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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
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 EC<sub>50</sub> 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
Neurotoxic consequences of GluN2A-P552R expression and rescue pharmacology.
<p><b>A</b>, Schematic of experimental timeline indicates the relative dates of neuronal cell culture from embryonic day 16/17 (E16/17), transfection along with memantine/vehicle treatment, and toxicity studies (luciferase assays, cell counts, and confocal imaging). <b>B</b>, Confocal images of cortical neurons transfected with a GFP-expressing construct, with various concentrations of the human GluN2A-P552R-expressing plasmid illustrate the cell morphology. Images were acquired 24 h post-transfection at 20× magnification (scale bars 10 μm). <b>C</b>, Confocal images of cortical neurons transfected with GFP-expressing construct with either 0.6 μg cDNA per well (40% of total transfection cDNA) of WT GluN2A or GluN2A-P552R, the latter in the absence or presence of memantine (50 μM). Images were acquired 24 h post-transfection at 20× magnification, with the exception of the bottom left panel (40×), which highlights GluN2A-P552R-induced dendritic swelling and blebs (scale bar 10 μm). <b>D</b>, The mean cell viability values are shown as a percent of control. Luciferase assays: neuronal cultures were transfected with GFP-N1 plasmid (0.525 μg or 0.825 μg per well) luciferase cDNA (0.375 μg/well) for cell viability assaying, with varied concentrations (0.3 μg or 0.6 μg per well) of pCIneo-vector, WT GluN2A, or GluN2A-P552R cDNA (1.5 μg total DNA per well). Each transfection condition was performed in pairs, either supplemented with vehicle (–) or memantine (20 μM for 0.3 μg; 50 μM for 0.6 μg) treatment (+). Luciferase assays were performed 48 h following transfection and treatment. Experiments were performed in quadruplicate, and independent experiments were repeated (0.3 μg cDNA, n = 7; 0.6 μg cDNA, n = 8). Each condition was normalized to its relevant vector-transfected group to obtain relative viability values, expressed as % control. Data are mean ± SEM of viability (% control) for each condition (ANOVA/Bonferroni; *p <0.05, **p < 0.01, ***p < 0.001). Cell counts: Neuronal cultures were transfected with GFP-N1 plasmid for cell visualization, with either 0.6 μg pCIneo including vector, WT GluN2A, or GluN2A-P552R cDNA (40% of total transfection cDNA). Each transfection condition was performed in duplicate. Cell counts were performed 48 h post-transfection (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006536#sec013" target="_blank">Methods</a>). Data are mean ± SEM of viability (% control) for each condition in 6 independent experiments. ANOVA/Bonferroni (**p < 0.01). See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006536#pgen.1006536.s017" target="_blank">S9 Table</a> for statistics.</p
Locations of pre-M1 mutations.
<p><b>A</b>, Domain architecture of NMDARs and protein sequence alignment showing pre-M1 helix across NMDAR subunits. <b>B-D</b>, Ribbon structure of homology model of GluN1/GluN2A built from GluN1/GluN2B [<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>]. Five residues harboring mutations in human patients are highlighted: GluN1-D552E (in GOLD), GluN1-P557R (in LIGHT BLUE), GluN2A-A548T (in BLUE), GluN2A-P552R (in MAGENTA), and GluN2B-P553L (in MAGENTA). The mutation labels refer to GluN1 as N1 and GluN2 as N2. ATD—amino terminal domain, S1 and S2 –first and second polypeptide sequences comprising the agonist binding domain (ABD), M1, M3, and M4 –transmembrane helices, and M2 –re-entrant pore loop. The mutation information is given in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006536#pgen.1006536.t002" target="_blank">Table 2</a>.</p
Potency and efficacy of NMDAR inhibitors on GluN1/GluN2A-WT and -P552R.
<p>Potency and efficacy of NMDAR inhibitors on GluN1/GluN2A-WT and -P552R.</p