Iizuka M, Nishimura S, Wakamori M, Akiba I, Imoto K, Barsoumian E L
Department of Molecular and Cellular Biology, Nippon Boehringer Ingelheim Co., Ltd Kawanishi Pharma Research Institute, 3-10-1, Yato, Kawanishi, 666-0193, Japan.
Eur J Neurosci. 2000 Nov;12(11):3900-8. doi: 10.1046/j.1460-9568.2000.00270.x.
alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) -type glutamate receptors play a critical role in excitotoxicity associated with cerebral hypoxia, ischaemia and other acute brain insults. AMPA receptors are composed of GluR1-GluR4 subunits in homomeric and heteromeric assemblies, forming nonselective cation channels. In addition, each subunit has alternative splice variants, flip and flop forms. Heterologous expression studies showed that the AMPA receptor channels exhibit diverse properties depending on subunit/variant composition. For example, the absence of the GluR2 subunit makes AMPA receptor assemblies Ca2+-permeable. Excitotoxicity induced by activating AMPA receptor channels has been linked to excessive Ca2+ influx through the GluR2-lacking channels. Here we demonstrate that coexpression of the AMPA receptor GluR2flip and GluR4flip subunits exerts a lethal effect on HEK293 cells, whereas no lethal activity is observed in other homomeric or heteromeric combinations of AMPA receptor subunits. Patch clamp recordings and Ca2+ imaging analyses have revealed that this GluR2flip/GluR4flip receptor exhibits a low Ca2+ permeability. This subunit combination, however, showed prolonged Na+ influx following AMPA stimulation, even in the absence of cyclothiazide, which attenuates AMPA receptor desensitization. Furthermore, the GluR2flip/GluR4flip-mediated lethality was potentiated by the interruption of cellular Na+ extrusion mechanisms using ouabain or benzamil. These observations suggest that the GluR2flip/GluR4flip receptor-mediated excitotoxicity is attributed to Na+ overload, but not Ca2+ influx.
α-氨基-3-羟基-5-甲基-4-异恶唑丙酸（AMPA）型谷氨酸受体在与脑缺氧、缺血及其他急性脑损伤相关的兴奋毒性中起关键作用。AMPA受体由GluR1 - GluR4亚基以同聚体和异聚体形式组装而成，形成非选择性阳离子通道。此外，每个亚基都有可变剪接变体，即翻转和摇摆形式。异源表达研究表明，AMPA受体通道根据亚基/变体组成表现出不同特性。例如，缺乏GluR2亚基会使AMPA受体组装体对Ca2+通透。通过激活AMPA受体通道诱导的兴奋毒性与通过缺乏GluR2的通道过度Ca2+内流有关。在此我们证明，AMPA受体GluR2flip和GluR4flip亚基的共表达对HEK293细胞具有致死作用，而在AMPA受体亚基的其他同聚体或异聚体组合中未观察到致死活性。膜片钳记录和Ca2+成像分析表明，这种GluR2flip/GluR4flip受体表现出低Ca2+通透性。然而，即使在没有可减弱AMPA受体脱敏的环噻嗪的情况下，这种亚基组合在AMPA刺激后仍显示出延长的Na+内流。此外，使用哇巴因或苯甲酰米阻断细胞Na+外排机制可增强GluR2flip/GluR4flip介导的致死性。这些观察结果表明，GluR2flip/GluR4flip受体介导的兴奋毒性归因于Na+过载，而非Ca2+内流。
