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 seven de novo missense mutations in NMDAR subunit genes, GRIN2A and GRIN2B, located in the M2 loop that lines the NMDAR channel pore and identified in children with epileptic encephalopathy. We also screened several existing FDA-approved medications for potential utility as patient-specific therapy. Methods: The mutant was introduced into human NMDAR GluN2A or GluN2B cDNA construct 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), and potency of FDA-approved NMDAR antagonists. Current-voltage relationships were also compared to evaluate changes in voltage-dependent Mg2+ block.  Results: The resulting amino acid exchange for seven missense mutations (GluN2A: p.Leu611Glu, p.Leu614Ser, p.Asn615Lys; GluN2B: p.Gly611Val, p.Asn615Ile, p.Asn616Lys, and p.Val618Gly) occurs in the M2 transmembrane domain.  Compared to WT receptors, the mutant NMDARs exhibited either no or modest changes in potency (EC50 values) for both glutamate and glycine, as well as no or modest changes in proton inhibition (percentage current inhibition at pH 6.8 compared with the pH 7.6). Mg2+ inhibition was significantly reduced in all seven mutants, with significantly increased IC50 values and decreased degree of Mg2+ inhibition of agonist-evoked current response. Current-voltage curves revealed marked reduction in Mg2+ inhibition in all mutant receptors. Since the patient’s seizures had proven refractory to conventional antiepileptic medications, mutant NMDAR sensitivity was evaluated in response to three FDA-approved NMDAR antagonists (memantine, dextromethorphan, and ketamine), which reverse the loss of Mg2+ inhibition in mutant receptors in certain degree.   Conclusions: Overall, these results suggest that M2 mutations produce a gain-of-function through reduced Mg2+ channel blockage, which is consistent with its location in the M2 loop of the channel pore. These functional changes may contribute to over-activation of NMDAR which may underlie the patients’ phenotypes. FDA-approved NMDAR antagonists reverse the loss of Mg2+ inhibition in mutant receptors, and may serve as an effective personalized treatment option for the affected children.  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 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