Summary of pharmacological data for GluN2A(D731N).

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

Summary of channel open probability for GluN2A(D731N).

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

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

GluN2A(D731N) enhances sensitivity to endogenous proton and zinc ions.

<p>(<b>A</b>) The composite proton concentration-response curves show an enhanced sensitivity of the GluN2A(D731N)-containing receptors to proton compared to the WT NMDA receptors; the abscissa shows hydrogen ion activity. (<b>B</b>) Summary of proton sensitivity of WT GluN2A and mutant receptors, evaluated by current ratio at pH 6.8 to pH 7.6 (in the presence of 30 mM glutamate and 100 μM glycine). Di-heteromeric (h2A-D731N), one-copy and two-copy mutant tri-heteromeric (D731N/2A and D731N/D731N) receptors show a decreased current ratio, indicating enhanced proton sensitivity. (<b>C</b>) The composite zinc concentration-response curves show an enhanced sensitivity of the GluN2A(D731N)-containing receptors to zinc compared to the WT NMDA receptors. (<b>D</b>) Composite Mg²⁺ concentration-response curves for di-heteromeric receptors indicate a similar Mg²⁺ inhibition of GluN2A(D731N)-containing receptors. The data were generated by TEVC recordings on <i>Xenopus</i> oocyte at holding potential of -40 mV for proton concentration-response curves, and -20 mV for zinc, and -60 mV for Mg²⁺ concentration-response curves. Fitted parameters are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170818#pone.0170818.t002" target="_blank">Table 2</a>.</p

EEG of patient 6245 at 6y7m.

<p>(A) During awake period, spike and spike-wave complexes (arrow heads) in Rolandic region, more obvious on the left side (blue). (B) During sleep period, NREM (non-rapid eye movement) index was about 85%.</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 patients' information.

<p>Summary of patients' information.</p

Agonist potency of GluN2A-P552R equivalent mutations in GluN2B, GluN2C, and GluN2D.

<p>Agonist potency of GluN2A-P552R equivalent mutations in GluN2B, GluN2C, and GluN2D.</p