Functional analysis of GABA_B receptors using RT-PCR in snail neurons

神经递质GABA （γ -氨基丁酸）参与中枢神经系统多种功能的抑制调节。GAGA受体主要包括GABA-A和GABA-B受体。GABA-A受体长期以来一直是深入研究的主题，因此它们的结构和功能特性现在已经得到了很好的理解。相反，GABA-B受体的作用仍有待阐明。本项目的目的是：1，研究GABA-B受体对电压门控钙通道功能的调节作用；2，开发一个模型系统，在这个模型系统中，GABA-B受体的功能可以表达，用于GABA-B受体的电生理分析。关于第一个目的，我使用膜片钳技术研究了GABA-B激动剂巴氯芬对高压活化（HVA）钙通道的作用。实验结果表明，GABA-B受体的激活不仅对钙离子打开有抑制作用，而且对hva -钙离子通道电流有短暂的增强作用。提示这种效应可能是由细胞内se信使系统的变化介导的。关于第二个目的，我建立了可以作为GABA-B受体表达系统的模型系统。培养的颈上神经节神经元具有许多优点，例如，这些细胞缺乏固有的GABA-B受体。通过将突变基因与荧光绿色荧光蛋白（GFP）结合的显微注射技术，外源突变基因可以在鉴定的培养盘细胞中表达。

The neurotransmitter GABA (gamma -aminobutyric acid) is involved in the inhibitory regulation of a variety of functions in the central nervous system. The receptors for GAGA comprise mainly GABA-A and GABA-B receptors. The GABA-A receptors has long a subject of intensive investigation and thus their structural and functional properties are now well understood. On the contrary, the role of GABA-B receptors still remains be elucidated. The purpose of this project was 1, to examine a modulatory role of GA B receptors on the function of voltage-gated calcium channels, and 2, to develop a model system in which functional GABA-B receptors can be expressed for electrophysiological analysis of GABA-B receptors. Regarding the first purpose, I have investigated the actions of GABA-B agonist, baclofen, on the High voltage activated (HVA)-calcium channels using patch clamp technique. The experimental results show that an activation of GABA-B receptors causes not only well-known inhibitory effect on the calcium cha opening, but also a transient augmentation of the HVA-calcium channel currents. suggested that this effect was probably mediated by a change in the intracellular se messenger system. Regarding the second purpose, I have established the model system that can be used as the expression system of GABA-B receptors. Cultured neurons of superior cervical ganglion provided a number of advantages, e.g., these cells are devoid inherent GABA-B receptors. By the microinjection technique of the mutated genes bined with the use of fluorescent GFP (green-fluorescent protein), exogenous mutated genes could be expressed in an identified cells of the culture disc.