Search

N-methyl-D-aspartate receptors (NMDARs) are ligand-gated ionotropic glutamate receptors that mediate a calcium-permeable component to fast excitatory neurotransmission. NMDARs are heterotetrameric assemblies of two obligate GluN1 subunits (GRIN1) and two GluN2 subunits (GRIN2A-GRIN2D). Sequencing data shows that 43% (297/679) of all currently known NMDAR disease-associated genetic variants are within the GRIN2A gene, which encodes the GluN2A subunit. Here, we show that unlike missense GRIN2A variants, individuals affected with disease-associated null GRIN2A variants demonstrate a transient period of seizure susceptibility that begins during infancy and diminishes near adolescence. We show increased circuit excitability and CA1 pyramidal cell output in juvenile mice of both Grin2a+/− and Grin2a−/− mice. These alterations in somatic spiking are not due to global upregulation of most Grin genes (including Grin2b). Deeper evaluation of the developing CA1 circuit led us to uncover age- and Grin2a gene dosing-dependent transient delays in the electrophysiological maturation programs of parvalbumin (PV) interneurons. We report that Grin2a+/+ mice reach PV cell electrophysiological maturation between the neonatal and juvenile neurodevelopmental timepoints, with Grin2a+/− mice not reaching PV cell electrophysiological maturation until preadolescence, and Grin2a−/− mice not reaching PV cell electrophysiological maturation until adulthood. Overall, these data may represent a molecular mechanism describing the transient nature of seizure susceptibility in disease-associated null GRIN2A patients. Null GRIN2A human patients display a largely transient seizure burden that resolves with age, which may be attributable to a transient delay in the developmental maturation of parvalbumin-positive interneurons in CA1 as is observed in Grin2a+/− and Grin2a-/- mice. [ABSTRACT FROM AUTHOR]

Glutamate receptors are essential ligand‐gated ion channels in the central nervous system that mediate excitatory synaptic transmission in response to the release of glutamate from presynaptic terminals. The structural and biophysical basis underlying the function of these receptors has been studied for decades by a wide range of approaches. However recent structural, pharmacological and genetic studies have provided new insight into the regions of this protein that are critical determinants of receptor function. Lack of variation in specific areas of the protein amino acid sequences in the human population has defined three regions in each receptor subunit that are under selective pressure, which has focused research efforts and driven new hypotheses. In addition, these three closely positioned elements reside near a cavity that is shown by multiple studies to be a likely site of action for allosteric modulators, one of which is currently in use as an FDA‐approved anticonvulsant. These structural elements are capable of controlling gating of the pore, and appear to permit some modulators bound within the cavity to also alter permeation properties. This creates a new precedent whereby features of the channel pore can be modulated by exogenous drugs that bind outside the pore. The convergence of structural, genetic, biophysical and pharmacological approaches is a powerful means to gain insight into the complex biological processes defined by neurotransmitter receptor function. [ABSTRACT FROM AUTHOR]

N-methyl- d -aspartate receptors (NMDARs), a subset of ligand-gated ionotropic glutamate receptors, are critical for learning, memory, and neuronal development. However, when NMDAR subunits are mutated, a host of neuropathological conditions can occur, including epilepsy. Recently, genetic variation within the GRIN2D gene, which encodes the GluN2D subunit of the NMDAR, has been associated with a set of early-onset neurological diseases, notably developmental and epileptic encephalopathy (DEE). Importantly, patients with GRIN2D variants are largely refractory to conventional anti-epileptic drug (AED) treatment, highlighting the need to further understand the distinctive characteristics of GluN2D in neurological and pathological functions. In this review, we first summarize GluN2D's unique spatial and temporal expression patterns, electrophysiological profiles, and contributions to both pre- and postsynaptic signaling. Next, we review thirteen unique case studies from DEE patients harboring ten different causal GRIN2D variants. These patients are highly heterogenous, manifesting multiple seizure types, electroencephalographic recordings, and neurological and developmental outcomes. Lastly, this review concludes by highlighting the difficulty in treating patients with DEE-associated GRIN2D variants, and stresses the need for selective therapeutic agents delivered within a precise time window. [ABSTRACT FROM AUTHOR]

N-methyl-D-aspartate receptors (NMDARs), ligand-gated ionotropic glutamate receptors, play key roles in normal brain development and various neurological disorders. Here we use standing variation data from the human population to assess which protein domains within NMDAR GluN1, GluN2A and GluN2B subunits show the strongest signal for being depleted of missense variants. We find that this includes the GluN2 pre-M1 helix and linker between the agonist-binding domain (ABD) and first transmembrane domain (M1). We then evaluate the functional changes of multiple missense mutations in the NMDAR pre-M1 helix found in children with epilepsy and developmental delay. We find mutant GluN1/GluN2A receptors exhibit prolonged glutamate response time course for channels containing 1 or 2 GluN2A-P552R subunits, and a slow rise time only for receptors with 2 mutant subunits, suggesting rearrangement of one GluN2A pre-M1 helix is sufficient for rapid activation. GluN2A-P552R and analogous mutations in other GluN subunits increased the agonist potency and slowed response time course, suggesting a functionally conserved role for this residue. Although there is no detectable change in surface expression or open probability for GluN2A-P552R, the prolonged response time course for receptors that contained GluN2A-P552R increased charge transfer for synaptic-like activation, which should promote excitotoxic damage. Transfection of cultured neurons with GluN2A-P552R prolonged EPSPs, and triggered pronounced dendritic swelling in addition to excitotoxicity, which were both attenuated by memantine. These data implicate the pre-M1 region in gating, provide insight into how different subunits contribute to gating, and suggest that mutations in the pre-M1 helix can compromise neuronal health. Evaluation of FDA-approved NMDAR inhibitors on the mutant NMDAR-mediated current response and neuronal damage provides a potential clinical path to treat individuals harboring similar mutations in NMDARs. [ABSTRACT FROM AUTHOR]

N -methyl- d -aspartate receptors (NMDARs) mediate a slow component of excitatory synaptic transmission that plays important roles in normal brain function and development. A large number of disease-associated variants in the GRIN gene family encoding NMDAR GluN subunits have been identified in patients with various neurological and neuropsychiatric disorders. Many of these variants reduce the function of NMDARs by a range of different mechanisms, including reduced glutamate potency, reduced glycine potency, accelerated deactivation time course, decreased surface expression, and/or reduced open probability. We have evaluated whether three positive allosteric modulators of NMDAR receptor function (24(S)-hydroxycholesterol, pregnenolone sulfate, tobramycin) and three co-agonists (d -serine, l -serine, and d -cycloserine) can mitigate the diminished function of NMDARs harboring GRIN variants. We examined the effects of these modulators on NMDARs that contained 21 different loss-of-function variants in GRIN1 , GRIN2A , or GRIN2B, identified in patients with epilepsy, intellectual disability, autism, and/or movement disorders. For all variants, some aspect of the reduced function was partially restored. Moreover, some variants showed enhanced sensitivity to positive allosteric modulators compared to wild type receptors. These results raise the possibility that enhancement of NMDAR function by positive allosteric modulators may be a useful therapeutic strategy. • Effects of PAMs and co-agonists on disease-associated GRIN variants were evaluated. • PAMs can enhance function of NMDARs harboring loss-of-function GRIN variants. • Co-agonists can augment glycine to enhance NMDAR function. • Enhancement of NMDAR function by PAMs may be a useful therapeutic strategy. [ABSTRACT FROM AUTHOR]

NMDA receptors are ligand-gated ion channels that mediate excitatory neurotransmission. Most native NMDA receptors are tetrameric assemblies of two glycine-binding GluN1 and two glutamate-binding GluN2 subunits. Co-assembly of the glycine-binding GluN1 with glycine-binding GluN3 subunits (GluN3A-B) creates glycine activated receptors that possess strikingly different functional and pharmacological properties compared to GluN1/GluN2 NMDA receptors. The role of GluN1/GluN3 receptors in neuronal function remains unknown, in part due to lack of pharmacological tools with which to explore their physiological roles. We have identified the negative allosteric modulator EU1180-438, which is selective for GluN1/GluN3 receptors over GluN1/GluN2 NMDA receptors, AMPA, and kainate receptors. EU1180-438 is also inactive at GABA, glycine, and P2X receptors, but displays inhibition of some nicotinic acetylcholine receptors. Furthermore, we demonstrate that EU1180-438 produces robust inhibition of glycine-activated current responses mediated by native GluN1/GluN3A receptors in hippocampal CA1 pyramidal neurons. EU1180-438 is a non-competitive antagonist with activity that is independent of membrane potential (i.e. voltage-independent), glycine concentration, and extracellular pH. Non-stationary fluctuation analysis of neuronal current responses provided an estimated weighted mean unitary conductance of 6.1 pS for GluN1/GluN3A channels, and showed that EU1180-438 has no effect on conductance. Site-directed mutagenesis suggests that structural determinants of EU1180-438 activity reside near a short pre-M1 helix that lies parallel to the plane of the membrane below the agonist binding domain. These findings demonstrate that structural differences between GluN3 and other glutamate receptor subunits can be exploited to generate subunit-selective ligands with utility in exploring the roles GluN3 in neuronal function. • EU1180-438 is negative allosteric modulator selective for GluN1/GluN3 receptors. • EU1180-438 inhibits current responses mediated by neuronal GluN1/GluN3A receptors. • EU1180-438 activity is independent of membrane potential and agonist concentration. • Structural determinants of EU1180-438 activity reside near the GluN3A pre-M1 helix. • EU1180-438 is a tool to investigate the physiology of native GluN1/GluN3 receptors. [ABSTRACT FROM AUTHOR]

Summary NMDA-type glutamate receptors (NMDARs) are ligand-gated ion channels that mediate excitatory neurotransmission in the CNS. Here we describe functional and single-channel properties of triheteromeric GluN1/GluN2A/GluN2C receptors, which contain two GluN1, one GluN2A, and one GluN2C subunits. This NMDAR has three conductance levels and opens in bursts similar to GluN1/GluN2A receptors but with a single-channel open time and open probability reminiscent of GluN1/GluN2C receptors. The deactivation time course of GluN1/GluN2A/GluN2C receptors is intermediate to GluN1/GluN2A and GluN1/GluN2C receptors and is not dominated by GluN2A or GluN2C. We show that triheteromeric GluN1/GluN2A/GluN2C receptors are the predominant NMDARs in cerebellar granule cells and propose that co-expression of GluN2A and GluN2C in cerebellar granule cells occludes cell surface expression of diheteromeric GluN1/GluN2C receptors. This new insight into neuronal GluN1/GluN2A/GluN2C receptors highlights the complexity of NMDAR signaling in the CNS. [ABSTRACT FROM AUTHOR]