N-Methyl-d-aspartate receptors (NMDARs), encoded by GRIN genes, are ionotropic glutamate receptors playing a critical role in synaptic transmission, plasticity, and synapse development. Genome sequence analyses have identified variants in GRIN genes in patients with neurodevelopmental disorders, but the underlying disease mechanisms are not well understood. Here, we have created and evaluated a transgenic mouse line carrying a missense variant Grin2bL825V , corresponding to a de novo GRIN2B variant encoding GluN2B(L825V) found in a patient with intellectual disability (ID) and autism spectrum disorder (ASD). We used HEK293T cells expressing recombinant receptors and primary hippocampal neurons prepared from heterozygous Grin2bL825V/+ (L825V/+) and wild-type (WT) Grin2b+/+ (+/+) male and female mice to assess the functional impact of the variant. Whole-cell NMDAR currents were reduced in neurons from L825V/+ compared with +/+ mice. The peak amplitude of NMDAR-mediated evoked excitatory postsynaptic currents (NMDAR-eEPSCs) was unchanged, but NMDAR-eEPSCs in L825V/+ neurons had faster deactivation compared with +/+ neurons and were less sensitive to a GluN2B-selective antagonist ifenprodil. Together, these results suggest a decreased functional contribution of GluN2B subunits to synaptic NMDAR currents in hippocampal neurons from L825V/+ mice. The analysis of the GluN2B(L825V) subunit surface expression and synaptic localization revealed no differences compared with WT GluN2B. Behavioral testing of mice of both sexes demonstrated hypoactivity, anxiety, and impaired sensorimotor gating in the L825V/+ strain, particularly affecting males, as well as cognitive symptoms. The heterozygous L825V/+ mouse offers a clinically relevant model of GRIN2B-related ID/ASD, and our results suggest synaptic-level functional changes that may contribute to neurodevelopmental pathology.

• Keywords
• GluN2B, NMDA receptors, autism spectrum disorder, mouse model, synaptic transmission,
• MeSH
• Excitatory Postsynaptic Potentials physiology   MeSH
• HEK293 Cells MeSH
• Hippocampus metabolism   MeSH
• Humans MeSH
• Mutation, Missense MeSH
• Mice, Inbred C57BL MeSH
• Mice, Transgenic * MeSH
• Mice MeSH
• Neurons metabolism   MeSH
• Neurodevelopmental Disorders * genetics   physiopathology   metabolism   MeSH
• Receptors, N-Methyl-D-Aspartate * genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• NR2B NMDA receptor MeSH Browser
• Receptors, N-Methyl-D-Aspartate * MeSH

N-methyl-D-aspartate receptors (NMDARs) play a critical role in normal brain function, and variants in genes encoding NMDAR subunits have been described in individuals with various neuropsychiatric disorders. We have used whole-cell patch-clamp electrophysiology, fluorescence microscopy and in-silico modeling to explore the functional consequences of disease-associated nonsense and frame-shift variants resulting in the truncation of GluN2A or GluN2B C-terminal domain (CTD). This study characterizes variant NMDARs and shows their reduced surface expression and synaptic localization, altered agonist affinity, increased desensitization, and reduced probability of channel opening. We also show that naturally occurring and synthetic steroids pregnenolone sulfate and epipregnanolone butanoic acid, respectively, enhance NMDAR function in a way that is dependent on the length of the truncated CTD and, further, is steroid-specific, GluN2A/B subunit-specific, and GluN1 splice variant-specific. Adding to the previously described effects of disease-associated NMDAR variants on the receptor biogenesis and function, our results improve the understanding of the molecular consequences of NMDAR CTD truncations and provide an opportunity for the development of new therapeutic neurosteroid-based ligands.

• Keywords
• Channelopathy, Endogenous neuroactive steroid, GRIN2 genes, Glutamate receptors, Rescue pharmacology, Surface expression,
• MeSH
• Electrophysiological Phenomena MeSH
• Humans MeSH
• Neurosteroids * MeSH
• Receptors, N-Methyl-D-Aspartate * genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• N-methyl D-aspartate receptor subtype 2A MeSH Browser
• Neurosteroids * MeSH
• NR2B NMDA receptor MeSH Browser
• Receptors, N-Methyl-D-Aspartate * MeSH

NMDARs are ligand-gated ion channels that cause an influx of Na+ and Ca2+ into postsynaptic neurons. The resulting intracellular Ca2+ transient triggers synaptic plasticity. When prolonged, it may induce excitotoxicity, but it may also activate negative feedback to control the activity of NMDARs. Here, we report that a transient rise in intracellular Ca2+ (Ca2+ challenge) increases the sensitivity of NMDARs but not AMPARs/kainate receptors to the endogenous inhibitory neurosteroid 20-oxo-5β-pregnan-3α-yl 3-sulfate and to its synthetic analogs, such as 20-oxo-5β-pregnan-3α-yl 3-hemipimelate (PAhPim). In cultured hippocampal neurons, 30 μm PAhPim had virtually no effect on NMDAR responses; however, following the Ca2+ challenge, it inhibited the responses by 62%; similarly, the Ca2+ challenge induced a 3.7-fold decrease in the steroid IC50 on recombinant GluN1/GluN2B receptors. The increase in the NMDAR sensitivity to PAhPim was dependent on three cysteines (C849, C854, and C871) located in the carboxy-terminal domain of the GluN2B subunit, previously identified to be palmitoylated (Hayashi et al., 2009). Our experiments suggested that the Ca2+ challenge induced receptor depalmitoylation, and single-channel analysis revealed that this was accompanied by a 55% reduction in the probability of channel opening. Results of in silico modeling indicate that receptor palmitoylation promotes anchoring of the GluN2B subunit carboxy-terminal domain to the plasma membrane and facilitates channel opening. Depalmitoylation-induced changes in the NMDAR pharmacology explain the neuroprotective effect of PAhPim on NMDA-induced excitotoxicity. We propose that palmitoylation-dependent changes in the NMDAR sensitivity to steroids serve as an acute endogenous mechanism that controls NMDAR activity.SIGNIFICANCE STATEMENT There is considerable interest in negative allosteric modulators of NMDARs that could compensate for receptor overactivation by glutamate or de novo gain-of-function mutations in neurodevelopmental disorders. By a combination of electrophysiological, pharmacological, and computational techniques we describe a novel feedback mechanism regulating NMDAR activity. We find that a transient rise in intracellular Ca2+ increases NMDAR sensitivity to inhibitory neurosteroids in a process dependent on GluN2B subunit depalmitoylation. These results improve our understanding of the molecular mechanisms of steroid action at the NMDAR and indeed of the basic properties of this important glutamate-gated ion channel and may aid in the development of therapeutics for treating neurologic and psychiatric diseases related to overactivation of NMDARs without affecting normal physiological functions.

• Keywords
• NMDAR, carboxy-terminal domain, molecular dynamics simulation, neurosteroid, palmitoylation, single-channel recording,
• MeSH
• HEK293 Cells MeSH
• Hippocampus physiology   MeSH
• Rats MeSH
• Humans MeSH
• Lipoylation drug effects   physiology   MeSH
• Neuroprotection physiology   MeSH
• Rats, Wistar MeSH
• Pregnanes metabolism   pharmacology   MeSH
• Receptors, N-Methyl-D-Aspartate metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 20-oxo-5beta-pregnan-3alpha-yl sulfate MeSH Browser
• NR2B NMDA receptor MeSH Browser
• Pregnanes MeSH
• Receptors, N-Methyl-D-Aspartate MeSH

N-methyl-D-aspartate receptor (NMDAR) hypofunction has been implicated in several neurodevelopmental disorders. NMDAR function can be augmented by positive allosteric modulators, including endogenous compounds, such as cholesterol and neurosteroid pregnenolone sulfate (PES). Here we report that PES accesses the receptor via the membrane, and its binding site is different from that of cholesterol. Alanine mutagenesis has identified residues that disrupt the steroid potentiating effect at the rat GluN1 (G638; I642) and GluN2B (W559; M562; Y823; M824) subunit. Molecular dynamics simulation indicates that, in the absence of PES, the GluN2B M1 helix residue W559 interacts with the M4 helix residue M824. In the presence of PES, the M1 and M4 helices of agonist-activated receptor rearrange, forming a tighter interaction with the GluN1 M3 helix residues G638 and I642. This stabilizes the open-state position of the GluN1 M3 helices. Together, our data identify a likely binding site for the NMDAR-positive allosteric modulator PES and describe a novel molecular mechanism by which NMDAR activity can be augmented.SIGNIFICANCE STATEMENT There is considerable interest in drugs that enhance NMDAR function and could compensate for receptor hypofunction associated with certain neuropsychiatric disorders. Positive allosteric modulators of NMDARs include an endogenous neurosteroid pregnenolone sulfate (PES), but the binding site of PES on the NMDAR and the molecular mechanism of potentiation are unknown. We use patch-clamp electrophysiology in combination with mutagenesis and in silico modeling to describe the interaction of PES with the NMDAR. Our data indicate that PES binds to the transmembrane domain of the receptor at a discrete group of residues at the GluN2B membrane helices M1 and M4 and the GluN1 helix M3, and that PES potentiates NMDAR function by stabilizing the open-state position of the GluN1 M3 helices.

• Keywords
• glutamate receptors, neurosteroids, patch clamp, structure,
• MeSH
• Alanine genetics   MeSH
• Cell Membrane drug effects   MeSH
• Cholesterol metabolism   MeSH
• Electrophysiological Phenomena MeSH
• HEK293 Cells MeSH
• Protein Conformation MeSH
• Rats MeSH
• Humans MeSH
• Patch-Clamp Techniques MeSH
• Pregnenolone pharmacology   MeSH
• Receptors, N-Methyl-D-Aspartate drug effects   MeSH
• Molecular Dynamics Simulation MeSH
• Binding Sites MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Alanine MeSH
• Cholesterol MeSH
• NMDA receptor A1 MeSH Browser
• NR2B NMDA receptor MeSH Browser
• Pregnenolone MeSH
• pregnenolone sulfate MeSH Browser
• Receptors, N-Methyl-D-Aspartate MeSH

Cholesterol is a structural component of cellular membranes particularly enriched in synapses but its role in synaptic transmission remains poorly understood. We used rat hippocampal cultures and their acute cholesterol depletion by methyl-β-cyclodextrin as a tool to describe the physiological role of cholesterol in glutamatergic synaptic transmission. Cholesterol proved to be a key molecule for the function of synapses as its depletion resulted in a significant reduction of both NMDA receptor (NMDAR) and AMPA/kainate receptor-mediated evoked excitatory postsynaptic currents (eEPSCs), by 94% and 72%, respectively. We identified two presynaptic and two postsynaptic steps of synaptic transmission which are modulated by cholesterol and explain together the above-mentioned reduction of eEPSCs. In the postsynapse, we show that physiological levels of cholesterol are important for maintaining the normal probability of opening of NMDARs and for keeping NMDARs localized in synapses. In the presynapse, our results favour the hypothesis of a role of cholesterol in the propagation of axonal action potentials. Finally, cholesterol is a negative modulator of spontaneous presynaptic glutamate release. Our study identifies cholesterol as an important endogenous regulator of synaptic transmission and provides insight into molecular mechanisms underlying the neurological manifestation of diseases associated with impaired cholesterol synthesis or decomposition.

• MeSH
• Receptors, AMPA metabolism   MeSH
• Cholesterol pharmacology   MeSH
• Excitatory Postsynaptic Potentials drug effects   MeSH
• Hippocampus drug effects   metabolism   MeSH
• Rats MeSH
• Glutamic Acid metabolism   MeSH
• Cerebral Cortex drug effects   metabolism   MeSH
• Synaptic Transmission * MeSH
• Neurons drug effects   metabolism   MeSH
• Rats, Wistar MeSH
• Presynaptic Terminals drug effects   metabolism   MeSH
• Receptors, N-Methyl-D-Aspartate metabolism   MeSH
• Synapses drug effects   metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Receptors, AMPA MeSH
• Cholesterol MeSH
• Glutamic Acid MeSH
• Receptors, N-Methyl-D-Aspartate MeSH

We report the first complete description of the molecular mechanisms behind the transition of the N-methyl-d-aspartate (NMDA) receptor from the state where the transmembrane domain (TMD) and the ion channel are in the open configuration to the relaxed unliganded state where the channel is closed. Using an aggregate of nearly 1 µs of unbiased all-atom implicit membrane and solvent molecular dynamics (MD) simulations we identified distinct structural states of the NMDA receptor and revealed functionally important residues (GluN1/Glu522, GluN1/Arg695, and GluN2B/Asp786). The role of the "clamshell" motion of the ligand binding domain (LBD) lobes in the structural transition is supplemented by the observed structural similarity at the level of protein domains during the structural transition, combined with the overall large rearrangement necessary for the opening and closing of the receptor. The activated and open states of the receptor are structurally similar to the liganded crystal structure, while in the unliganded receptor the extracellular domains perform rearrangements leading to a clockwise rotation of up to 45 degrees around the longitudinal axis of the receptor, which closes the ion channel. The ligand-induced rotation of extracellular domains transferred by LBD-TMD linkers to the membrane-anchored ion channel is responsible for the opening and closing of the transmembrane ion channel, revealing the properties of NMDA receptor as a finely tuned molecular machine.

• Keywords
• NMDA receptor transition, glutamate receptor gating, molecular dynamics simulations, molecular modeling, open and closed state,
• MeSH
• Rats MeSH
• Receptors, N-Methyl-D-Aspartate chemistry   metabolism   MeSH
• Molecular Dynamics Simulation * MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Receptors, N-Methyl-D-Aspartate MeSH