Abstracts

Rationale: N-methyl-D-aspartate receptors (NMDARs) are ligand-gated cation channels that mediate excitatory synaptic transmission.  Loss-of functional genetic mutations in multiple NMDAR subunits cause various childhood epilepsy syndromes.  Here, we evaluated several NMDAR-targeted drugs, including existing FDA-approved medication, for potential utility to rectify the dysregulated NMDAR caused by loss-of-function GRIN mutations as potential patient-specific therapy. Methods: Two-electrode voltage clamp current recordings (holding potential -40 mV) of oocytes were performed to evaluate the sensitivity to positive allosteric modulators of loss-of-function GRIN variants that were introduced by site-directed mutagenesis into cDNAs encoding human NMDAR subunits. Results: Two neurosteroids, pregnenolone sulfate and 24(S)-hydroxycholesterol, are positive allosteric modulators on GluN2A- and GluN2B-containing NMDARs.  We evaluated these modulators on epilepsy-associated loss-of-function GRIN mutations (GluN2A-C436R, -G483R, -V685G, -M705V, -A716T, -A727T, -V734L, -G760V, -K772E, and GluN2B-C461F, -S541S). In vitro analysis on Xenopus oocytes expressing recombinant NMDARs harboring these variants showed that pregnenolone sulfate (100 µM) potentiated current response amplitude (1000 µM glutamate and 100 µM glycine) of the loss-of-function mutants by 1.5~9.0-fold. The maximum potentiation by 10 µM 24(S)-hydroxycholesterol was 1.5~2.0-fold. One additional positive allosteric modulator, FDA-approved tobramycin (300 µM), potentiated GluN2B-C461F and GluN2B-S541S by 2.0-fold and 3.8-fold, respectively.  In addition, concentration-effect curves of these positive modulators were assessed on several selected loss-of-function mutations (e.g. GluN2A-V685G and GluN2B-C461F), which showed a comparable EC₅₀ values to the wild type receptors.   Conclusions: Overall, these results suggest that positive allosteric modulators can potentiate the current response of epilepsy-related loss-of-function GRIN mutations, which provides a strategy for enhancing mutant NMDAR function that could be beneficial. 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 SAGE to H.Y., 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