Integrated Study On Molecular Function And Animal Model Of Variants Of NMDA Receptor

Background and Objectives:N-methyl-D-aspartate（NMDA）receptors are ligand-gated ion channels that mediates excitatory synaptic transmission in the brain.Functional NMDA receptors are typically assembled by two GluN1subunits（encoded by GRIN1）and two GluN2 subunits（encoded by GRIN2A-D）.Mutations of highly conserved agonist binding domain（ABD）are associated with a variety of neurological diseases.Due to the diversity of gene sequences and the complexity of receptor structures,there are numerous discovered disease-related GRIN mutations,and reported mutations require further study to reveal their impacts in neurological diseases.It is necessary to explore the functions of NMDA receptor mutations both in vivo and in vitro.In this study,we focused on two mutations located at S1 segment of ABD in NMDA receptors and conducted functional experiments.One mutation is a de novo GRIN2A missense mutation（c.1608G>T,p.Met536Ile,GluN2A-M536I）,and the other is a GRIN2B missense mutation（c.1238A>G,p.Glu413Gly,GluN2B-E413G）.Methods:1.We used Quik-Change technology to construct GluN2A-M536I mutant cDNA with wild-type GRIN gene,synthesized c RNA in vitro and injected them into Xenopus oocytes.Two electrode voltage clamp was used to assess the potency changes of the mutation.2.We transfected human embryonic kidney cells 293（HEK293）with wild-type and GluN2A-M536I mutant cDNA respectively,and whole-cell voltage clamp was used to evaluate current amplitudes and channel opening response times.3.We co-cultured HEK293 transfected with wild-type and GluN2B-E413G mutant cDNA with primary neurons,and whole-cell voltage clamp was used to evaluate excitatory postsynaptic currents.4.We established GluN2B-E413G mutant mouse model with CRISPR,and evaluated effects on electrophysiological functions,synaptic development and behaviors with slice voltage clamp,dendritic spine count and behavioral experiments.Results:1.The patient with GluN2A-M536I suffered from seizure and abnormal EEG.Agonist potency evaluation showed that GluN2A-M536I exhibited stronger glycine potency than wild type（P=0.01）,while no significant difference for glutamate potency（P=0.28）.Negative allosteric sensitivity(H⁺,Mg²⁺,Zn²⁺)showed no significant difference（P=0.80,P=0.93,P=0.86）.Open probability of ion channel of the mutation was smaller than that of wild type（P=0.03）.2.There was no statistical difference of the current amplitudes between GluN2A-M536I and wild type（P=0.96）.There was no statistical difference of channel opening times between the mutant and wildtype（P=0.74）.3.The excitatory postsynaptic currents showed smaller peaks,fast rise time and fast decay time of co-cultured HEK cells transfected with GluN2B-E413G,while there is no statistical difference of the current frequency and half-width.4.Micro-EPSCs（m EPSCs）generated from hippocampal CA1neurons showed significant difference between weighted Tau values of GluN2B-E413G and wild type（P=0.0078）.The density of total dendritic spines and mushroom-type dendritic spines decreased in the hippocampal CA1 of mutant mice.The mutant mice exhibited normal exercise coordination ability in rotarod experiment,anxiety-like behavior in elevated zero-maze experiment,and exhibited anxiety-like behaviors and memory impairment in the locomotor experiment.Conclusion:1.GluN2A-M536I enhanced the glycine efficacy of NMDA receptors and reduced the opening probability of ion channels,while GluN2B-E413G produced excitatory postsynaptic currents with smaller amplitude and faster decay in electrophysiological experiments in vitro.2.The mutant mice with GluN2B-E413G showed obvious anxiety behaviors and memory impairment and reduced the number of dendritic spines in the hippocampus CA1 region,which affected abilities of learning,memory and neurodevelopment.