus, Primary hippocampal neurons, Surface expression,
• MeSH
• Gene Expression MeSH
• Hippocampus * metabolism   cytology   MeSH
• Rats MeSH
• Cells, Cultured MeSH
• Lentivirus genetics   MeSH
• Humans MeSH
• Neurons * metabolism   MeSH
• Protein Subunits metabolism   genetics   MeSH
• Primary Cell Culture methods   MeSH
• Receptors, N-Methyl-D-Aspartate * metabolism   genetics   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• N-methyl D-aspartate receptor subtype 2A MeSH Browser
• Protein Subunits MeSH
• Receptors, N-Methyl-D-Aspartate * MeSH

N-methyl-D-aspartate receptors (NMDARs) are a subtype of ionotropic glutamate receptors critical for synaptic transmission and plasticity, and for the development of neural circuits. Rare or de-novo variants in GRIN genes encoding NMDAR subunits have been associated with neurodevelopmental disorders characterized by intellectual disability, developmental delay, autism, schizophrenia, or epilepsy. In recent years, some disease-associated variants in GRIN genes have been characterized using recombinant receptors expressed in non-neuronal cells, and a few variants have also been studied in neuronal preparations or animal models. Here we review the current literature on the functional evaluation of human disease-associated variants in GRIN1, GRIN2A and GRIN2B genes at all levels of analysis. Focusing on the impact of different patient variants at the level of receptor function, we discuss effects on receptor agonist and co-agonist affinity, channel open probability, and receptor cell surface expression. We consider how such receptor-level functional information may be used to classify variants as gain-of-function or loss-of-function, and discuss the limitations of this classification at the synaptic, cellular, or system level. Together this work by many laboratories worldwide yields valuable insights into NMDAR structure and function, and represents significant progress in the effort to understand and treat GRIN disorders. Keywords: NMDA receptor , GRIN genes, Genetic variants, Electrophysiology, Synapse, Animal models.

• MeSH
• Genetic Predisposition to Disease MeSH
• Genetic Variation MeSH
• Humans MeSH
• Neurodevelopmental Disorders genetics   MeSH
• Nerve Tissue Proteins genetics   metabolism   MeSH
• Receptors, N-Methyl-D-Aspartate * genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• GRIN1 protein, human MeSH Browser
• N-methyl D-aspartate receptor subtype 2A MeSH Browser
• NR2B NMDA receptor MeSH Browser
• Nerve Tissue Proteins MeSH
• Receptors, N-Methyl-D-Aspartate * MeSH

NMDA receptors (NMDARs) are ionotropic glutamate receptors that play a key role in excitatory neurotransmission. The number and subtype of surface NMDARs are regulated at several levels, including their externalization, internalization, and lateral diffusion between the synaptic and extrasynaptic regions. Here, we used novel anti-GFP (green fluorescent protein) nanobodies conjugated to either the smallest commercially available quantum dot 525 (QD525) or the several nanometer larger (and thus brighter) QD605 (referred to as nanoGFP-QD525 and nanoGFP-QD605, respectively). Targeting the yellow fluorescent protein-tagged GluN1 subunit in rat hippocampal neurons, we compared these two probes to a previously established larger probe, a rabbit anti-GFP IgG together with a secondary IgG conjugated to QD605 (referred to as antiGFP-QD605). The nanoGFP-based probes allowed faster lateral diffusion of the NMDARs, with several-fold increased median values of the diffusion coefficient (D). Using thresholded tdTomato-Homer1c signals to mark synaptic regions, we found that the nanoprobe-based D values sharply increased at distances over 100 nm from the synaptic edge, while D values for antiGFP-QD605 probe remained unchanged up to a 400 nm distance. Using the nanoGFP-QD605 probe in hippocampal neurons expressing the GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits, we detected subunit-dependent differences in the synaptic localization of NMDARs, D value, synaptic residence time, and synaptic-extrasynaptic exchange rate. Finally, we confirmed the applicability of the nanoGFP-QD605 probe to study differences in the distribution of synaptic NMDARs by comparing to data obtained with nanoGFPs conjugated to organic fluorophores, using universal point accumulation imaging in nanoscale topography and direct stochastic optical reconstruction microscopy.SIGNIFICANCE STATEMENT Our study systematically compared the localization and mobility of surface NMDARs containing GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits expressed in rodent hippocampal neurons, using anti-green fluorescent protein (GFP) nanobodies conjugated to the quantum dot 605 (nanoGFP-QD605), as well as nanoGFP probes conjugated with small organic fluorophores. Our comprehensive analysis showed that the method used to delineate the synaptic region plays an important role in the study of synaptic and extrasynaptic pools of NMDARs. In addition, we showed that the nanoGFP-QD605 probe has optimal parameters for studying the mobility of NMDARs because of its high localization accuracy comparable to direct stochastic optical reconstruction microscopy and longer scan time compared with universal point accumulation imaging in nanoscale topography. The developed approaches are readily applicable to the study of any GFP-labeled membrane receptors expressed in mammalian neurons.

• Keywords
• GluN subunit, excitatory synapse, glutamate receptor, lateral diffusion, live microscopy, mammalian neuron,
• MeSH
• Hippocampus metabolism   MeSH
• Immunoglobulin G metabolism   MeSH
• Single-Domain Antibodies * metabolism   MeSH
• Rabbits MeSH
• Rats MeSH
• Neurons metabolism   MeSH
• Receptors, N-Methyl-D-Aspartate * metabolism   MeSH
• Mammals MeSH
• Synapses physiology   MeSH
• Animals MeSH
• Check Tag
• Rabbits MeSH
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Immunoglobulin G MeSH
• Single-Domain Antibodies * MeSH
• Receptors, N-Methyl-D-Aspartate * MeSH