Authors :
Presenting Author: Lu Zhang, PhD – Emory University School of Medicine
Ethan Shi, Mr – Emory University; Andrew Nie, Mr – Emory University; Yuchen Xu, PhD – Emory University; Wenshu XiangWei, PhD – Emory University; Eva Sarai Diaz, BS – Department of Pharmacology and Chemical Biology – Emory University School of Medicine; Johannes Lemke, MD – University of Leipzig Hospitals and Clinics; Guojun Zhang, MD/PhD – Children’s Healthcare of Atlanta – Emory University; Ammar Kheder, MD – Emory University School of Medicine; Stephen Traynelis, PhD – Department of Pharmacology and Chemical Biology – Emory University School of Medicine; Hongjie Yuan, MD/PhD – Department of Pharmacology and Chemical Biology – Emory University School of Medicine
Rationale:
N-methyl-D-aspartate receptors (NMDARs) are ligand-gated ionotropic receptors mediating a slow, calcium-permeable component of excitatory synaptic transmission in the brain. Variants in genes encoding NMDAR subunits have been associated with a spectrum of neurodevelopmental disorders. Genetic variation in the
GRIN2D gene encoding the GluN2D subunit of NMDAR has been associated with a spectrum of neurological and neurodevelopmental conditions, such as developmental and epileptic encephalopathy (DEE). In this study, we describe seven
GRIN2D missense variants and evaluate their impact on the pharmacological and biophysical properties of NMDAR function.
Methods:
Each variant was introduced into a cDNA encoding human NMDAR GluN2D 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 assess the effects of the variants on agonist potency, sensitivity to negative modulators (magnesium and proton), channel open probability, and the sensitivity to FDA-approved NMDAR channel blockers. Whole-cell voltage clamp current recordings were performed to evaluate synaptic-like response time course.
Results:
Each variant was introduced into a cDNA encoding human NMDAR GluN2D 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 assess the effects of the variants on agonist potency, sensitivity to negative modulators (magnesium and proton), channel open probability, and the sensitivity to FDA-approved NMDAR channel blockers. Whole-cell voltage clamp current recordings were performed to evaluate synaptic-like response time course.
Conclusions:
Overall, this study emphasizes the importance of functional and biochemical validation for each individual variant. The combination of a rapidly growing array of genomic sequencing data with functional evaluation of specific molecular mechanism and potential rescue pharmacology
in vitro may provide a better understanding of the underlying disease mechanism and diagnosis, as well as present an opportunity for precision medicine for a subset of severe pediatric neurodevelopmental diseases related to
GRIN variants.
Funding:
This work was supported by the Simon’s Foundation (SFT), the CureGRIN Foundation (SFT), the NIH (NINDS NS111619 to SFT; NICHD HD082373 to HY; NIAAG075444 to HY) .