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Modulation of the N-methyl-D-aspartate (NMDA)-selective glutamate receptors by extracellular protons and Zn 2؉ may play important roles during ischemia in the brain and during seizures. Recombinant NR1͞ NR2A receptors exhibit a much higher apparent affinity for voltageindependent Zn 2؉ inhibition than receptors with other subunit combinations. Here, we show that the mechanism of this apparent high-affinity, voltage-independent Zn 2؉ inhibition for NR2A-containing receptors results from the enhancement of proton inhibition. We also show that the N-terminal leucine͞isoleucine͞valine binding protein (LIVBP)-like domain of the NR2A subunit contains critical determinants of the apparent high-affinity, voltage-independent Zn 2؉ inhibition. Mutations H42A, H44G, or H128A greatly increase the Zn 2؉ IC50 (by up to Ϸ700-fold) with no effect on the potencies of glutamate and glycine or on voltage-dependent block by Mg 2؉ . Furthermore, the amino acid residue substitution H128A, which mediates the largest effect on the apparent high-affinity Zn 2؉ inhibition among all histidine substitutions we tested, is also critical to the pH-dependency of Zn 2؉ inhibition. Our data revealed a unique interaction between two important extracellular modulators of NMDA receptors.G lutamate receptors are ligand-gated ion channels that mediate excitatory synaptic transmission in the central nervous system. Although overactivation of the N-methyl-D-aspartate (NMDA)-selective glutamate receptors can trigger neurodegeneration in neuropathological conditions such as stroke, NMDA receptor function is regulated under normal conditions by several extracellular ions (Mg 2ϩ , H ϩ , and Zn 2ϩ ), which exert strong tonic inhibition in a subunit selective manner (1). Inhibition by extracellular protons is particularly relevant for the neuropathological consequences of occlusive stroke because acidification of the extracellular environment during ischemia has been hypothesized to inhibit NMDA receptor overactivation by extrasynaptic glutamate that accumulates following metabolic failure (2, 3). The proton inhibition of NMDA receptors could delay their contribution to subsequent neuronal death until the pH gradients are restored, and this delay may provide a therapeutic window for postinsult treatment with NMDA receptor antagonists. Acidification of the extracellular space during electrographic seizure may also contribute to seizure termination through inhibition of NMDA receptor function (4, 5). Because of these potentially important aspects of the pH sensitivity of the NMDA receptor, we have sought to understand the structural basis by which the receptor might control its regulation by extracellular protons.In the central nervous system, the extracellular Zn 2ϩ concentration has been shown to vary under normal brain function as well as neuropathological conditions (6, 7). In addition, there are large amounts of chelatable Zn 2ϩ in the glutamatergic terminals of hippocampus (8-10), which are released in a Ca 2ϩ -dependent manner (for review, see ref. 6). ...

The advent of whole exome/genome sequencing and the technology-driven reduction in the cost of next-generation sequencing as well as the introduction of diagnostic-targeted sequencing chips have resulted in an unprecedented volume of data directly linking patient genomic variability to disorders of the brain. This information has the potential to transform our understanding of neurologic disorders by improving diagnoses, illuminating the molecular heterogeneity underlying diseases, and identifying new targets for therapeutic treatment. There is a strong history of mutations in GABA receptor genes being involved in neurologic diseases, particularly the epilepsies. In addition, a substantial number of variants and mutations have been found in GABA receptor genes in patients with autism, schizophrenia, and addiction, suggesting potential links between the GABA receptors and these conditions. A new and unexpected outcome from sequencing efforts has been the surprising number of mutations found in glutamate receptor subunits, with the GRIN2A gene encoding the GluN2A N-methyl-D-aspartate receptor subunit being most often affected. These mutations are associated with multiple neurologic conditions, for which seizure disorders comprise the largest group. The GluN2A subunit appears to be a locus for epilepsy, which holds important therapeutic implications. Virtually all a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor mutations, most of which occur within GRIA3, are from patients with intellectual disabilities, suggesting a link to this condition. Similarly, the most common phenotype for kainate receptor variants is intellectual disability. Herein, we summarize the current understanding of disease-associated mutations in ionotropic GABA and glutamate receptor families, and discuss implications regarding the identification of human mutations and treatment of neurologic diseases.

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Extracellular protons inhibit N-methyl-D-aspartate (NMDA) receptors with an IC 50 value in the physiological pH range. To identify the molecular determinants of proton sensitivity, we used scanning mutagenesis of the NR1 subunit to search for residues that control proton inhibition of NMDA receptors. Homology modeling of the extracellular domains suggested that residues at which mutations perturbed pH sensitivity were localized in discrete regions. The majority of mutations that strongly affected proton sensitivity were clustered in the extracellular end of the second transmembrane domain (M3) and adjacent linker leading to the S2 portion of the glycine-binding domain of NR1. Mutations in NR2A confirmed that the analogous region controls the pH sensitivity of this subunit and also identified the linker region between the third transmembrane domain (M4) and the S2 portion of the NR2 glutamate binding domain as an additional determinant of proton sensitivity. One mutant receptor, NR1(A649C)/NR2A(A651T), showed a 145-fold reduction in the IC 50 for protons (IC 50 , 17.3 M corresponding to pH 4.9). The M3-S2 linker region has been suggested to control NMDA receptor gating, leading to the hypothesis that the proton sensor and receptor gate may be structurally and functionally integrated.

Fast desensitization is an important regulatory mechanism of neuronal NMDA receptor function. Only recombinant NMDA receptors composed of NR1/NR2A exhibit a fast component of desensitization similar to neuronal NMDA receptors. Here we report that the fast desensitization of NR1/NR2A receptors is caused by ambient zinc, and that a positive allosteric interaction occurs between the extracellular zinc-binding site located in the amino terminal domain and the glutamate-binding domain of NR2A. The relaxation of macroscopic currents reflects a shift to a new equilibrium due to increased zinc affinity after binding of glutamate. We also show a similar interaction between the ifenprodil binding site and the glutamate binding site of NR1/NR2B receptors. These data raise the possibility that there is an allosteric interaction between the amino terminal domain and the ligand-binding domain of other glutamate receptors. Our findings may provide insight into how zinc and other extracellular modulators regulate NMDA receptor function.

Rapid advances in sequencing technology have led to an explosive increase in the number of genetic variants identified in patients with neurological disease and have also enabled the assembly of a robust database of variants in healthy individuals. A surprising number of variants in the GRIN genes that encode N-methyl-D-aspartate (NMDA) glutamatergic receptor subunits have been found in patients with various neuropsychiatric disorders, including autism spectrum disorders, epilepsy, intellectual disability, attention-deficit/hyperactivity disorder, and schizophrenia. This review compares and contrasts the available information describing the clinical and functional consequences of genetic variations in GRIN2A and GRIN2B. Comparison of clinical phenotypes shows that GRIN2A variants are commonly associated with an epileptic phenotype but that GRIN2B variants are commonly found in patients with neurodevelopmental disorders. These observations emphasize the distinct roles that the gene products serve in circuit function and suggest that functional analysis of GRIN2A and GRIN2B variation may provide insight into the molecular mechanisms, which will allow more accurate subclassification of clinical phenotypes. Furthermore, characterization of the pharmacological properties of variant receptors could provide the first opportunity for translational therapeutic strategies for these GRIN-related neurological and psychiatric disorders.

The deregulation of cyclin-dependent kinase 5 (Cdk5) by p25 has been shown to contribute to the pathogenesis in a number of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD) and Alzheimer's disease (AD). In particular, p25/Cdk5 has been shown to produce hyperphosphorylated tau, neurofibrillary tangles as well as aberrant amyloid precursor protein processing found in AD. Neuroinflammation has been observed alongside the pathogenic process in these neurodegenerative diseases, however the precise mechanism behind the induction of neuroinflammation and the significance in the AD pathogenesis has not been fully elucidated. In this report, we uncover a novel pathway for p25-induced neuroinflammation where p25 expression induces an early trigger of neuroinflammation in vivo in mice. Lipidomic mass spectrometry, in vitro coculture and conditioned media transfer experiments show that the soluble lipid mediator lysophosphatidylcholine (LPC) is released by p25 overexpressing neurons to initiate astrogliosis, neuroinflammation and subsequent neurodegeneration. Reverse transcriptase PCR and gene silencing experiments show that cytosolic phospholipase 2 (cPLA2) is the key enzyme mediating the p25-induced LPC production and cPLA2 upregulation is critical in triggering the p25-mediated inflammatory and neurodegenerative process. Together, our findings delineate a potential therapeutic target for the reduction of neuroinflammation in neurodegenerative diseases including AD.