Epilepsy-associated GRIN2A mutations reduce NMDA receptor trafficking and agonist potency - molecular profiling and functional rescue.
Epilepsy-associated GRIN2A mutations reduce NMDA receptor trafficking and agonist potency - molecular profiling and functional rescue.
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DOI：
10.1038/s41598-017-00115-w
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发表时间：
2017-02-27
影响因子：
4.6
通讯作者：
Ursu D
中科院分区：
文献类型：
--
作者：
Addis L;Virdee JK;Vidler LR;Collier DA;Pal DK;Ursu D
N-甲基-D-天冬氨酸受体（NMDAR）基因GRIN 2A的突变导致癫痫-失语综合征（EAS），一系列癫痫、认知和语言障碍。使用生物信息学和患者数据，我们筛选出10种不同的错义突变进行表征。我们使用高通量钙流测定和膜片钳记录瞬时转染的HEK-293细胞的电生理特性，蛋白质印迹和共聚焦成像来测定表达和表面运输。突变P79 R、C231 Y、G483 R和M705 V导致谷氨酸和甘氨酸激动剂效力显著降低，而D 731 N无响应。这些突变体，沿着E714 K，也显示出显著降低的总蛋白水平和向细胞表面的运输，而C436 R根本不被运输。至关重要的是，这种表面表达的减少并没有导致激动剂反应的减少。我们能够挽救表型P79 R，C231 Y，G483 R和M705 V治疗后，与GluN 2A选择性正变构调节剂。使用我们的方法，我们无法鉴定位于谷氨酸结合结构域和C末端之间的突变I814 T、D933 N和N976 S中的任何功能缺陷。我们发现GRIN 2A突变以不同的方式影响受体的表达和功能。对患者进行仔细的分子分析对于未来有效的个性化治疗选择至关重要。
Mutations in the N-methyl-D-aspartate receptor (NMDAR) gene GRIN2A cause epilepsy-aphasia syndrome (EAS), a spectrum of epileptic, cognitive and language disorders. Using bioinformatic and patient data we shortlisted 10 diverse missense mutations for characterisation. We used high-throughput calcium-flux assays and patch clamp recordings of transiently transfected HEK-293 cells for electrophysiological characterization, and Western blotting and confocal imaging to assay expression and surface trafficking. Mutations P79R, C231Y, G483R and M705V caused a significant reduction in glutamate and glycine agonist potency, whilst D731N was non-responsive. These mutants, along with E714K, also showed significantly decreased total protein levels and trafficking to the cell surface, whilst C436R was not trafficked at all. Crucially this reduced surface expression did not cause the reduced agonist response. We were able to rescue the phenotype of P79R, C231Y, G483R and M705V after treatment with a GluN2A-selective positive allosteric modulator. With our methodology we were not able to identify any functional deficits in mutations I814T, D933N and N976S located between the glutamate-binding domain and C-terminus. We show GRIN2A mutations affect the expression and function of the receptor in different ways. Careful molecular profiling of patients will be essential for future effective personalised treatment options.