Abstracts

Rationale: N-methyl-D-aspartate receptors (NMDARs) are ligand-gated cation channels that mediate excitatory synaptic transmission.  Genetic mutations in multiple NMDAR subunits cause various childhood epilepsy syndromes.  Here, we evaluated functional changes for a de novo missense mutation (p.Ala678Asp) in NMDAR subunit gene GRIN2D, located in the M3 transmembrane domain and identified in children with epileptic encephalopathy.  Methods: The mutant was introduced into a cDNA encoding human NMDAR GluN2D using the QuikChange protocol. cRNAs were synthesized from cDNA and injected into Xenopus laevis oocytes. Two-electrode voltage clamp current recordings of oocytes were performed to evaluate agonist potency, sensitivity to negative modulators (magnesium and proton), channel open probability, and the sensitivity to FDA-approved NMDAR channel blockers.   Results: The missense mutation is located in the M3 transmembrane domain.  The mutation increases glutamate potency by 40-fold, decreasing the EC₅₀ from 0.44 ± 0.07 µM of the wild type receptors to 0.01 ± 0.003 µM. We could not detect a significant change in the sensitivity to extracellular Mg²⁺ and protons.  Moreover, this mutation increases channel open probability by 30-fold, from 0.01 ± 0.003 for WT receptors to 0.32 ± 0.05 for GluN1/GluN2D-A678D.  We subsequently evaluated the mutant NMDAR sensitivity in response to the FDA-approved NMDAR channel blockers memantine, dextromethorphan, and ketamine, which showed a comparable sensitivity compared to the wild type receptors.  Conclusions: Overall, these results suggest that this variant produces a gain-of-function and may contribute to over-activation of NMDARs. FDA-approved NMDAR antagonists can reduce mutant receptor function, and thus may be useful as potential adjuvant epilepsy therapy in patients with GRIN2D gain-of-function mutations.  Funding: This work was supported by the Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD) of the National Institutes of Health (NIH) under award number R01HD082373 to H.Y., by CSC scholarship to W.X., by the Beijing Municipal Natural Science Key Project under award numbers 15G10050 to YJ, and by the National Institute of Neurological Disorders and Stroke (NINDS) of the NIH under award numbers NIH-NINDS R01NS036654, R01NS065371, and R24NS092989 to S.F.T