Abstracts

Rationale: N-methyl-D-aspartate receptors (NMDARs) are densely expressed in brain and have been found to play an important role in neurodevelopment. We here report one female patient with myoclonus and severe intellectual disability associated with a de novo GRIN1 variant (p.Pro532His) located in agonist-binding domain. We evaluated functional properties of the mutant-containing recombinant NMDA receptors. Methods: The mutant was introduced into human NMDAR GluN1 using the QuikChange protocol. cRNAs were synthesized from cDNA and injected into Xenopus laevis oocytes. Two-electrode voltage clamp (TEVC) current recordings of oocytes were performed to evaluate the agonist potency, sensitivity to negative modulators (magnesium and protons), and channel open probability. Whole cell voltage clamp recordings on transfected HEK cells were performed to assess synaptic-like current response time course. Results: TEVC recordings on oocytes showed that GluN1-P532H co-expressed with GluN2A or GluN2B increased glutamate EC₅₀ values (decreased potency) by 15-fold and 43-fold, respectively, compared to the corresponding wild type (WT) receptors. The mutant-containing receptors exhibited either no or modest changes in glycine potency and sensitivity to Mg²⁺ and protons.  Whole cell recordings on transfected HEK cells revealed a brief synaptic-like response time course that was accelerated by 2.6-fold (weighed tau: 26 ± 7 ms vs. 67 ± 4 ms of WT) when co-expressed with GluN2A and 21-fold (weighed tau: 37 ± 3 ms vs. 780 ± 33 ms of WT) when co-expressed with GluN2B. Charge transfer was reduced by 8-fold and 29-fold, respectively. In addition, channel open probability was evaluated by measuring the degree of MTSEA potentiation on mutant receptors co-expressed with GluN2A-A651C subunit in TEVC recordings on Xenopus oocytes. The GluN1-P532H/GluN2A-A651C receptors showed a reduced channel open probability of 0.11 vs. 0.26 for GluN1/GluN2A-A651C. Conclusions: Overall, these results suggest that the higher concentrations of glutamate needed to activate the variant-containing NMDARs, which has a shortened synaptic response time course and reduced channel open probability. These data suggest this variant produces a loss-of-function.  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.