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

谷氨酸(Glu)是中枢神经系统(CNS)中的一种主要的兴奋性神经递质，它可以增强突触的功效，这一点在海马区最为明显，这表明Glu在学习和记忆功能中起着关键作用。另一方面，最近发现了抑制中枢神经系统膜电位超极化的谷氨酸受体(GluR)，这一发现为调节神经元网络的兴奋性提供了一种新的机制。然而，抑制性GluR的分子克隆还没有成功，因为它在中枢神经系统中的存在是分散的和有限的。在先前的研究中，我们已经证明了抑制性GluR的存在，它与钾通道有关，由未知的但可能是GQ蛋白控制的，在已鉴定的Euhadra神经节的神经元中。本研究的目的是利用膜片钳记录和RTPCR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。…用膜片钳技术进一步全细胞记录Glu诱导的神经元抑制反应，将细胞胞浆吸入膜片移液管，其内液含有逆转录酶和Aligo-T7等试剂以促进cDNAi合成。首次用GluR亚基(GluR1-4编码序列)的正、反义引物进行聚合酶链式反应后，用琼脂糖凝胶电泳法对扩增出的DNA进行分析。但没有检测到与GluR1-4相对应的条带。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性与那些离子型GluR亚型有很大的不同。另一方面，在与代谢型(mGluR1-6的共同片段)的第一次PCR反应后扩增的DNA分析中，发现了一些相似之处。这些聚合酶链式反应产物被亚克隆。在它们测序后，至少检测到两条未知条带。用这些聚合酶链式反应产物作为探针对获得的文库进行筛选。较少

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