1.Mutations of N-Methyl-D-Aspartate Receptor Subunits in Epilepsy.
Neuroscience Bulletin 2018;34(3):549-565
Epilepsy is one of the most common neurological diseases. Of all cases, 70%-80% are considered to be due to genetic factors. In recent years, a large number of genes have been identified as being involved in epilepsy. Among them, N-methyl-D-aspartate receptor (NMDAR) subunit-encoding genes represent a large proportion, suggesting an important role for NMDARs in epilepsy. In this review, we summarize and analyze the genotypes, functional alterations, and clinical aspects of NMDAR subunit mutations/variants identified from patients with epilepsy. These data will help to throw light upon the pathogenicity of these NMDAR mutations and advance our understanding of the subtle and complicated role of NMDARs in epilepsy. It will also offer new insights into precision therapy for this disorder.
Animals
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Epilepsy
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genetics
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Genetic Predisposition to Disease
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genetics
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Humans
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Mutation
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genetics
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Receptors, N-Methyl-D-Aspartate
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genetics
2.Functional Investigation of a GRIN2A Variant Associated with Rolandic Epilepsy.
Xing-Xing XU ; Xiao-Rong LIU ; Cui-Ying FAN ; Jin-Xing LAI ; Yi-Wu SHI ; Wei YANG ; Tao SU ; Jun-Yu XU ; Jian-Hong LUO ; Wei-Ping LIAO
Neuroscience Bulletin 2018;34(2):237-246
N-methyl-D-aspartate receptors (NMDARs), a subtype of glutamate-gated ion channels, play a central role in epileptogenesis. Recent studies have identified an increasing number of GRIN2A (a gene encoding the NMDAR GluN2A subunit) mutations in patients with epilepsy. Phenotypes of GRIN2A mutations include epilepsy-aphasia disorders and other epileptic encephalopathies, which pose challenges in clinical treatment. Here we identified a heterozygous GRIN2A mutation (c.1341T>A, p.N447K) from a boy with Rolandic epilepsy by whole-exome sequencing. The patient became seizure-free with a combination of valproate and lamotrigine. Functional investigation was carried out using recombinant NMDARs containing a GluN2A-N447K mutant that is located in the ligand-binding domain of the GluN2A subunit. Whole-cell current recordings in HEK 293T cells revealed that the N447K mutation increased the NMDAR current density by ~1.2-fold, enhanced the glutamate potency by 2-fold, and reduced the sensitivity to Mg inhibition. These results indicated that N447K is a gain-of-function mutation. Interestingly, alternative substitutions by alanine and glutamic acid at the same residue (N447A and N447E) did not change NMDAR function, suggesting a residual dependence of this mutation in altering NMDAR function. Taken together, this study identified human GluN2A N447K as a novel mutation associated with epilepsy and validated its functional consequences in vitro. Identification of this mutation is also helpful for advancing our understanding of the role of NMDARs in epilepsy and provides new insights for precision therapeutics in epilepsy.
Adolescent
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Epilepsy, Rolandic
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genetics
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Humans
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Male
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Mutation
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Receptors, N-Methyl-D-Aspartate
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genetics
3.Construction of expression vectors for C-terminally deleted NR2B subunit mutants and their application in the study of assembling of NMDA receptors.
Wei YANG ; Jian-hong LUO ; Ying GAO ; Man HUANG ; Chan-ying ZHENG
Journal of Zhejiang University. Medical sciences 2003;32(6):480-485
OBJECTIVETo investigate the role of NR2B subunit C-terminus in assembling and surface expression of NMDA receptor subtype composed of NR1-1a/NR2B subunits.
METHODSEight vectors NR2BDelta1-Delta8) expressing GFP-tagged NR2B subunit mutants with various deletion in the carboxyl-terminal region were generated by conventional molecular cloning techniques. Each of these vectors was transfected alone, or co-transfected with NR1-1a into HEK293 cells. NR1-1a/GFP-NR2B receptors on membrane surface of the living transfected cells were immuno-stained using rabbit antibody against GFP followed by Cy3 conjugated secondary antibody.
RESULTSThe eight vectors NR2BDelta1-Delta8 were successfully constructed. No surface labeling of GFP-tagged NMDA receptor was found for those transfected cells with NR1-1a, GFP-NR2B, and GFP-NR2BDelta1-Delta8 alone. GFP-tagged NMDA receptors were immuno-stained by anti-GFP for those cells co-transfected by NR1-1a and GFP-NR2B or GFP-NR2BDelta1-Delta6, which were mutants with partially deleted c-terminus at different region. However, positive stained was not found for those cells co-transfected by NR1-1a and GFP-NR2BDelta 7 (lack of most C-terminus and with PDZ binding motif fused with TM4) or GFP-NR2BDelta8 (lack of whole C-terminus).
CONCLUSIONSThe formation of NR1-1a/NR2B sub-type NMDA receptor requires co-expression and assembling of NR1-1a and NR2B subunits. Shield or inhibition of ER retention motif within C1 cassette of NR1-1a subunit by NR2B subunit when assembling is not dependent on any particular region in NR2B C-terminus.
Cell Line ; Genetic Vectors ; Humans ; Mutation ; Receptors, N-Methyl-D-Aspartate ; chemistry ; genetics ; Transfection
4.Inhibition of N-methyl-D-aspartate-activated current by bis(7)-tacrine in HEK-293 cells expressing NR1/NR2A or NR1/NR2B receptors.
Journal of Huazhong University of Science and Technology (Medical Sciences) 2012;32(6):793-797
In normal rat forebrain, the NR1/NR2A and NR1/NR2B dimmers are the main constitutional forms of NMDA receptors. The present study was carried out to determine the functional properties of the heteromeric NMDA receptor subunits and their inhibition by bis(7)-tacrine (B7T). Rat NR1, NR2A and NR2B cDNAs were transfected into human embryonic kidney 293 cells (HEK-293). The inhibition of NMDA-activated currents by B7T was detected in HEK-293 cell expressing NR1/NR2A or NR1/NR2B receptors by using whole-cell patch-clamp techniques. The results showed that in HEK-293 cells expressing NR1/NR2A receptor, 1 μmol/L B7T inhibited 30 μmol/L NMDA- and 1000 μmol/L NMDA-activated steady-state currents by 46% and 40%, respectively (P>0.05; n=5), suggesting that the inhibition of B7T on NR1/NR2A receptor doesn't depend on NMDA concentration, which is consistent with a non-competitive mechanism of inhibition. But for the NR1/NR2B receptor, 1 μmol/L B7T inhibited 30 μmol/L NMDA- and 1000 μmol/L NMDA-activated steady-state currents by 61% and 13%, respectively (P<0.05; n=6), showing that B7T appears to be competitive with NMDA. In addition, simultaneous application of 1 μmol/L B7T and 1000 μmol/L NMDA produced a moderate inhibition of peak NMDA-activated current, followed by a gradual decline of the current to a steady state. However, the gradual onset of inhibition produced by B7T applied simultaneously with NMDA was eliminated when B7T was given 5 s before NMDA. These results suggested that B7T inhibition of NMDA current mediated by NR1/NR2B receptor was slow onset, and it did not depend on the presence of the agonist. With holding potentials ranging from -50 to +50 mV, the B7T inhibition rate of NMDA currents didn't change significantly, and neither did the reversal potential. We are led to conclude that the NR1/NR2B recombinant receptor can serve as a very useful model for studying the molecular mechanism of NMDA receptor inhibition by B7T.
Cell Line
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HEK293 Cells
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Humans
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N-Methylaspartate
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pharmacology
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Receptors, N-Methyl-D-Aspartate
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genetics
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Tacrine
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analogs & derivatives
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pharmacology
6.Surface expression of NMDA receptors composed of NR1 subunit and NR2A subunit mutants with partially deleted C-terminus in HEK293 cells.
Chan-ying ZHENG ; Jian-hong LUO ; Ting FU ; Wei YANG ; Hai-qing SHEN
Journal of Zhejiang University. Medical sciences 2003;32(6):475-479
OBJECTIVETo examine the potential function of NMDA receptor NR2A subunit C-terminus in assembling and surface expression of the receptor in HEK293 cells.
METHODSFive vectors GFP- NR2ADeltaC1- DeltaC5 were constructed for expressing N-terminally GFP-tagged NR2A with C-terminal deletion at different regions by using conventional techniques of molecular cloning. The deleted region for NR2ADeltaC1-Delta C5 was 897L-1017S, 1024D-1142P, 1149D-1347G, 1354S-1464V, and 897L-1464V. These plasmids were transfected alone or co-transfected with NR1-1a into HEK293 cells. The surface NMDA receptors were immuno-stained using rabbit antibody against GFP and Cy3 conjugated secondary antibody in living cells.
RESULTThe vectors GFP-NR2ADeltaC1-DeltaC5 were generated and all of them expressed GFP fluorescence in the transfected cells. Surface NMDA receptors were detected by immuno-labeling with anti-GFP in the cells co-transfected by NR1-1a and any one of GFP-NR2ADeltaC1-DeltaC5. However, no surface expression of NR2A proteins was found in the transfected cells with any one of these plasmids alone.
CONCLUSIONWithin the region downstream from the 897L of NR2A subunit, neither a particular domain directly interacted with ER retention domain in NR1-1a C1 cassette, nor that determining ER retention of NR2A subunit itself has been found, indicating that more complicated mechanisms might exist in which the subunit assembling and targeting to plasma membrane of NMDA receptors undergo.
Cell Line ; Gene Deletion ; Green Fluorescent Proteins ; Humans ; Luminescent Proteins ; metabolism ; Mutation ; Receptors, N-Methyl-D-Aspartate ; analysis ; genetics
7.Influences of NR2B-containing NMDA receptors knockdown on neural activity in hippocampal newborn neurons.
Zhi-jun LI ; Hui-wen ZHANG ; Na TANG
Journal of Huazhong University of Science and Technology (Medical Sciences) 2013;33(4):457-462
Adult-born neurons undergo a transient period of plasticity during their integration into the neural circuit. This transient plasticity may involve NMDA receptors containing NR2B, the major subunit expressed at early developmental stages. The main objective of the present study was to investigate the effects of NR2B gene knockdown on the functional integration of the adult-born granule cells generated from the subgranule zone (SGZ) in the hippocampus. The small interfering RNA (siRNA) was used to knock down the NR2B gene in the adult-born hippocampal neurons. In the functional integration test, the mice were exposed to a novel environment (open field arena), and the expression of c-fos was immunohistochemically detected in the hippocampus. After exposure to the novel environment, siRNA-NR2B mice were significantly different from control mice in either the number of squares or the number of rears they crossed, showing decreased horizontal and vertical activity (P<0.05). Moreover, the c-fos expression was increased in both control and siRNA-NR2B mice after open field test. But, it was significantly lower in siRNA-NR2B neurons than in control neurons. It was concluded that the neural activity of newborn neurons is regulated by their own NR2B-containing NMDA glutamate receptors during a short, critical period after neuronal birth.
Animals
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Female
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Gene Knockdown Techniques
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Hippocampus
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physiology
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Male
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Mice
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Neurons
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physiology
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Receptors, N-Methyl-D-Aspartate
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genetics
8.Association of NMDAR, NR2A, and NR2B with cognitive impairment in diabetic patients.
Xiaopeng WANG ; Yongjie HUANG ; Fang WANG ; Yingying ZOU
Journal of Southern Medical University 2013;33(12):1848-1851
Cognitive impairment in diabetes (CID) is a severe chronic complication of diabetes mellitus, and its pathogenesis has not yet been fully understood. Increasing evidence has shown that the distribution and expression of N-methyl-D-aspartame receptor (NMDAR) and subunits NR2A and NR2B, which all participated in the development of the central nervous system and formation of learning and memory, are correlated with the occurrence and development of CID.
Central Nervous System
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Cognition Disorders
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complications
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genetics
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Diabetes Mellitus
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physiopathology
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Humans
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Memory
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Receptors, N-Methyl-D-Aspartate
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metabolism
9.Roles and expressions of the NMDA receptor subunits (NR2A and NR2B) in visual cortex area of kittens with the normal visual development and anisometropic amblyopia.
Haiwei LI ; Longqian LIU ; Xuyang LIU
Journal of Biomedical Engineering 2011;28(2):264-267
In order to understand the roles of the other subunits, we investigated expression of the NMDA receptor subunits (NR2A and NR2B) in visual cortex of normal and anisometropic amblyopia kittens with different ages in the present study. We examined the expressions of NR2A and NR2B in the visual cortex of the kittens by immunohistochemistry with polyclonal anti-NR2A antibody and anti-NR2B antibody, respectively. Using immunohisto-chemical Streptavidin Perosidase (SP) method, we observed the dynamic changes of NR2A and NR2B with microscope and computer-assisted image analyses. We found that NR2A and NR2B remained low expression after the peak of the critical period of kitten visual development; compared with normal group of the same age, NR2A expresses low. However, the difference is not significant for NR2B before maturation period of visual development. NR2B rises after the maturation period of visual development. According to this, the component of NR2A and NR2B can be affected by anisometropia. This research suggests that the difference of NR2A and NR2B expressions may affect the formation of amblyopia.
Amblyopia
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metabolism
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Animals
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Cats
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Female
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Male
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Receptors, N-Methyl-D-Aspartate
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genetics
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metabolism
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Vision, Ocular
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physiology
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Visual Cortex
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metabolism
10.A novel cell tool for α2δ-1-NMDAR target-based analgesic drug discovery.
Lin DONG ; Yiya ZHANG ; Jinjun CHEN
Chinese Journal of Biotechnology 2022;38(3):1149-1158
The α2δ-1 protein coded by Cacna2d1 is dramatically up-regulated in dorsal root ganglion (DRG) neurons and spinal dorsal horn following sensory nerve injury in various animal models of neuropathic pain. Cacna2d1 overexpression potentiates presynaptic and postsynaptic NMDAR activity of spinal dorsal horn neurons to cause pain hypersensitivity. The α2δ-1-NMDAR interaction promotes surface trafficking and synaptic targeting of NMDARs in neuropathic pain caused by chemotherapeutic agents and peripheral nerve injury, as well as in other pathological conditions such as in the paraventricular nucleus (PVN) with neurogenic hypertension and in the brain with ischemic stroke. The lentiviral transfection method was used to construct a human embryonic kidney HEK293T cell line that could stably express α2δ-1-NMDAR complex. A stably transfected cell line was observed by florescence microscope, and identified by RT-qPCR and Western blotting. The results showed that the HEK293T cell line was successfully transfected and the genes could be stably expressed. Subsequently, the transfected cell line was successfully developed into a target drug screening system using patch clamp techniques. It provides a promising cell model for further research on the interaction mechanism of α2δ-1-NMDAR complex and drug screening for chronic pain and related diseases with low side effects.
Analgesics/therapeutic use*
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Animals
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Drug Discovery
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HEK293 Cells
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Humans
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Neuralgia/metabolism*
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Receptors, N-Methyl-D-Aspartate/genetics*