Department of Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, King's College London, London, UK.
Neuroscience Discovery, Eli Lilly Research Centre, Windlesham, Surrey, UK.
Sci Rep. 2017 Feb 27;7(1):66. doi: 10.1038/s41598-017-00115-w.
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.
N-甲基-D-天冬氨酸受体（NMDAR）基因 GRIN2A 的突变导致癫痫伴失语综合征（EAS），这是一种癫痫、认知和语言障碍的谱系疾病。我们使用生物信息学和患者数据，对 10 种不同的错义突变进行了筛选，以进行特征描述。我们使用高通量钙通量测定和瞬时转染的 HEK-293 细胞的膜片钳记录进行电生理特性分析，并用 Western blot 和共聚焦成像检测表达和表面转运。突变 P79R、C231Y、G483R 和 M705V 导致谷氨酸和甘氨酸激动剂的效力显著降低，而 D731N 则没有反应。这些突变体，连同 E714K，也显示出总蛋白水平和表面转运的显著降低，而 C436R 则完全没有转运。至关重要的是，这种减少的表面表达并没有导致激动剂反应的减少。我们能够在用 GluN2A 选择性正变构调节剂治疗后，挽救 P79R、C231Y、G483R 和 M705V 的表型。通过我们的方法，我们无法在位于谷氨酸结合域和 C 末端之间的突变 I814T、D933N 和 N976S 中发现任何功能缺陷。我们表明，GRIN2A 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方案至关重要。
