Abstracts

Rationale: Using electrophysiology assays, we characterized the functional impact of a mutation in the GRIN2A gene of the NMDA receptor (NMDAR) that was identified by exome sequencing of a 3-yr old boy with severe drug-resistant epileptic encephalopathy. The mutation at position 611 is located in the M2 loop that lines the NMDAR channel pore, which is implicated in voltage-dependent channel block by Mg2+. In addition to elucidating the mechanism by which the mutation alters normal NMDAR activity, we screened several existing FDA-approved medications for potential utility as patient-specific therapy. Methods: The patient's mutation was introduced into human NMDAR cDNA constructs using site-directed mutagenesis (QuikChange protocol). Wild type (WT) and mutant NMDAR cRNAs were synthesized from cDNA and injected into Xenopus laevis oocytes. Two-electrode voltage clamp was used to compare current responses of WT and mutant oocytes to NMDAR agonists (glutamate, glycine), endogenous inhibitors (Mg2+, H+), and FDA-approved NMDAR antagonists (memantine, dextromethorphan, dextrorphan, amantadine, and ketamine). Current-voltage relationships were also compared to evaluate changes in voltage-dependent Mg2+ block. Results: Compared to WT receptors, the mutant NMDARs exhibited a modest increase in potency (decreased EC50 values) for both glutamate (2.6 uM vs. 4.5 uM of WT) and glycine (1.0 uM vs. 1.4 uM of WT), as well as a significant reduction in proton inhibition (percentage current inhibition at pH 6.8 compared with the pH 7.6: 42% vs. 56% of WT). Mg2+ inhibition was significantly reduced in mutants, with ~40-fold increase in IC50 value (1120 uM) compared to the WT (28 uM). Ketamine showed similar potency between WT and mutant receptors (7.5 uM vs. 5.1 uM of WT). Current-voltage curves revealed marked reduction in Mg2+ inhibition in mutants, which was reversed by subsequent administration of ketamine in a dose-dependent manner. Conclusions: This GRIN2A mutation most likely causes pathologic over-excitation (and ultimately seizures) through reduced Mg2+ channel blockage, which is consistent with its location in the M2 loop of the channel pore. Ketamine reverses the loss of Mg2+ inhibition in mutant receptors, and may serve as an effective personalized treatment option for the affected child. 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., and by the National Institute of Neurological Disorders and Stroke (NINDS) of the NIH under award number R01NS036654 to S.F.T. S.F.T. is a paid consultant for NeurOp, Pfizer, and Janssen