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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. [ABSTRACT FROM AUTHOR]

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 autismspectrum 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. [ABSTRACT FROM AUTHOR]

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 GluNl (G638; 1642) and GluN2B (W559; M562; Y823; M824) subunit. Molecular dynamics simulation indicates that, in the absence of PES, the GluN2B Ml helix residue W559 interacts with the M4 helix residue M824. In the presence of PES, the Ml and M4 helices of agonist-activated receptor rearrange, forming a tighter interaction with the GluNl M3 helix residues G638 and 1642. This stabilizes the open-state position of the GluNl 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. [ABSTRACT FROM AUTHOR]

Abstract: In order to interpret the effects of melatonin ligands in rats, we need to determine their activity at the receptor subtype level in the corresponding species. Thus, the rat melatonin rMT1 receptor was cloned using DNA fragments for exon 1 and 2 amplified from rat genomic DNA followed by screening of a rat genomic library for the full length exon sequences. The rat rMT2 receptor subtype was cloned in a similar manner with the exception of exon 1 which was identified by screening a rat genomic library with exon 1 of the human hMT2 receptor. The coding region of these receptors translates proteins of 353 and 364 amino acids, respectively, for rMT1 and rMT2. A 55% homology was observed between both rat isoforms. The entire contiguous rat MT1 and MT2 receptor coding sequences were cloned, stably expressed in CHO cells and characterized in binding assay using 2-[125I]-Iodomelatonin. The dissociation constants (K d) for rMT1 and rMT2 were 42 and 130 pM, respectively. Chemically diverse compounds previously characterized at human MT1 and MT2 receptors were evaluated at rMT1 and rMT2 receptors, for their binding affinity and functionality in [35S]-GTPγS binding assay. Some, but not all, compounds shared a similar binding affinity and functionality at both rat and human corresponding subtypes. A different pharmacological profile of the MT1 subtype has also been observed previously between human and ovine species. These in vitro results obtained with the rat melatonin receptors are thus of importance to understand the physiological roles of each subtype in animal models. [Copyright &y& Elsevier]

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 Grin2b L825V , 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 Grin2b L825V/+ (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., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)

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., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Multiple molecular targets have been identified to mediate membrane-delimited and nongenomic effects of natural and synthetic steroids, but the influence of steroid metabolism on neuroactive steroid signaling is not well understood. To begin to address this question, we set out to identify major metabolites of a neuroprotective synthetic steroid 20-oxo-5β-pregnan-3α-yl l-glutamyl 1-ester (pregnanolone glutamate, PAG) and characterize their effects on GABA A and NMDA receptors (GABARs, NMDARs) and their influence on zebrafish behavior. Gas chromatography-mass spectrometry was used to assess concentrations of PAG and its metabolites in the hippocampal tissue of juvenile rats following intraperitoneal PAG injection. PAG is metabolized in the peripheral organs and nervous tissue to 20-oxo-17α-hydroxy-5β-pregnan-3α-yl l-glutamyl 1-ester (17-hydroxypregnanolone glutamate, 17-OH-PAG), 3α-hydroxy-5β-pregnan-20-one (pregnanolone, PA), and 3α,17α-dihydroxy-5β-pregnan-20-one (17-hydroxypregnanolone, 17-OH-PA). Patch-clamp electrophysiology experiments in cultured hippocampal neurons demonstrate that PA and 17-OH-PA are potent positive modulators of GABARs, while PAG and 17-OH-PA have a moderate inhibitory effect at NMDARs. PAG, 17-OH-PA, and PA diminished the locomotor activity of zebrafish larvae in a dose-dependent manner. Our results show that PAG and its metabolites are potent modulators of neurotransmitter receptors with behavioral consequences and indicate that neurosteroid-based ligands may have therapeutic potential.

Pituitary gonadotrophs express non-desensitizing gonadotropin-releasing hormone (GnRH) receptors and their activations leads to inositol 1,4,5-trisphosphate (InsP3)-dependent Ca2+ mobilization. When added in physiological concentration range GnRH induces baseline Ca2+ oscillations, whereas in higher concentrations it induces a prolonged spike response accompanied with non-oscillatory or oscillatory plateau response. Here, we studied the recovery of calcium signaling during repetitive stimulation with short (10-30 s) GnRH pulses and variable interpulse intervals in neonatal gonadotrophs perfused with Ca2+/Na+ -containing, Ca2+ -deficient/Na+ -containing, and Ca2+ -containing/Na+ -deficient media. In Ca2+/Na+ -containing medium, baseline Ca2+ oscillations recovered without refractory period and with a time constant of approximately 20 s, whereas the recovery of spike response occurred after 25-35 s refractory period and with a time constant of approximately 30 s. During repetitive GnRH stimulation, removal of Ca2+ had only a minor effect on baseline oscillations but abolished spike response, whereas removal of Na+ slightly extended duration of baseline oscillations and considerably prolonged spike response. These results indicate that two calcium handling mechanisms are operative in gonadotrophs: redistribution of calcium within InsP3-sensitive and -insensitive pools and a sodium-dependent calcium efflux followed by calcium influx. Redistribution of Ca2+ within the cell leads to rapid recovery of InsP3-dependent pool, whereas the Na+ -dependent Ca2+ efflux pathway is activated by spike response and limits the time of exposure to elevated cytosolic Ca2+ concentrations.