Divergent Responses of Seizure-related GRIN Variants Located in Pore-forming M2 Domain of NMDA Receptor to FDA-approved Drugs
Abstract number :
2.467
Submission category :
12. Genetics / 12A. Human Studies
Year :
2023
Submission ID :
1354
Source :
www.aesnet.org
Presentation date :
12/3/2023 12:00:00 AM
Published date :
Authors :
Presenting Author: Hao Xing, PhD – Emory University
Stephen Traynelis, PhD – Emory University; Hongjie Yuan, PhD – Emory University; Chad Camp, PhD – Emory University; Jiahui Chen, PhD – Emory University; Varun Kannan, MD – Baylor College of Medicine; Jia Li, PhD – Emory University; Yinlong Li, PhD – Emory University; Steven Liang, PhD – University; Riley Perszyk, PhD – Emory University; Ethan Shi, Research Intern – Emory University; Rui Song, MD – Emory University; Weiting Tang, PhD – Emory University; Yuchen Xu, MD/PhD – Emory University; Jin Zhang, PhD – Emory University
Rationale:
N-methyl-D-aspartate receptors (NMDARs) mediate the slow component of excitatory synaptic transmission in brain. Genetic variations of GRIN genes situated in the M2 re-entrant loop result in a reduction in voltage-dependent Mg2+ block, which will have profound impact on synaptic function and may underlie the mechanisms of certain seizure syndromes. Here, we choose seven seizure-related missense GRIN variants that locate in the M2 loop and evaluate a set of FDA-approved NMDAR drugs for their ability to rectify dysregulated NMDAR function by supplementing the reduced voltage-dependent Mg2+ block.
Methods:
GRIN variants were introduced into cDNA encoding human NMDARs using the QuikChange protocol. cRNAs were synthesized from cDNA and injected into Xenopus oocytes. Two-electrode voltage clamp (TEVC) recordings were performed to assess the effects of the variants on sensitivity to three FDA-approved NMDAR drugs (memantine, dextromethorphan, and ketamine).
Results:
TEVC recordings (VHOLD −40 mV) revealed that the M2 variants have differential sensitivity (potency) to the channel blockers. In the presence of physiological concentration of Mg2+ (1.0 mM), memantine showed an enhanced potency on 2A-L611Q (3.7-fold lower IC50) and 2B-N615I (7.2-fold lower IC50), but reduced potency (2- to 24-fold higher IC50) on 2A-N614S, 2A-N615K, 2B-N616K, and 2B-V618G. The antitussive dextromethorphan showed an enhanced potency on 2A-N615K-, 2B-G611V-, and 2B-N616K by 3.5- to 8.3-fold lower IC50, but a reduced potency at 2B-N616K (14-fold higher IC50) and 2B-V618G (3.2-fold higher IC50). The anesthetic ketamine showed 4- to 17-fold enhanced potency (lower IC50) on 2A-L611Q, 2A-N615K, 2B-N615I, and 2B-N616K. We also repeated the experiments in the absence of Mg2+, which showed a divergent sensitivity to the FDA drugs (e.g., 2B-N615I presented an enhanced potency to memantine and ketamine by 4.3--fold, and 8.2-fold, respectively, but a reduced potency to dextromethorphan by 3.1-fold). In addition, analysis of the current-voltage curves revealed concentration-dependent inhibition on 2A-L611Q (associated with medically refractory epilepsy) by ketamine. However, the reduced affinity and voltage-dependence of Mg2+ binding (the apparent affinity KD,0 mV and the electric field zδ) caused by 2A-L611Q cannot be fully restored by the addition of the monovalent organic cation ketamine.
Conclusions:
Taken together, these in vitro analyses of missense GRIN variants revealed a wide range of effects on channel blocker potency and voltage dependence. Some variants showed a similar or modestly enhanced potency to FDA-approved NMDAR drugs compared to WT receptors, suggesting these drugs might help to reduce overexcitation produced by relief of extracellular Mg2+ block at resting membrane potentials against seizures for patients with validated gain-of-function variants. These results reinforce the importance of confirming drug actions and potency prior to consideration of clinical use.
Funding:
NIH R01HD082373, R01AG075444, R01AG080262, R01NS111619, R24NS092989
Genetics