A de novo loss-of-function GRIN2A mutation associated with childhood focal epilepsy and acquired epileptic aphasia.

OBJECTIVE:N-methyl-D-aspartate receptors (NMDAR) subunit GRIN2A/GluN2A mutations have been identified in patients with various neurological diseases, such as epilepsy and intellectual disability / developmental delay (ID/DD). In this study, we investigated the phenotype and underlying molecular mechanism of a GRIN2A missense mutation identified by next generation sequencing on idiopathic focal epilepsy using in vitro electrophysiology. METHODS:Genomic DNA of patients with epilepsy and ID/DD were sequenced by targeted next-generation sequencing within 300 genes related to epilepsy and ID/DD. The effects of one missense GRIN2A mutation on NMDAR function were evaluated by two-electrode voltage clamp current recordings and whole cell voltage clamp current recordings. RESULTS:We identified one de novo missense GRIN2A mutation (Asp731Asn, GluN2A(D731N)) in a child with unexplained epilepsy and DD. The D731N mutation is located in a portion of the agonist-binding domain (ABD) in the GluN2A subunit, which is the binding pocket for agonist glutamate. This residue in the ABD is conserved among vertebrate species and all other NMDAR subunits, suggesting an important role in receptor function. The proband shows developmental delay as well as EEG-confirmed seizure activity. Functional analyses reveal that the GluN2A(D731N) mutation decreases glutamate potency by over 3,000-fold, reduces amplitude of current response, shortens synaptic-like response time course, and decreases channel open probability, while enhancing sensitivity to negative allosteric modulators, including extracellular proton and zinc inhibition. The combined effects reduce NMDAR function. SIGNIFICANCE:We identified a de novo missense mutation in the GRIN2A gene in a patient with childhood focal epilepsy and acquired epileptic aphasia. The mutant decreases NMDAR activation suggesting NMDAR hypofunction may contribute to the epilepsy pathogenesis

GluN2A(D731N) reduces the agonist potency.

<p>(<b>A,B</b>) Representative TEVC recordings obtained from oocytes expressing WT GluN1/GluN2A (WT 2A) receptors (<b>A</b>) and GluN1/GluN2A(D731N) (2A-D731N) receptors (<b>B</b>) in which the currents were evoked by increasing concentrations (μM) of glutamate (in the presence of 100 μM glycine) at the holding potential of -40 mV. (<b>C,D</b>) Composite concentration-response curves of glutamate and glycine for di-heteromeric receptors GluN1/GluN2A (WT 2A) and GluN1/GluN2A-D731N (2A-D731N). (<b>E,F</b>) Composite concentration-response curves of glutamate and glycine for tri-heteromeric receptors GluN1/GluN2A/GluN2A (2A/2A), GluN1/GluN2A(D731N)/GluN2A (D731N/2A) and GluN1/GluN2A(D731N)/GluN2A(D731N) (D731N/D731N). (<b>C,E</b>) The composite glutamate (in the presence of 100 μM glycine) concentration-response curves reveal a significant decrease in glutamate potency in both di-heteromeric (<b>C</b>) and tri-heteromeric (<b>E</b>) GluN2A(D731N)-containing NMDARs compared to wild type receptors. A single copy D731N-containing receptor (D731N/2A) (<b>E</b>) showed an intermediate but a dominantly negative effect on glutamate potency. The traces for D731N-contianing receptors (dash lines in panels <b>C</b> and <b>E</b>) were fitted by predicted glutamate concentrations of maximal responses. (<b>D,F</b>) The composite glycine (in the presence of 3–30 mM glutamate) concentration-response curves indicate a mild, but significantly reduced glycine potency in both di-heteromeric (<b>D</b>) and tri-heteromeric (<b>F</b>) GluN2A(D731N) receptors. Error bars are SEM, and are shown when larger than symbol size.</p

GluN2A(D731N) decreases current amplitudes and shortens synaptic-like response time course.

<p>The representative current response time course was generated by the whole cell voltage clamp recording (V_HOLD -60 mV) of GluN1/GluN2A (WT 2A, in BLACK) and GluN1/GluN2A-D731N (2A-D731N, in RED) receptor responses to rapid application of long (1.5 sec) (<b>A,B</b>) and brief (5 ms) (<b>C,D</b>) application of 30 mM glutamate. Panels <b>B</b> and <b>D</b> showed normalized responses to the WT response at the moment glutamate were removed. The mutant D731N-containing receptors showed an accelerated deactivation time course (<i>right panel</i> in <b>B,D</b>). Saturating glycine (100 μM) was present in all of solutions. Fitted parameters describing the response time course are given in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170818#pone.0170818.t003" target="_blank">Table 3</a>.</b></p

Genetic and protein changes of <i>GRIN2A</i> and GluN2A.

<p>(<b>A</b>) Family pedigree and genotypes (indicated by *) reveal a <i>de novo</i> mutation (affected proband is indicated by arrow; parentage was confirmed by Sanger sequencing). (<b>B</b>) Schematic representation of GluN2A subunit (asterisk indicates the position of the D731N mutation). The residue aspartic acid at position 731 is highly conserved across vertebrate species, and other GluN subunits. (<b>C</b>) A homology model of GluN1/GluN2A complex built from the GluN2B crystallographic data [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170818#pone.0170818.ref032" target="_blank">32</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170818#pone.0170818.ref033" target="_blank">33</a>] with Asp731 shown as spacefill in red. The red asterisk in the cartoon illustrating the domain arrangement of an individual GluN subunit (right panel) shows the position of Asp731 in the agonist-binding domain (S2, ABD). Panel <b>D</b> shows glutamate binding pocket depicting the position of D731 (in GREEN) and D731N (in RED) in the GluN2A ABD structure in complex with ligand glutamate (in CYAN).</p

Summary of response time course for GluN2A(D731N).

<p>Summary of response time course for GluN2A(D731N).</p

Summary of channel open probability for GluN2A(D731N).

<p>Summary of channel open probability for GluN2A(D731N).</p

Summary of pharmacological data for GluN2A(D731N).

<p>Summary of pharmacological data for GluN2A(D731N).</p

Molecular Mechanism of Disease-Associated Mutations in the Pre-M1 Helix of NMDA Receptors and Potential Rescue Pharmacology

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