Korczak B, Nutt S L, Fletcher E J, Hoo K H, Elliott C E, Rampersad V, McWhinnie E A, Kamboj R K
Allelix Biopharmaceuticals Inc., Mississauga, Ontario, Canada.
Recept Channels. 1995;3(1):41-9.
We have isolated a new member of the human glutamate receptor family from a fetal brain cDNA library. This cDNA clone, designated EAA3a, shares a 90% nucleotide identity with the previously reported rat GluR5-2b cDNA splice variant and differed from human GluR5-1d in the amino and carboxy terminal regions. Cell lines stably expressing EAA3a protein formed homomeric ligand-gated ion channels responsive, in order of decreasing affinity to domoate, kainate, L-glutamate and (RS)-alpha-amino-3-hydroxy-5- methylisoxazole-propionate (AMPA). Kainate-evoked currents showed partial desensitization that was reduced on incubation with concanavalin A (conA) but not cyclothiazide and were attenuated by the non-N-methyl-D-aspartate (NMDA) receptor antagonist CNQX (6-cyano-7-nitro-quinoxalinedione). Coexpression of EAA3a and human EAA1 cDNAs in HEK 293 cells formed a heteromeric channel with unique properties. Kainate and AMPA activated the heteromeric channel with significantly higher affinities than observed for EAA3a alone. Ligand binding studies with the recombinant EAA3a receptor expressed in mammalian cells indicated a high affinity kainate binding site (Kd = 120 +/- 15.0 nM). The relative potency of compounds in displacing [3H]-kainate binding to EAA3a receptor was: domoate > kainate > L-glutamate = quisqualate > 6,7-dinitroquinoxaline-2,3-dione (DNQX) = CNQX > AMPA > dihydrokainate > NMDA.
我们从胎儿脑cDNA文库中分离出了人类谷氨酸受体家族的一个新成员。这个cDNA克隆被命名为EAA3a，与先前报道的大鼠GluR5-2b cDNA剪接变体有90%的核苷酸同一性，并且在氨基和羧基末端区域与人类GluR5-1d不同。稳定表达EAA3a蛋白的细胞系形成了同源配体门控离子通道，对软骨藻酸、海人藻酸、L-谷氨酸和（RS）-α-氨基-3-羟基-5-甲基异恶唑-4-丙酸（AMPA）的亲和力依次降低。海人藻酸诱发的电流显示出部分脱敏，用伴刀豆球蛋白A（conA）孵育后脱敏程度降低，但用环噻嗪孵育则无此现象，并且被非N-甲基-D-天冬氨酸（NMDA）受体拮抗剂CNQX（6-氰基-7-硝基喹喔啉-2,3-二酮）减弱。在HEK 293细胞中共表达EAA3a和人类EAA1 cDNA形成了具有独特性质的异源通道。海人藻酸和AMPA激活异源通道的亲和力明显高于单独观察到的EAA3a。对在哺乳动物细胞中表达的重组EAA3a受体进行的配体结合研究表明存在一个高亲和力的海人藻酸结合位点（Kd = 120 +/- 15.0 nM）。化合物取代[3H]-海人藻酸与EAA3a受体结合的相对效力为：软骨藻酸>海人藻酸>L-谷氨酸 = 喹啉酸>6,7-二硝基喹喔啉-2,3-二酮（DNQX） = CNQX>AMPA>二氢海人藻酸>NMDA。
