Abstracts

Rationale: N-methyl-D-aspartate receptors (NMDARs) are ligand-gated cation channels that mediate excitatory synaptic transmission. Twelve missense mutations have been identified in the M2 transmembrane domain of GRIN1, GRIN2A, and GRIN2B genes in patients with epilepsy and/or intellectual disability.  Here, we evaluated several FDA-approved NMDAR-targeted drugs for potential utility to restore the reduced/abolished Mg²⁺ block caused by GRIN mutations located in M2 re-entrant loop. Methods: The mutant was introduced into cDNAs encoding human NMDAR subunits GluN1, GluN2A or GluN2B using the QuikChange protocol. cRNAs were synthesized from cDNA and injected into Xenopus laevis oocytes. Two-electrode voltage clamp (TEVC) current recordings (holding potential -40 mV) from oocytes were performed to evaluate potency of NMDAR-targeted drugs. Current-voltage relationships were also evaluated to assess potentials changes in voltage-dependent Mg²⁺ block. Results: Our functional evaluation showed all the mutant receptors showed significantly reduced Mg²⁺ inhibition in all mutant receptors, with significantly increased IC₅₀ values and decreased degree of Mg²⁺ inhibition of agonist-evoked current response. Since several patients’ seizures had proven refractory to conventional antiepileptic medications, variant NMDAR sensitivity was evaluated in response to three FDA-approved NMDAR antagonists (memantine, dextromethorphan, and ketamine). In vitro analysis by TEVC recordings revealed that the TM2 variants have differential sensitivity (potency, IC₅₀) to these channel blockers. In the absence of Mg²⁺, memantine showed enhanced potency on GluN1-G620R- and GluN2B-N615I-containing NMDARs (2.6-fold and 4.3-fold compared to wild-type NMDARs, respectively), but decreased potency by 2~14-fold for GluN1-G618R, GluN2A-L611Q, GluN2A-N614S, GluN2A-N615K, GluN2B-W607C, GluN2B-N615K, GluN2B-N616K, and GluN2B-V618G. Dextromethorphan showed enhanced potency on GluN1-G620R-, GluN2A-N615K-, and GluN2B-V620M (2.5-fold, 2.9-fold, and 2.4-fold compared to WT, respectively). Dextromethorphan exhibited reduced potency by ~3-fold on GluN1-G618R, GluN2A-L611Q, and GluN2B-N615I.  The anesthetic ketamine showed an enhanced potency on GluN1-G620R-, GluN2B-N615I-, GluN2B-N615K-, and GluN2B-V620M (2.5-fold, 9.8-fold, 2.3-fold, and 2.7-fold, respectively), but a 3-fold reduced potency on GluN2B-W607C, and GluN2B-V618G.  We subsequently repeated the experiments in the presence of extracellular Mg²⁺. For all three channel blockers evaluated, the WT receptors showed 2~14-fold reduced potency in the presence of 1 mM Mg²⁺. By contrast, almost all M2 variants evaluated showed a similar or enhanced potency to at least one channel blockers evaluated compared to the WT receptors, except for GluN2A-L614S, which had reduced the potency for all three channel blockers. Conclusions: Overall, the differential responses of these M2 variants-containing NMDARs to channel blockers suggest that these blockers may engage different structural determinants at their binding sites in the pore, which reverse the loss of Mg²⁺ inhibition in mutant receptors in certain degree. These FDA-approved NMDAR antagonists may serve as an effective personalized treatment option for the affected children. Funding: This work was supported by CSC to LJ, 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