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

尝试建立表达谷氨酸受体通道的克隆细胞株。

基本信息

批准号：
03557005
负责人：
OZAWA Seiji
金额：
$ 6.66万
依托单位：
Gunma University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03557005/
关键词：
Glutamate receptor AMPA Kainate Expression vector Transfection Clonal cell strain Patch-clamp Fura-2 cDNA 発現プロモーター fura-2 cDNA発現

项目摘要

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.

编码非 NMDA 谷氨酸受体亚基的 GluR1-GluR3 cDNA 已从大鼠脑中分离出来（Boulter al., Science, 249:1033, 1990）。由这些亚基组成的同聚或异聚受体不仅对 AMPA 和使君子酸盐有反应，而且对红藻氨酸有反应。本研究的目的是建立表达由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。GluR1和GluR3 cDNA由Jim Boulter博士（The Salk）友情提供研究所，美国）。我们已经证实，AMPA、红藻氨酸和谷氨酸会在注射了来自 GluR1 或 GluR3 cDNA 的体外转录 RNA 的非洲爪蟾卵母细胞中诱导电流反应。首先将 GluR1 或 GluR3 cDNA 克隆到带有新霉素抗性基因的 pKan 2 载体中。然后将该有义方向含有 GluR1 或 GluR3 cDNA 的载体亚克隆到 pcDL-sR α 296 表达载体中。通过电穿孔将该表达载体构建体转染至大鼠神经胶质瘤C6细胞中。这些C6细胞被置于新霉素选择之下。对选定的 C6 细胞进行非 NMDA 受体的放射性配体结合测定。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功能性非 NMDA 受体。全细胞膜片钳研究表明 AMPA 红藻氨酸和谷氨酸不能诱导电生理反应。由于已知同聚GluR1或GluR3受体对Ca 2+ 具有高度渗透性，因此我们还尝试使用fura-2显微荧光测定法来检测响应这些激动剂的胞质Ca 2+ 浓度的增加。然而，这些激动剂未能改变这些细胞中的胞质Ca 2+ 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。