Abstracts

Rationale: N-methyl-D-aspartate receptors (NMDAR) 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 in a NMDAR subunit gene, GRIN2D, identified in a child with severe epileptic encephalopathy. Methods: The mutant was introduced into human NMDAR GluN2D cDNA construct using QuikChange protocol. cRNAs were synthesized from cDNA and injected into Xenopus laevis oocytes. Two-electrode voltage clamp current recordings on oocytes were performed to evaluate agonist potency, sensitivity to negative modulators (magnesium and proton) and FDA-approved NMDAR antagonists. Whole-cell voltage clamp current recordings and outside out single channel patch recordings were performed to evaluate response time course and channel open time. The effect of the mutant on neurotoxicity was evaluated on transiently transfected rat cortical neurons in vitro. Results: The resulting GluN2D p.V667I amino acid exchange occurs in the M3 transmembrane domain. The mutation increases glutamate and glycine potency by 2-fold, increases channel open probability by 10-fold, and reduces receptor sensitivity to endogenous negative modulators such as extracellular protons. Moreover, this mutation prolongs the deactivation time course following glutamate removal, which controls the synaptic time course. Outside out single channel recording showed that the mean open channel time was increased 6-fold to 5.2 +- 0.03 ms (n = 4) for the mutant compared to 0.76 +- 0.03 ms (n = 3) for the WT receptors. Transfection of cultured neurons with cDNA encoding human GluN2D harboring this mutation leads to dendritic swelling and neuronal cell death, findings suggestive of excitotoxicity mediated by NMDAR over-activation. Since the patient's seizures had proven refractory to conventional antiepileptic medications, mutant NMDAR sensitivity was evaluated in response to FDA-approved NMDAR antagonists (memantine, ketamine, and dextromethorphan). Conclusions: Overall, these results suggest this mutant is a gain-of-function mutation and may contribute to over-activation of NMDAR. FDA-approved NMDAR antagonists may be used 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., the National Center for Advancing Translational Sciences of the National Institutes of Health under award number UL1TR000454 to H.Y, by the National Institute of Neurological Disorders and Stroke (NINDS) of the NIH under award number R01NS036654 to S.F.T. and NS043277 to E.A., and by Institutional Development Funds to the Center for Applied Genomics (CAG) at the Children's Hospital of Philadelphia (CHOP). SFT is a paid consultant for NeurOp, Pfizer, and Janssen