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Objective Genetic variants in the GRIN genes that encode N‐methyl‐D‐aspartate receptor (NMDAR) subunits have been identified in various neurodevelopmental disorders, including epilepsy. We identified a GRIN1 variant from an individual with early‐onset epileptic encephalopathy, evaluated functional changes to NMDAR properties caused by the variant, and screened FDA‐approved therapeutic compounds as potential treatments for the patient. Methods Whole exome sequencing identified a missense variant in GRIN1. Electrophysiological recordings were made from Xenopus oocytes and transfected HEK cells to determine the NMDAR biophysical properties as well as the sensitivity to agonists and FDA‐approved drugs that inhibit NMDARs. A beta‐lactamase reporter assay in transfected HEK cells evaluated the effects of the variant on the NMDAR surface expression. Results A recurrent de novo missense variant in GRIN1 (c.1923G>A, p.Met641Ile), which encodes the GluN1 subunit, was identified in a pediatric patient with drug‐resistant seizures and early‐onset epileptic encephalopathy. In vitro analysis indicates that GluN1‐M641I containing NMDARs showed enhanced agonist potency and reduced Mg2+ block, which may be associated with the patient’s phenotype. Results from screening FDA‐approved drugs suggested that GluN1‐M641I containing NMDARs are more sensitive to the NMDAR channel blockers memantine, ketamine, and dextromethorphan compared to the wild‐type receptors. The addition of memantine to the seizure treatment regimen significantly reduced the patient’s seizure burden. Interpretation Our finding contributes to the understanding of the phenotype–genotype correlations of patients with GRIN1 gene variants, provides a molecular mechanism underlying the actions of this variant, and explores therapeutic strategies for treating GRIN1‐related neurological conditions.

Background: Mutations in the GRIN2A gene, which encodes the GluN2A (glutamate [NMDA] receptor subunit epsilon-1) subunit of the N-methyl-d-aspartate receptor, have been identified in patients with epilepsy-aphasia spectrum disorders, idiopathic focal epilepsies with centrotemporal spikes, and epileptic encephalopathies with severe developmental delay. However, thus far, mutations in this gene have not been associated with a nonepileptic neurodevelopmental disorder with dystonia. Objectives: The objective of this study was to identify the disease-causing gene in 2 siblings with neurodevelopmental and movement disorders with no epileptiform abnormalities. Methods: The study method was targeted next-generation sequencing panel for neuropediatric disorders and subsequent electrophysiological studies. Results: The 2 siblings carry a novel missense mutation in the GRIN2A gene (p.Ala643Asp) that was not detected in genomic DNA isolated from blood cells of their parents, suggesting that the mutation is the consequence of germinal mosaicism in 1 progenitor. In functional studies, the GluN2A-A643D mutation increased the potency of the agonists L-glutamate and glycine and decreased the potency of endogenous negative modulators, including protons, magnesium and zinc but reduced agonist-evoked peak current response in mammalian cells, suggesting that this mutation has a mixed effect on N-methyl-d-aspartate receptor function. Conclusion: De novo GRIN2A mutations can give rise to a neurodevelopmental and movement disorder without epilepsy. © 2018 International Parkinson and Movement Disorder Society.

Objective Early-onset epileptic encephalopathies have been associated with de novo mutations of numerous ion channel genes. We employed techniques of modern translational medicine to identify a disease-causing mutation, analyze its altered behavior, and screen for therapeutic compounds to treat the proband. Methods Three modern translational medicine tools were utilized: (1) high-throughput sequencing technology to identify a novel de novo mutation; (2) in vitro expression and electrophysiology assays to confirm the variant protein's dysfunction; and (3) screening of existing drug libraries to identify potential therapeutic compounds. Results A de novo GRIN2A missense mutation (c.2434C>A; p.L812M) increased the charge transfer mediated by N-methyl-D-aspartate receptors (NMDAs) containing the mutant GluN2A-L812M subunit. In vitro analysis with NMDA receptor blockers indicated that GLuN2A-L812M-containing NMDARs retained their sensitivity to the use-dependent channel blocker memantine; while screening of a previously reported GRIN2A mutation (N615K) with these compounds produced contrasting results. Consistent with these data, adjunct memantine therapy reduced our proband's seizure burden. Interpretation This case exemplifies the potential for personalized genomics and therapeutics to be utilized for the early diagnosis and treatment of infantile-onset neurological disease.

Astrocytes express a wide range of G-protein coupled receptors that trigger release of intracellular Ca2+, including P2Y, bradykinin and protease activated receptors (PARs). By using the highly sensitive sniffer-patch technique, we demonstrate that the activation of P2Y receptors, bradykinin receptors and protease activated receptors all stimulate glutamate release from cultured or acutely dissociated astrocytes. Of these receptors, we have utilized PAR1 as a model system because of favourable pharmacological and molecular tools, its prominent expression in astrocytes and its high relevance to neuropathological processes. Astrocytic PAR1-mediated glutamate release in vitro is Ca2+ dependent and activates NMDA receptors on adjacent neurones in culture. Activation of astrocytic PAR1 in hippocampal slices induces an APV-sensitive inward current in CA1 neurones and causes APV-sensitive neuronal depolarization in CA1 neurones, consistent with release of glutamate from astrocytes. PAR1 activation enhances the NMDA receptor-mediated component of synaptic miniature EPSCs, evoked EPSCs and evoked EPSPs in a Mg2+-dependent manner, which may reflect spine head depolarization and consequent reduction of NMDA receptor Mg2+ block during subsequent synaptic currents. The release of glutamate from astrocytes following PAR1 activation may also lead to glutamate occupancy of some perisynaptic NMDA receptors, which pass current following relief of tonic Mg2+ block during synaptic depolarization. These results suggest that astrocytic G-protein coupled receptors that increase intracellular Ca2+ can tune synaptic NMDA receptor responses.

We have compared the potencies of structurally distinct channel blockers at recombinant NR1/NR2A, NR1/NR2B, NR1/NR2C and NR1/NR2D receptors. The IC50 values varied with stereochemistry and subunit composition, suggesting that it may be possible to design subunit-selective channel blockers. For dizocilpine (MK-801), the differential potency of MK-801 stereoisomers determined at recombinant NMDA receptors was confirmed at native receptors in vitro and in vivo. Since the proton sensor is tightly linked both structurally and functionally to channel gating, we examined whether blocking molecules that interact in the channel pore with the gating machinery can differentially sense protonation of the receptor. Blockers capable of remaining trapped in the pore during agonist unbinding showed the strongest dependence on extracellular pH, appearing more potent at acidic pH values that promote channel closure. Determination of pKa values for channel blockers suggests that the ionization of ketamine but not of other blockers can influence its pH-dependent potency. Kinetic modelling and single channel studies suggest that the pH-dependent block of NR1/NR2A by (−)MK-801 but not (+)MK-801 reflects an increase in the MK-801 association rate even though protons reduce channel open probability and thus MK-801 access to its binding site. Allosteric modulators that alter pH sensitivity alter the potency of MK-801, supporting the interpretation that the pH sensitivity of MK-801 binding reflects the changes at the proton sensor rather than a secondary effect of pH. These data suggest a tight coupling between the proton sensor and the ion channel gate as well as unique subunit-specific mechanisms of channel block.