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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质或异质受体不仅对AMPA和准质酸有反应，而且对盐酸盐也有反应。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、生理和药理研究。GluR1和GluR3 cdna由Dr. Jim Boulter （The Salk Institute， USA）提供。我们已经证实，AMPA， kainate和谷氨酸盐在注射GluR1或GluR3 cDNA体外转录RNA的爪蟾卵母细胞中诱导电流反应。首先将GluR1或GluR3 cDNA克隆到带有新霉素耐药基因的pKan 2载体上。该载体在义方向上含有GluR1或GluR3 cDNA，然后将其亚克隆到pcDL-sR α 296表达载体中。将该表达载体通过电穿孔转染到大鼠胶质瘤C6细胞中。这些C6细胞置于新霉素选择下。选择的C6细胞进行非nmda受体的放射配体结合试验。在这些细胞中检测到[^3H] AMPA的特异性结合。接下来，我们检查了功能性非nmda受体是否在这些细胞中表达。全细胞膜片钳研究表明AMPA、kainate和谷氨酸不能诱导电生理反应。由于已知同质GluR1或GluR3受体对Ca^<2+>具有高度渗透性，我们也试图使用fura-2微荧光法检测这些激动剂对细胞内Ca^<2+>浓度的反应。然而，这些激动剂未能改变这些细胞的胞浆Ca^<2+>浓度。因此，需要更多的工作来建立表达功能GluR受体通道的克隆细胞株。