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Molecular Cloning of a Novel Type of Glutamate Receptor Eliciting Membrane Hyperpolarization in a Single Cell Level.

新型谷氨酸受体的分子克隆在单细胞水平上引发膜超极化。

基本信息

批准号：
09680762
负责人：
WATANABE Kazuko
金额：
$ 0.7万
依托单位：
Gifu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

Glutamate (Glu), a major excitatory neurotransmitter in the central nervous system (CNS), has been known to enhance synaptic efficacy that is most pronounced in the hippocampus, indicating that Glu has a key role in learning and memory function. Recently, on the other hand, the inhibitory Glu receptor (GluR) which hyperpolarizes membrane potential in the CNS has founded and this finding suggests a new mechanism for modulating excitability in the neuronal networks. However, molecular cloning of the inhibitory GluR has not yet succeeded because of its scattered and limited presence in the CNS.In the previous study we have demonstrated the presence of the inhibitory GluR, which is linked to potassium channel controlled by unknown but probably a Gq protein, in the identified neurons of Euhadra ganglia. The purpose of the present study is to detect the genes coding this inhibitory GluR using the combination technique of patch-clamp recording and RT-PCR analysis in single live neurons.Follow … More ing whole-cell recording of the inhibitory response induced by Glu in the identified neuron using patch-clamp method, the cytoplasma of the cell was aspirated into the patch pipette whose internal solution contains required reagents including reverse transcriptase and aligo-T7 to facilitate cDNA synthesis. After the first PCR reaction with sense and antisense primers from subunits of ionotropic type of GluRs (GluR1-4 coding sequence), the amplified DNA was analysed on agarose gel elctrophoresis. But no band corresponding to the GluR1-4 was detected. This result was expected because that the electrophysiological and molecular biological characteristics of the inhibitory GluR was quite different from those ionotropic subtypes of GluRs. On the other hand, in the analysis of amplified DNA followed the first PCR reaction with the primers from metabotropic types (common segment of mGluR1-6), some similarity was founded. These PCR products were subcloned. Following their sequencing, at least two unknown bands were detected. A screening test of cDNA library obtained using these PCR products as a probe is continued. Less

谷氨酸（Glu）是中枢神经系统（CNS）中一种主要的兴奋性神经递质，已知可以增强突触效能，在海马中最为明显，这表明谷氨酸在学习和记忆功能中起关键作用。另一方面，近年来，抑制Glu受体（GluR）在中枢神经系统中引起膜电位的超极化，这一发现提示了一种调节神经元网络兴奋性的新机制。然而，由于其在中枢神经系统中的分散和有限存在，抑制GluR的分子克隆尚未成功。在之前的研究中，我们已经证明了在已识别的尾神经节神经元中存在抑制性GluR，它与由未知但可能是Gq蛋白控制的钾通道有关。本研究的目的是利用膜片钳记录和RT-PCR分析相结合的技术在单个活神经元中检测编码这种抑制性GluR的基因。利用膜片钳法对所鉴定的神经元中Glu诱导的抑制反应进行全细胞记录，将细胞的胞浆吸入膜片移液管，膜片移液管内液中含有逆转录酶和aligo-T7等促进cDNA合成所需的试剂。与GluRs嗜离子型亚基（GluR1-4编码序列）的正义和反义引物进行首次PCR反应后，用琼脂糖凝胶电泳分析扩增的DNA。但未检测到GluR1-4对应的条带。这一结果是意料之中的，因为抑制性GluR的电生理和分子生物学特征与那些嗜离子型GluR有很大不同。另一方面，在与代谢型引物（mGluR1-6共同片段）进行首次PCR反应后的扩增DNA分析中，发现了一定的相似性。这些PCR产物被亚克隆。在测序之后，至少发现了两个未知的条带。继续以这些PCR产物为探针对cDNA文库进行筛选试验。少