An attempt to establish clonal cell strains expressing glutamate receptor channels.

编码非NMDA谷氨酸受体亚单位的GluR1-GluR3 cDNA已经从大鼠脑中分离出来(Boulter al.，Science，249：1033,1990)。由这些亚基组成的同体或异体受体不仅对AMPA和奎斯奎宁有反应，而且对海人藻酸也有反应。本研究的目的是建立表达这些亚基组成的功能通道的克隆细胞株，以促进谷氨酸受体的生化、生理和药理学研究。我们已经证实，AMPA、海人藻酸和谷氨酸对注射GluR1或GluR3体外转录RNA的非洲爪哇卵母细胞产生电流反应。首次将GluR1或GluR3基因克隆到带有新霉素抗性基因的pKan 2载体中。该载体在正义方向含有GluR1或GluR3，然后将其亚克隆到PCDL-srα296表达载体中。用电穿孔法将该表达载体导入大鼠脑胶质瘤C6细胞。将这些C6细胞置于新霉素筛选下。对选定的C6细胞进行非NMDA受体的放射配基结合分析。在这些细胞中检测到[^~3H]AMPA的特异性结合。接下来，我们检查了这些细胞中是否表达了功能性的非NMDA受体。全细胞膜片钳研究表明，AMPA、海人藻酸和谷氨酸不能诱发电生理反应。由于同分异构体GluR1或GluR3受体对Ca^&lt；2+&gt；有很高的通透性，我们也尝试用Fura-2微量荧光分光光度法检测这些激动剂引起的胞内Ca+&gt；浓度的升高。然而，这些激动剂不能改变这些细胞内的胞浆钙离子浓度。因此，需要更多的工作来建立表达功能性GluR受体通道的克隆细胞株。

GluR1-GluR3 cDNAs encoding non-NMDA glutamate receptor subunits have been isolated from the rat brain (Boulter al., Science, 249:1033, 1990). Either homomeric or heteromeric receptors composed of these subunits respond to not only AMPA and quisqualate, but also kainate. The purpose of the present study is to establish clonal cell strains that express the functional channels composed of these subunits in order to facilitate biochemical, physilogical and pharmacological studies of glutamate receptors.GluR1 and GluR3 cDNAs were kindly provided by Dr. Jim Boulter (The Salk Institute, USA). We have confirmed that AMPA, kainate and glutamate induce current responses in Xenopus oocytes injected with in vitro transcribed RNA from either GluR1 or GluR3 cDNA. GluR1 or GluR3 cDNA was first cloned into pKan 2 vector with the neomycine-resistant gene. This vector containing either GluR1 or GluR3 cDNA in the sense direction was then subcloned into a pcDL-sR alpha 296 expression vector. This expression vector construct was transfected into the rat glioma C6 cells by electroporation. These C6 cells were placed under the neomycin selection. The selected C6 cells were subjected to radioligand binding assay for the non-NMDA receptor. The specific binding of [^3H] AMPA was detected in these cells. Next, we examined whether functional non-NMDA receptors were expressed in these cells. Whole-cell patch-clamp studies indicated that AMPA kainate and glutamate failed to induce electrophysiological responses. Since homomeric GluR1 or GluR3 receptors are known to be highly permeable to Ca^<2+>, we also tried to detect an increase in cytosolic Ca^<2+> concentration in response to these agonists with the use of fura-2 microfluorometry. However, these agonists failed to change the cytosolic Ca^<2+> concentration in these cells. Thus, more work is needed to establish clonal cell strains expressing functional GluR receptor channels.