Conserved effects of the Pro552Arg mutation across GluN2 subunits.

<p><b>A</b>,<b>B</b>, Composite concentration-response curves of glutamate in the presence of 100 μM glycine (<b>A</b>) and glycine in the presence of 100 μM glutamate (<b>B</b>) for human GluN1-P557R/GluN2A, GluN1-P557R/GluN2B, GluN1/GluN2B-P553R, and rat GluN1/GluN2C-P550R, and GluN1/GluN2D-P577R. The graph legends refer to GluN1 as N1 and GluN2 as N2. Fitted EC₅₀ values are summarized in Tables <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006536#pgen.1006536.t003" target="_blank">3</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006536#pgen.1006536.t005" target="_blank">5</a>. <b>C</b>,<b>D</b>, human GluN1-P557R/GluN2A significantly prolongs deactivation time course after removal of glutamate (<b>C</b>) or removal of glycine (<b>D</b>) on transfected HEK293 cells, but does not slow the rise time when the receptors were activated by the agonists. <b>E</b>, GluN1/GluN2B-P553R significantly slows rise time and prolongs deactivation time course. Fitted parameters describing the response time course are given in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006536#pgen.1006536.t006" target="_blank">Table 6</a>.</p

Potential interaction between the pre-M1 and M3 helices.

<p><b>A</b>,<b>B</b>, Ribbon structures of the GluN1/GluN2A (<b><i>A</i></b>) and GluN1/GluN2B (<b><i>B</i></b>) receptors without the amino terminal domain is shown. GluN1 is tan and GluN2 is light blue; regions with an OE-ratio below the 5^th percentile are colored purple, and indicate the regions under the strongest purifying selection. <b>C</b>, Side and top down view of the pore forming elements M1, M3, M4 in GluN1/GluN2A receptors colored as in (<b><i>A</i></b>), with regions of purifying selection shown in purple. <b>D</b>, Expanded view of the pre-M1 helix for GluN1 (<i>left panel</i>) and for GluN2A (<i>right panel</i>).</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

Analysis of genetic variation on S1-M1 linker in <i>GRIN1</i>, <i>GRIN2A</i>, <i>and GRIN2B</i>.

<p>Analysis of genetic variation on S1-M1 linker in <i>GRIN1</i>, <i>GRIN2A</i>, <i>and GRIN2B</i>.</p

Patients’ and mutations’ information.

<p>Patients’ and mutations’ information.</p

Pharmacology and response time course for substitution of GluN2A-ProP552 with Ala, Gly, Lys, Gln, Ile, or Leu.

<p>Pharmacology and response time course for substitution of GluN2A-ProP552 with Ala, Gly, Lys, Gln, Ile, or Leu.</p

Summary of pharmacological and functional properties of pre-M1 mutations.

<p>Summary of pharmacological and functional properties of pre-M1 mutations.</p

Assessment of alternative Pro552 substitutions in GluN2A.

<p><b>A,B</b>, the composite glutamate (in the presence of 100 μM glycine) and glycine (in the presence of 100 μM glutamate) concentration-response curves of GluN2A- P552A, P552G, P552I, P552K, P552Q, P552L constructs. Error bars are SEM and shown when larger than symbol. <b>C,D,E</b>, The response time courses are shown of GluN1/GluN2A(P552K), GluN1/GluN2A(P552G), and GluN1/GluN2A(P552L) receptors activated by rapid application of 100 μM glutamate; 100 μM glycine was present in all solutions. For panel <b>D</b> the rise time is expanded as an inset. The data (mean ± SEM) are given in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006536#pgen.1006536.t007" target="_blank">Table 7</a>.</p

Glutamate response time course of GluN2A-P552R equivalent amino acid changes in GluN1 and GluN2B.

<p>Glutamate response time course of GluN2A-P552R equivalent amino acid changes in GluN1 and GluN2B.</p

Intolerance analysis of genetic variation across functional domains of GluN1, GluN2A and GluN2B.

<p><b>A</b>, Ribbon structure of homology model of a tetrameric NMDAR and a single GluN subunit [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006536#pgen.1006536.ref010" target="_blank">10</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006536#pgen.1006536.ref012" target="_blank">12</a>]. <b>B</b>, A cartoon illustrating the domain arrangement of an individual GluN subunit. ATD—amino terminal domain (in GREEN), S1 and S2 –first and second polypeptide sequences comprising the agonist binding domain (ABD, in BLUE), linker regions (S1-M1 linker, M3-S2 linker, and S2-M4 linker; in GRAY), M1, M3, and M4 –transmembrane domains (TM, in ORANGE), M2 –re-entrant pore loop (in ORANGE), and CTD—carboxy-terminal domain (in PINK). <b>C</b>, <i>GRIN1</i>; <b>D</b>, <i>GRIN2A</i>; <b>E</b>, <i>GRIN2B</i>: Sliding window OE-ratio estimates (black full line), Neutrality expected OE-ratio estimate (blue full line), Median OE-ratio for the gene (dark grey dashed line), 25^th percentile of OE-ratio (green dashed line), 5^th percentile of OE-ratio (red dashed line).</p