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N -methyl- D -aspartate receptors (NMDARs) belong to a family of ionotropic glutamate receptors that play essential roles in excitatory neurotransmission and synaptic plasticity in the mammalian central nervous system (CNS). Functional NMDARs consist of heterotetramers comprised of GluN1, GluN2A-D, and/or GluN3A-B subunits, each of which contains four membrane domains (M1 through M4), an intracellular C-terminal domain, a large extracellular N-terminal domain composed of the amino-terminal domain and the S1 segment of the ligand-binding domain (LBD), and an extracellular loop between M3 and M4, which contains the S2 segment of the LBD. Both the number and type of NMDARs expressed at the cell surface are regulated at several levels, including their translation and posttranslational maturation in the endoplasmic reticulum (ER), intracellular trafficking via the Golgi apparatus, lateral diffusion in the plasma membrane, and internalization and degradation. This review focuses on the roles played by the extracellular regions of GluN subunits in ER processing. Specifically, we discuss the presence of ER retention signals, the integrity of the LBD, and critical N -glycosylated sites and disulfide bridges within the NMDAR subunits, each of these steps must pass quality control in the ER in order to ensure that only correctly assembled NMDARs are released from the ER for subsequent processing and trafficking to the surface. Finally, we discuss the effect of pathogenic missense mutations within the extracellular domains of GluN subunits with respect to ER processing of NMDARs.

N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors essential for excitatory neurotransmission. Previous studies proposed the existence of four disulfide bonds in the GluN1 subunit; however, their role in NMDAR trafficking remains unclear. Our study first confirmed the existence of four disulfide bonds in the GluN1 subunit using biochemistry in human embryonic kidney 293T (HEK293T) cells. Disrupting the individual disulfide bonds by serine replacements produced the following surface expression trend for GluN1/GluN2A, GluN1/GluN2B, and GluN1/GluN3A receptors: wild-type (WT) > GluN1-C744S-C798S > GluN1-C79S-C308S > GluN1-C420S-C454S > GluN1-C436S-C455S subunits. Electrophysiology revealed altered functional properties of NMDARs with disrupted disulfide bonds, specifically an increased probability of opening (Po) at the GluN1-C744S-C798S/GluN2 receptors. Synchronized release from the endoplasmic reticulum confirmed that disruption of disulfide bonds impaired early trafficking of NMDARs in HEK293T cells and primary hippocampal neurons prepared from Wistar rats of both sexes (embryonic day 18). The pathogenic GluN1-C744Y variant, associated with neurodevelopmental disorder and seizures, caused reduced surface expression and increased Po at GluN1/GluN2 receptors, consistent with findings for the GluN1-C744S-C798S subunit. The FDA-approved memantine inhibited GluN1-C744Y/GluN2 receptors more potently and with distinct kinetics compared to WT GluN1/GluN2 receptors. We also observed enhanced NMDA-induced excitotoxicity in hippocampal neurons expressing the GluN1-C744Y subunit, which memantine reduced more effectively compared to the WT GluN1 subunit. Lastly, we demonstrated that the presence of the hGluN1-1a-C744Y subunit counteracted the effect of the hGluN3A subunit on decreasing dendritic spine maturation, consistent with the reduced surface delivery of the NMDARs carrying this variant. Our findings highlight the critical role of disulfide bonds in the GluN1 subunit in regulating trafficking and function of major conventional (GluN1/GluN2A, GluN1/GluN2B) and unconventional (GluN1/GluN3A) diheteromeric NMDAR subtypes in the postnatal forebrain. We further demonstrated that the pathogenic GluN1-C744Y variant reduces surface expression of all studied NMDARs, as well as increases the probability of opening (Po) of the GluN1/GluN2 receptors, leading to heightened NMDA-induced excitotoxicity in hippocampal neurons. Additionally, we introduced an ARIAD-based system for the synchronized release of NMDARs from the endoplasmic reticulum in hippocampal neurons. This system provides a powerful tool for studying pathogenic GRIN gene variants and addresses the current lack of molecular methods for analyzing the early trafficking of NMDARs.

The essential role of N-methyl-D-aspartate receptors (NMDARs) in excitatory neurotransmission is underscored by numerous pathogenic variants in the GluN subunits, including those identified in their ligand-binding domains (LBDs). The prevailing hypothesis postulates that the endoplasmic reticulum (ER) quality control machinery verifies the agonist occupancy of NMDARs; however, whether it controls the structure of LBDs or the functionality of NMDARs is unknown. Using alanine substitutions combined with microscopy and electrophysiology, we found that surface expression of GluN1/GluN2A receptors, the primary NMDAR subtype in the adult forebrain, strongly correlates with EC50 values for glycine and L-glutamate. Interestingly, co-expression of both GluN1 and GluN2A subunits with alanine substitutions led to an additive reduction in the surface number of GluN1/GluN2A receptors, as did co-expression of both GluN1 and GluN2A subunits containing closed cleft conformation of LBDs. The synchronized ER release confirmed the altered regulation of early trafficking of GluN1/GluN2A receptors bearing alanine substitutions in the LBDs. Furthermore, the human versions of GluN1/GluN2A receptors containing pathogenic GluN1-S688Y, GluN1-S688P, GluN1-D732E, GluN2A-S511L, and GluN2A-T690M variants exhibited distinct surface expression compared to the corresponding alanine substitutions. Mutant cycles of GluN1-S688, GluN1-D732, GluN2A-S511, and GluN2A-T690 residues revealed, in most cases, a weak correlation between surface expression of the mutant GluN1/GluN2A receptors and their EC50 values for glycine or L-glutamate. Consistent with our experimental data, molecular modeling and dynamics showed that the ER quality control machinery likely perceives structural changes of the LBDs but not the functionality of GluN1/GluN2A receptors. Our study showed that structural changes in LBDs independently regulate the early trafficking of GluN1/GluN2A receptors and that the surface numbers of mutant GluN1/GluN2A receptors do not necessarily correlate with their agonist sensitivity. In addition, we validated a novel system of synchronized release of GluN1/GluN2A receptors from the ER. Together, our experimental and in silico findings support the urgency of further detailed research on the regulation of early trafficking of NMDARs, as it may open the avenue to targeted therapeutic intervention of CNS disorders associated with pathogenic variants in GluN subunits.

N-methyl-D-aspartate receptors (NMDARs) play a significant role in developing several central nervous system (CNS) disorders. Currently, memantine, used for treating Alzheimer’s disease, and ketamine, known for its anesthetic and antidepressant properties, are two clinically used NMDAR open-channel blockers. However, despite extensive research into NMDAR modulators, many have shown either harmful side effects or inadequate effectiveness. For instance, dizocilpine (MK-801) is recognized for its powerful psychomimetic effects due to its high-affinity and nearly irreversible inhibition of the GluN1/GluN2 NMDAR subtypes. Unlike ketamine, memantine and MK-801 also act through a unique, low-affinity “membrane-to-channel inhibition” (MCI). We aimed to develop an open-channel blocker based on MK-801 with distinct inhibitory characteristics from memantine and MK-801. Our novel compound, K2060, demonstrated effective voltage-dependent inhibition in the micromolar range at key NMDAR subtypes, GluN1/GluN2A and GluN1/GluN2B, even in the presence of Mg2+. K2060 showed reversible inhibitory dynamics and a partially trapping open-channel blocking mechanism with a significantly stronger MCI than memantine. Using hippocampal slices, 30 µM K2060 inhibited excitatory postsynaptic currents in CA1 hippocampal neurons by ∼51%, outperforming 30 µM memantine (∼21% inhibition). K2060 exhibited No Observed Adverse Effect Level (NOAEL) of 15 mg/kg upon intraperitoneal administration in mice. Administering K2060 at a 10 mg/kg dosage resulted in brain concentrations of approximately 2 µM, with peak concentrations (Tmax) achieved within 15 minutes. Finally, applying K2060 with trimedoxime and atropine in mice exposed to tabun improved treatment outcomes. These results underscore K2060’s potential as a therapeutic agent for CNS disorders linked to NMDAR dysfunction.