Abstracts

Rationale:
N-methyl-D-aspartate receptors (NMDARs) are highly expressed in central nervous system and have been found to play an important role in neurodevelopment and pathological conditions. We here report a de novo GRIN1 variant (p.Pro532His) identified in a female patient with myoclonus and severe intellectual disability. We evaluated functional properties of the variant receptors and explore rescue pharmacology to rectify the altered function.
Method:
The mutagenesis using the QuikChange protocol was performed to introduce the variant into human NMDAR GluN1. cRNAs were synthesized from cDNA and injected into Xenopus laevis oocytes. Two-electrode voltage clamp (TEVC) current recordings of oocytes were applied to assess the agonist potency, sensitivity to negative modulators, channel open probability, and effects of positive allosteric modulators and co-agonists. Whole cell voltage clamp recordings and beta-lactamase assay on transfected HEK cells were performed to evaluate current response time course and receptor surface expression, respectively.
Results:
When co-expression with GluN2A and Glun2B in TEVC current recordings of oocytes, the variant receptor increased glutamate EC50 values (decreased potency) by 15-fold and 43-fold, respectively, compared to the corresponding wild type (WT) receptors. Whole cell recordings on transfected HEK cells showed an accelerated synaptic-like response time course by 2.6-fold when co-expressed with GluN2A and 21-fold when co-expressed with GluN2B. Charge transfer was reduced by 8-fold and 29-fold, respectively. Channel open probability evaluated by measuring the degree of MTSEA potentiation on variant receptors co-expressed with GluN2A-A651C subunit showed a reduced channel open probability of 0.11 vs. 0.26 for the WT. The beta-lactamase assay showed a significant decrease in surface-to-total protein level in the variant receptors when co-expressed with GluN2A and GluN2B by 36% and 33%, respectively. Three NMDAR positive allosteric modulators (24(S)-hydroxycholesterol, pregnenolone sulfate, and tobramycin) and three co-agonists at glycine site (L-serine, D-serine, and D-cycloserine) showed a similar potentiation effects or a similar potency for WT and variant GluN1-P532H-containing NMDARs.
Conclusion:
Taken together, these data suggest the variant receptor is a loss-of-function variant with decreased agonist potency, shortened synaptic response time course, and reduced channel open probability. Evaluation of positive allosteric modulators may explore the possibility to use these compounds to partially rectify some functional deficits of the loss-of-function variant.
Funding:
:During this work, HY was supported by NIH - the Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD) [grant R01HD082373], by the University Research Committee (URC; #00085889) from Emory University, and by a research grant from Sage Therapeutics to Emory University School of Medicine. SFT was supported by the NIH- National Institute of Neurologic Disorders and Stroke (NINDS) [grants R35NS111619and R24NS092989] and Austin Purpose.