Fletcher E J, Nutt S L, Hoo K H, Elliott C E, Korczak B, McWhinnie E A, Kamboj R K
Allelix Biopharmaceuticals Inc., Mississauga, ON, Canada.
Recept Channels. 1995;3(1):21-31.
A member of the ionotropic family of glutamate receptors, hGluR4, was isolated from a human cDNA library and characterized following expression in mammalian cell lines. Human GluR4 possessed a 99% amino acid and 92% nucleotide homology to that of its rat counterpart with sequence differences restricted to the carboxy and amino terminal regions of the molecule. Transfection of simian kidney cells (COS-1) with an hGluR4 expression plasmid resulted in the transient formation of a membrane protein that possessed high specific binding for 3H-alpha-amino- 3-hydroxy-5-methylisoxazole-4-propionic acid ([3H]AMPA) but not [3H]kainate. Competition studies yielded a displacement profile of AMPA = quisqualate > glutamate > domoate > kainate >> N-methyl-D-aspartate (NMDA) or dihydrokainate. Whole-cell, voltage-clamp recordings from a human embryonic kidney cell line (HEK 293) stably expressing hGluR4 confirmed the presence of constitutively active, ligand-gated ion channels activated by AMPA, glutamate and kainate but not N-methyl-D-aspartate. Kainate-evoked currents were reversibly attenuated by 6-cyano-7-nitro- quinoxaline-2,3-dione (CNQX) but not DL-2-amino-5- phosphonovalerate (DL-AP5). Agonist-evoked currents exhibited inward rectification and ion substitution experiments indicated that hGluR4 receptor-linked ion channels in their homomeric state are permeable to both CA2+ and Na+ ions. In the same cell line antibody to rat GluR4 immunoprecipitated a major protein band at approximately 108 kDa and a minor one at approximately 340 kDa. The immunoblot analysis of membranes chemically crosslinked with dithiobis(succinimidylpropionate) showed a broad band at 550-600 kDa suggesting that the GluR4 receptor forms a pentamer in situ. This is the first report of the cloning of hGluR4 receptor and its stable expression in a human cell line.
离子型谷氨酸受体家族的成员hGluR4，是从人cDNA文库中分离出来的，并在哺乳动物细胞系中表达后进行了特性鉴定。人GluR4与其大鼠对应物具有99%的氨基酸同源性和92%的核苷酸同源性，序列差异仅限于分子的羧基和氨基末端区域。用hGluR4表达质粒转染猴肾细胞（COS-1）导致瞬时形成一种膜蛋白，该蛋白对3H-α-氨基-3-羟基-5-甲基异恶唑-4-丙酸（[3H]AMPA）具有高特异性结合，但对[3H]海人藻酸没有。竞争研究得出的置换曲线为AMPA = quisqualate > 谷氨酸 > 软骨藻酸 > 海人藻酸 >> N-甲基-D-天冬氨酸（NMDA）或二氢海人藻酸。对稳定表达hGluR4的人胚肾细胞系（HEK 293）进行全细胞电压钳记录，证实存在组成型激活的配体门控离子通道，该通道可被AMPA、谷氨酸和海人藻酸激活，但不能被N-甲基-D-天冬氨酸激活。海人藻酸诱发的电流可被6-氰基-7-硝基喹喔啉-2,3-二酮（CNQX）可逆性减弱，但不能被DL-2-氨基-5-磷酸戊酸（DL-AP5）减弱。激动剂诱发的电流表现出内向整流，离子置换实验表明，处于同源状态的hGluR4受体连接的离子通道对Ca2+和Na+离子均具有通透性。在同一细胞系中，抗大鼠GluR4抗体免疫沉淀出一条约108 kDa的主要蛋白带和一条约340 kDa的次要蛋白带。用二硫代双（琥珀酰亚胺丙酸酯）化学交联的膜的免疫印迹分析显示在550-600 kDa处有一条宽带，表明GluR4受体在原位形成五聚体。这是关于hGluR4受体克隆及其在人细胞系中稳定表达的首次报道。
