NMDA Receptor Channel Blocker Research Articles

De novo GRIN variants in M3 helix associated with neurological disorders control channel gating of NMDA receptor

N-methyl-d-aspartate receptors (NMDARs) are members of the glutamate receptor family and participate in excitatory postsynaptic transmission throughout the central nervous system. Genetic variants in GRIN genes encoding NMDAR subunits are associated with a spectrum of neurological disorders. The M3 transmembrane helices of the NMDAR couple directly to the agonist-binding domains and form a helical bundle crossing in the closed receptors that occludes the pore. The M3 functions as a transduction element whose conformational change couples ligand binding to opening of an ion conducting pore. In this study, we report the functional consequences of 48 de novo missense variants in GRIN1, GRIN2A, and GRIN2B that alter residues in the M3 transmembrane helix. These de novo variants were identified in children with neurological and neuropsychiatric disorders including epilepsy, developmental delay, intellectual disability, hypotonia and attention deficit hyperactivity disorder. All 48 variants in M3 for which comprehensive testing was completed produce a gain-of-function (28/48) compared to loss-of-function (9/48); 11 variants had an indeterminant phenotype. This supports the idea that a key structural feature of the M3 gate exists to stabilize the closed state so that agonist binding can drive channel opening. Given that most M3 variants enhance channel gating, we assessed the potency of FDA-approved NMDAR channel blockers on these variant receptors. These data provide new insight into the structure–function relationship of the NMDAR gate, and suggest that variants within the M3 transmembrane helix produce a gain-of-function.

Esmethadone (REL-1017) and Other Uncompetitive NMDAR Channel Blockers May Improve Mood Disorders via Modulation of Synaptic Kinase-Mediated Signaling.

This article presents a mechanism of action hypothesis to explain the rapid antidepressant effects of esmethadone (REL-1017) and other uncompetitive N-methyl-D-aspartate receptor (NMDAR) antagonists and presents a corresponding mechanism of disease hypothesis for major depressive disorder (MDD). Esmethadone and other uncompetitive NMDAR antagonists may restore physiological neural plasticity in animal models of depressive-like behavior and in patients with MDD via preferential tonic block of pathologically hyperactive GluN2D subtypes. Tonic Ca2+ currents via GluN2D subtypes regulate the homeostatic availability of synaptic proteins. MDD and depressive behaviors may be determined by reduced homeostatic availability of synaptic proteins, due to upregulated tonic Ca2+ currents through GluN2D subtypes. The preferential activity of low-potency NMDAR antagonists for GluN2D subtypes may explain their rapid antidepressant effects in the absence of dissociative side effects.

Pharmacological Comparative Characterization of REL-1017 (Esmethadone-HCl) and Other NMDAR Channel Blockers in Human Heterodimeric N-Methyl-D-Aspartate Receptors.

Excessive Ca2+ currents via N-methyl-D-aspartate receptors (NMDARs) have been implicated in many disorders. Uncompetitive NMDAR channel blockers are an emerging class of drugs in clinical use for major depressive disorder (MDD) and other neuropsychiatric diseases. The pharmacological characterization of uncompetitive NMDAR blockers in clinical use may improve our understanding of NMDAR function in physiology and pathology. REL-1017 (esmethadone-HCl), a novel uncompetitive NMDAR channel blocker in Phase 3 trials for the treatment of MDD, was characterized together with dextromethorphan, memantine, (±)-ketamine, and MK-801 in cell lines over-expressing NMDAR subtypes using fluorometric imaging plate reader (FLIPR), automated patch-clamp, and manual patch-clamp electrophysiology. In the absence of Mg2+, NMDAR subtypes NR1-2D were most sensitive to low, sub-μM glutamate concentrations in FLIPR experiments. FLIPR Ca2+ determination demonstrated low μM affinity of REL-1017 at NMDARs with minimal subtype preference. In automated and manual patch-clamp electrophysiological experiments, REL-1017 exhibited preference for the NR1-2D NMDAR subtype in the presence of 1 mM Mg2+ and 1 μM L-glutamate. Tau off and trapping characteristics were similar for (±)-ketamine and REL-1017. Results of radioligand binding assays in rat cortical neurons correlated with the estimated affinities obtained in FLIPR assays and in automated and manual patch-clamp assays. In silico studies of NMDARs in closed and open conformation indicate that REL-1017 has a higher preference for docking and undocking the open-channel conformation compared to ketamine. In conclusion, the pharmacological characteristics of REL-1017 at NMDARs, including relatively low affinity at the NMDAR, NR1-2D subtype preference in the presence of 1 mM Mg2+, tau off and degree of trapping similar to (±)-ketamine, and preferential docking and undocking of the open NMDAR, could all be important variables for understanding the rapid-onset antidepressant effects of REL-1017 without psychotomimetic side effects.

Inhibition of NMDA receptors through a membrane-to-channel path

N-methyl-d-aspartate receptors (NMDARs) are transmembrane proteins that are activated by the neurotransmitter glutamate and are found at most excitatory vertebrate synapses. NMDAR channel blockers, an antagonist class of broad pharmacological and clinical significance, inhibit by occluding the NMDAR ion channel. A vast literature demonstrates that NMDAR channel blockers, including MK-801, phencyclidine, ketamine, and the Alzheimer’s disease drug memantine, can bind and unbind only when the NMDAR channel is open. Here we use electrophysiological recordings from transfected tsA201 cells and cultured neurons, NMDAR structural modeling, and custom-synthesized compounds to show that NMDAR channel blockers can enter the channel through two routes: the well-known hydrophilic path from extracellular solution to channel through the open channel gate, and also a hydrophobic path from plasma membrane to channel through a gated fenestration (“membrane-to-channel inhibition” (MCI)). Our demonstration that ligand-gated channels are subject to MCI, as are voltage-gated channels, highlights the broad expression of this inhibitory mechanism.

Structural insights into binding of therapeutic channel blockers in NMDA receptors.

Excitatory signaling mediated by N-methyl-D-aspartate receptor (NMDAR) is critical for brain development and function, as well as for neurological diseases and disorders. Channel blockers of NMDARs are of medical interest owing to their potential for treating depression, Alzheimer's disease, and epilepsy. However, precise mechanisms underlying binding and channel blockade have remained limited owing to challenges in obtaining high-resolution structures at the binding site within the transmembrane domains. Here, we monitor the binding of three clinically important channel blockers: phencyclidine, ketamine, and memantine in GluN1-2B NMDARs at local resolutions of 2.5-3.5 Å around the binding site using single-particle electron cryo-microscopy, molecular dynamics simulations, and electrophysiology. The channel blockers form different extents of interactions with the pore-lining residues, which control mostly off-speeds but not on-speeds. Our comparative analyses of the three unique NMDAR channel blockers provide a blueprint for developing therapeutic compounds with minimal side effects.

Effects of NMDA receptor antagonists on behavioral economic indices of cocaine self-administration

BackgroundCurrently, there are no FDA-approved medications for the treatment of psychostimulant (e.g., cocaine) use disorders. Because the GluN2B subunit of the glutamate N-methyl-D-aspartate (NMDA) receptor is an important mediator of addiction-like behaviors, the goal of the current study was to determine if the GluN2B-selective antagonist Ro 63–1908 is efficacious in attenuating cocaine self-administration. MethodsAdult Sprague Dawley rats (24 males and 11 females) were implanted with indwelling catheters and were trained to self-administer cocaine (0.75 mg/kg/inf). Rats were then trained in a threshold procedure, in which the dose of cocaine decreased across six 6-min blocks (0.75, 0.27, 0.08, 0.03, 0.01, 0.003 mg/kg/inf). This procedure allowed for the quantification of behavioral economic indices of drug self-administration. Following training in the threshold procedure, rats were treated with the GluN2B-selective antagonist Ro 63–1908 (0, 0.1, 0.3, 1.0 mg/kg; s.c.). Rats also received treatments of the NMDA receptor channel blocker MK-801 (0, 0.01, 0.03, 0.06 mg/kg; s.c.). ResultsBlocking NMDA receptors decreased initial intake (i.e., consumption during the first block), although Ro 63–1908 and MK-801 increased area under the curve (global measure of demand) and decreased demand elasticity, an effect observed primarily in males. Neither drug affected demand intensity (i.e., consumption of cocaine at a minimally constrained price). ConclusionsWhile blocking the NMDA receptor decreases initial intake of cocaine, NMDA receptor antagonists make cocaine more inelastic with increasing price. These results suggest that NMDA receptor antagonists can exacerbate addiction-like behaviors during self-administration during extinction-like conditions that are observed in later blocks of the threshold procedure.

Imprecision in Precision Medicine: Differential Response of a Disease-Linked GluN2A Mutant to NMDA Channel Blockers.

Mutations in N-methyl-d-aspartate receptors (NMDAR) subunits have been implicated in a growing number of human neurodevelopmental disorders. Previously, a de novo mutation in GRIN2A, encoding the GluN2A subunit, was identified in a patient with severe epilepsy and developmental delay. This missense mutation, which leads to GluN2A-P552R, produces significant dendrotoxicity in transfected rodent cortical neurons, as evidenced by pronounced dendritic blebbing. This injurious process can be prevented by treatment with the NMDA antagonist memantine. Given the increasing use of FDA approved NMDA antagonists to treat patients with GRIN mutations, who may have seizures refractory to traditional anti-epileptic drugs, we investigated whether additional NMDA antagonists were effective in attenuating neurotoxicity associated with GluN2A-P552R expression. Intriguingly, we found that while treatment with memantine can effectively block GluN2A-P552R-mediated dendrotoxicity, treatment with ketamine does not, despite the fact that both drugs work as open NMDAR channel blockers. Interestingly, we found that neurons expressing GluN2A-P552R were more vulnerable to an excitotoxic insult—an effect that, in this case, could be equally rescued by both memantine and ketamine. These findings suggest that GluN2A-P552R induced dendrotoxicity and increased vulnerability to excitotoxic stress are mediated through two distinct mechanisms. The differences between memantine and ketamine in halting GluN2A-P552R dendrotoxicity could not be explained by NMDA antagonist induced changes in MAP or Src kinase activation, previously shown to participate in NMDA-induced excitotoxicity. Our findings strongly suggest that not all NMDA antagonists may be of equal clinical utility in treating GRIN2A-mediated neurological disorders, despite a shared mechanism of action.

Adenosine and Kynurenic Acid Interactions: Possible Relevance for Schizophrenia Treatment?

Adenosine and Kynurenic Acid Interactions: Possible Relevance for Schizophrenia Treatment?

Agmatine requires GluN2B-containing NMDA receptors to inhibit the development of neuropathic pain.

A decarboxylated form of L-arginine, agmatine, preferentially antagonizes NMDArs containing Glun2B subunits within the spinal cord and lacks motor side effects commonly associated with non-subunit-selective NMDAr antagonism, namely sedation and motor impairment. Spinally delivered agmatine has been previously shown to reduce the development of tactile hypersensitivity arising from spinal nerve ligation. The present study interrogated the dependence of agmatine’s alleviation of neuropathic pain (spared nerve injury (SNI) model) on GluN2B-containing NMDArs. SNI-induced hypersensitivity was induced in mice with significant reduction of levels of spinal GluN2B subunit of the NMDAr and their floxed controls. Agmatine reduced development of SNI-induced tactile hypersensitivity in controls but had no effect in subjects with reduced levels of GluN2B subunits. Ifenprodil, a known GluN2B-subunit-selective antagonist, similarly reduced tactile hypersensitivity in controls but not in the GluN2B-deficient mice. In contrast, MK-801, an NMDA receptor channel blocker, reduced hypersensitivity in both control and GluN2B-deficient mice, consistent with a pharmacological pattern expected from a NMDAr antagonist that does not have preference for GluN2B subtypes. Additionally, we observed that spinally delivered agmatine, ifenprodil and MK-801 inhibited nociceptive behaviors following intrathecal delivery of NMDA in control mice. By contrast, in GluN2B-deficient mice, MK-801 reduced NMDA-evoked nociceptive behaviors, but agmatine had a blunted effect and ifenprodil had no effect. These results demonstrate that agmatine requires the GluN2B subunit of the NMDA receptor for inhibitory pharmacological actions in pre-clinical models of NMDA receptor-dependent hypersensitivity.

Roles of N-methyl-D-aspartate receptors and D-amino acids in cancer cell viability.

N-methyl-D-aspartate (NMDA) receptors, which are widely present in the central nervous system, have also been found to be up-regulated in a variety of cancer cells and tumors and they can play active roles in cancer cell growth regulation. NMDA receptor antagonists have been found to affect cancer cell viability and interfere with tumor growth. Moreover, cancer cells also have been shown to have elevated levels of some D-amino acids. Two human skin cell lines: Hs 895.T skin cancer and Hs 895.Sk skin normal cells were investigated. They were derived from the same patient to provide tumor and normal counterparts for comparative studies. The expression of specific NMDA receptors was confirmed for the first time in both skin cell lines. Dizocilpine (MK-801) and memantine, NMDA receptor channel blockers, were found to inhibit the growth of human skin cells by reducing or stopping NMDA receptor activity. Addition of D-Ser, D-Ala, or D-Asp, however, significantly reversed the antiproliferative effect on the human skin cells triggered by MK-801 or memantine. Even more interesting was the finding that the specific intracellular composition of a few relatively uncommon amino acids was selectively elevated in skin cancer cells when exposed to MK-801. It appears that a few specific and upregulated D-amino acids can reverse the drug-induced antiproliferative effect in skin cancer cells via the reactivation of NMDA receptors. This study provides a possible innovative anticancer therapy by acting on the D-amino acid pathway in cancer cells either blocking or activating their regulatory enzymes.

Leptin Sensitizes NTS Neurons to Vagal Input by Increasing Postsynaptic NMDA Receptor Currents.

Leptin signaling within the nucleus of the solitary tract (NTS) contributes to the control of food intake, and injections of leptin into the NTS reduce meal size and increase the efficacy of vagus-mediated satiation signals. Leptin receptors (LepRs) are expressed by vagal afferents as well as by a population of NTS neurons. However, the electrophysiological properties of LepR-expressing NTS neurons have not been well characterized, and it is unclear how leptin might act on these neurons to reduce food intake. To address this question, we recorded from LepR-expressing neurons in horizontal brain slices containing the NTS from male and female LepR-Cre X Rosa-tdTomato mice. We found that the vast majority of NTS LepR neurons received monosynaptic innervation from vagal afferent fibers and LepR neurons exhibited large synaptic NMDA receptor (NMDAR)-mediated currents compared with non-LepR neurons. During high-frequency stimulation of vagal afferents, leptin increased the size of NMDAR-mediated currents, but not AMPAR-mediated currents. Leptin also increased the size of evoked EPSPs and the ability of low-intensity solitary tract stimulation to evoke action potentials in LepR neurons. These effects of leptin were blocked by bath applying a competitive NMDAR antagonist (DCPP-ene) or by an NMDAR channel blocker applied through the recording pipette (MK-801). Last, feeding studies using male rats demonstrate that intra-NTS injections of DCPP-ene attenuate reduction of overnight food intake following intra-NTS leptin injection. Our results suggest that leptin acts in the NTS to reduce food intake by increasing NMDAR-mediated currents, thus enhancing NTS sensitivity to vagal inputs.SIGNIFICANCE STATEMENT Leptin is a hormone that critically impacts food intake and energy homeostasis. The nucleus of the solitary tract (NTS) is activated by vagal afferents from the gastrointestinal tract, which promotes termination of a meal. Injection of leptin into the NTS inhibits food intake, while knockdown of leptin receptors (LepRs) in NTS neurons increases food intake. However, little was known about how leptin acts in the NTS neurons to inhibit food intake. We found that leptin increases the sensitivity of LepR-expressing neurons to vagal inputs by increasing NMDA receptor-mediated synaptic currents and that NTS NMDAR activation contributes to leptin-induced reduction of food intake. These findings suggest a novel mechanism by which leptin, acting in the NTS, could potentiate gastrointestinal satiation signals.

Differential effects of traxoprodil and S-ketamine on quantitative EEG and auditory event-related potentials as translational biomarkers in preclinical trials in rats and mice

Quantitative Electroencephalography (qEEG) and event-related potential (ERP) assessment have emerged as powerful tools to unravel translational biomarkers in preclinical and clinical psychiatric drug discovery trials. The aim of the present study was to compare the GluN2B negative allosteric modulator (NAM) traxoprodil (CP-101,606) with the unselective NMDA receptor channel blocker S-ketamine to give insight into central target engagement and differentiation on multiple EEG readouts.For qEEG recordings telemetric transmitters were implanted in male Wistar rats. Recorded EEG data were analyzed using fast Fourier transformation to determine power spectra and vigilance states. Additionally, body temperature and locomotor activity were assessed via telemetry. For recordings of auditory event-related potentials (AERP) male C57Bl/6J mice were chronically implanted with deep electrodes using a tethered system.Power spectral analysis revealed a significant increase in gamma power following ketamine treatment, whereas traxoprodil (6&18 mg/kg) induced an overall decrease primarily within alpha and beta bands. Additionally, ketamine disrupted sleep and enhanced time spent in wake vigilance states, whereas traxoprodil did not alter sleep-wake architecture. AERP and mismatch negativity (MMN) revealed that ketamine (10 mg/kg) selectively disrupts auditory deviance detection, whereas traxoprodil (6 mg/kg) did not alter MMN at clinically relevant doses. In contrast to ketamine treatment, traxoprodil did not produce hyperactivity and hypothermia.In conclusion, ketamine and traxoprodil showed very different effects on diverse EEG readouts differentiating selective GluN2B antagonism from non-selective pan-NMDA-R antagonists like ketamine. These readouts are thus perfectly suited to support drug discovery efforts on NMDA-R and understanding the different functions of NMDA-R subtypes.

Identification of tetracyclic lactams as NMDA receptor antagonists with potential application in neurological disorders

N-Methyl-d-aspartate receptors (NMDARs) are crucial for the normal function of the central nervous system (CNS), and fundamental in memory and learning-related processes. The overactivation of these receptors is associated with numerous neurodegenerative and psychiatric disorders. Therefore, NMDAR is considered a relevant therapeutic target for many CNS disorders.Herein, we report the synthesis and pharmacological evaluation of a new scaffold with antagonistic activity for NMDAR. Specifically, a chemical library of eighteen 1-aminoindan-2-ol tetracyclic lactams was synthesized and screened as NMDAR antagonists. The compounds were obtained by chiral pool synthesis using enantiomerically pure 1-aminoindan-2-ols as chiral inductors, and their stereochemistry was proven by X-ray crystallographic analysis of two target compounds. Most compounds reveal NMDAR antagonism, and eleven compounds display IC50 values in a Ca2+ entry-sensitive fluo-4 assay in the same order of magnitude of memantine, a clinically approved NMDAR antagonist. Docking studies suggest that the novel compounds can act as NMDAR channel blockers since there is a compatible conformation with MK-801 co-crystallized with NMDAR channel. In addition, we show that the tetracyclic 1-aminoindan-2-ol derivatives are brain permeable and non-toxic, and we identify promising hits for further optimization as modulators of the NMDAR function.

The NMDA Receptor Channel Blockers Memantine and IEM-1921 Decrease the Duration of Status Epilepticus in Wistar and Krushinskii–Molodkina Rats in a Lithium-Pilocarpine Model

Chronic experiments on Wistar and Krushinskii–Molodkina rats, which have an inherited predisposition to audiogenic convulsions, addressed the effects of preliminary administration of memantine and IEM-1921 – NMDA glutamate receptor channel blockers – on brain electrical activity on development of status epilepticus (SE) induced by pilocarpine. Electrograms were recorded from the hippocampus, caudate nucleus, and the somatosensory, visual, and auditory areas of the cortex. The nature and sequence of changes in the phases of SE identifi ed from electrogram patterns were not altered by blockers. In addition, the latent period of the onset of epileptiform activity on the EEG after administration of pilocarpine to Wistar rats decreased almost two-fold (to 10–15 min) after administration of memantine (5 mg/kg) or IEM-1921 (5 mg/kg). In Krushinskii–Molodkina rats, the latent period of development of epileptiform activity was initially shorter (13 ± 2 min) and was not altered by use of blockers. The mean durations of SE after administration of memantine to Wistar and Krushinskii–Molodkina rats were 2 and 3 times, respectively, shorter than without blocker, while durations were reduced seven-fold by IEM-1921. NMDA receptor blockers also signifi cantly weakened behavioral motor convulsions, decreasing the intensity of convulsions from 8 to 4 points on the Pinel and Rovner [1978] scale. These data provide evidence that blockade of NMDA receptors did not prevent the typical development of pilocarpine-induced SE, though duration was signifi cantly reduced.

NMDA receptors containing GluN2C and GluN2D subunits have opposing roles in modulating neuronal oscillations; potential mechanism for bidirectional feedback

NMDA receptor (NMDAR) antagonists such as ketamine, can reproduce many of the symptoms of schizophrenia. A reliable indicator of NMDAR channel blocker action in vivo is the augmentation of neuronal oscillation power. Since the coordinated and rhythmic activation of neuronal assemblies (oscillations) is necessary for perception, cognition and working memory, their disruption (inappropriate augmentation or inhibition of oscillatory power or inter-regional coherence) both in psychiatric conditions and with NMDAR antagonists may reflect the underlying defects causing schizophrenia symptoms. NMDAR antagonists and knockout (KO) mice were used to evaluate the role of GluN2C and GluN2D NMDAR subunits in generating NMDAR antagonist-induced oscillations. We find that basal oscillatory power was elevated in GluN2C-KO mice, especially in the low gamma frequencies while there was no statistically significant difference in basal oscillations between WT and GluN2D-KO mice. Compared to wildtype (WT) mice, NMDAR channel blockers caused a greater increase in oscillatory power in GluN2C-KO mice and were relatively ineffective in inducing oscillations in GluN2D-KO mice. In contrast, preferential blockade of GluN2A- and GluN2B-containing receptors induced oscillations that did not appear to be changed in either KO animal. We propose a model wherein NMDARs containing GluN2C in astrocytes and GluN2D in interneurons serve to detect local cortical excitatory synaptic activity and provide excitatory and inhibitory feedback, respectively, to local populations of postsynaptic excitatory neurons and thereby bidirectionally modulate oscillatory power.

NYX-2925 induces metabotropic N-methyl-d-aspartate receptor (NMDAR) signaling that enhances synaptic NMDAR and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor.

N‐methyl‐d‐aspartate receptors (NMDARs) mediate both physiological and pathophysiological processes, although selective ligands lack broad clinical utility. NMDARs are composed of multiple subunits, but N‐methyl‐d‐aspartate receptor subunit 2 (GluN2) is predominately responsible for functional heterogeneity. Specifically, the GluN2A‐ and GluN2B‐containing subtypes are enriched in adult hippocampus and cortex and impact neuronal communication via dynamic trafficking into and out of the synapse. We sought to understand if ((2S, 3R)‐3‐hydroxy‐2‐((R)‐5‐isobutyryl‐1‐oxo‐2,5‐diazaspiro[3,4]octan‐2‐yl) butanamide (NYX‐2925), a novel NMDAR modulator, alters synaptic levels of GluN2A‐ or GluN2B‐containing NMDARs. Low‐picomolar NYX‐2925 increased GluN2B colocalization with the excitatory post‐synaptic marker post‐synaptic density protein 95 (PSD‐95) in rat primary hippocampal neurons within 30 min. Twenty‐four hours following oral administration, 1 mg/kg NYX‐2925 increased GluN2B in PSD‐95‐associated complexes ex vivo, and low‐picomolar NYX‐2925 regulated numerous trafficking pathways in vitro. Because the NYX‐2925 concentration that increases synaptic GluN2B was markedly below that which enhances long‐term potentiation (mid‐nanomolar), we sought to elucidate the basis of this effect. Although NMDAR‐dependent, NYX‐2925‐mediated colocalization of GluN2B with PSD‐95 occurred independent of ion flux, as colocalization increased in the presence of either the NMDAR channel blocker (5R,10S)‐(–)‐5‐Methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine hydrogen maleate or glycine site antagonist 7‐chlorokynurenic acid. Moreover, while mid‐nanomolar NYX‐2925 concentrations, which do not increase synaptic GluN2B, enhanced calcium transients, functional plasticity was only enhanced by picomolar NYX‐2925. Thus, NYX‐2925 concentrations that increase synaptic GluN2B facilitated the chemical long‐term potentiation induced insertion of synaptic α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor GluA1 subunit levels. Basal (unstimulated by chemical long‐term potentiation) levels of synaptic GluA1 were only increased by mid‐nanomolar NYX‐2925. These data suggest that NYX‐2925 facilitates homeostatic plasticity by initially increasing synaptic GluN2B via metabotropic‐like NMDAR signaling. Cover Image for this issue: doi: 10.1111/jnc.14735.

The human NMDA receptor GluN2AN615K variant influences channel blocker potency.

N‐methyl‐D‐aspartate (NMDA) receptors are glutamate receptors with key roles in synaptic plasticity, due in part to their Mg2+ mediated voltage‐dependence. A large number of genetic variants affecting NMDA receptor subunits have been found in people with a range of neurodevelopmental disorders, including GluN2AN615K (GRIN2A C1845A) in two unrelated individuals with severe epileptic encephalopathy. This missense variant substitutes a lysine in place of an asparagine known to be important for blockade by Mg2+ and other small molecule channel blockers. We therefore measured the impact of GluN2AN615K on a range of NMDA receptor channel blockers using two‐electrode voltage clamp recordings made in Xenopus oocytes. We found that GluN2AN615K resulted in block by Mg2+ 1 mmol/L being greatly reduced (89% vs 8%), block by memantine 10 μmol/L (76% vs 27%) and amantadine 100 μmol/L (45% vs 17%) being substantially reduced, block by ketamine 10 μmol/L being modestly reduced (79% vs 73%) and block by dextromethorphan 10 μmol/L being enhanced (45% vs 55%). Coapplying Mg2+ with memantine or amantadine did not reduce the GluN2AN615K block seen with either small molecule. In addition, we measured single–channel conductance of GluN2AN615K–containing NMDA receptors in outside‐out patches pulled from Xenopus oocytes, finding a 4‐fold reduction in conductance (58 vs 15 pS). In conclusion, the GluN2AN615K variant is associated with substantial changes to important physiological and pharmacological properties of the NMDA receptor. Our findings are consistent with GluN2AN615K having a disease–causing role, and inform potential therapeutic strategies.

Differential effect of NMDA receptor GluN2C and GluN2D subunit ablation on behavior and channel blocker-induced schizophrenia phenotypes

The GluN2C- and GluN2D-containing NMDA receptors are distinct from GluN2A- and GluN2B-containing receptors in many aspects including lower sensitivity to Mg2+ block and lack of desensitization. Recent studies have highlighted the unique contribution of GluN2C and GluN2D subunits in various aspects of neuronal and circuit function and behavior, however a direct comparison of the effect of ablation of these subunits in mice on pure background strain has not been conducted. Using knockout-first strains for the GRIN2C and GRIN2D produced on pure C57BL/6N strain, we compared the effect of partial or complete ablation of GluN2C and GluN2D subunit on various behaviors relevant to mental disorders. A large number of behaviors described previously in GluN2C and GluN2D knockout mice were reproduced in these mice, however, some specific differences were also observed possibly representing strain effects. We also examined the response to NMDA receptor channel blockers in these mouse strains and surprisingly found that unlike previous reports GluN2D knockout mice were not resistant to phencyclidine-induced hyperlocomotion. Interestingly, the GluN2C knockout mice showed reduced sensitivity to phencyclidine-induced hyperlocomotion. We also found that NMDA receptor channel blocker produced a deficit in prepulse inhibition which was prevented by a GluN2C/2D potentiator in wildtype and GluN2C heterozygous mice but not in GluN2C knockout mice. Together these results demonstrate a unique role of GluN2C subunit in schizophrenia-like behaviors.

Investigation of NMDA Receptor Channel Blockers in a Series of Methylene Blue Conjugates Using QSAR and Molecular Modeling

29 conjugates of methylene blue and four chemical structures, including derivatives of carbazole, tetrahydrocarbazole, substituted indoles and γ-carboline, combined with a 1-oxopropylene spacer have been studied as channel blockers of the NMDA receptor (binding site of MK-801) by using four QSAR methods (multiple linear regression, random forest, support vector machine, Gaussian process) and molecular docking. QSAR models have satisfactory characteristics. The analysis of regression models at the statistical level revealed an important role of the hydrogen bond in the complex formation. This was also confirmed by the study of modeled by docking complexes. It was found that the increase in the inhibitory activity of the part of compounds could be attributed to appearance of additional H bonds between the ligands and the receptor.

Correction: A Metabotropic-Like Flux-Independent NMDA Receptor Regulates Ca2+ Exit from Endoplasmic Reticulum and Mitochondrial Membrane Potential in Cultured Astrocytes

[This corrects the article DOI: 10.1371/journal.pone.0126314.].