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性调节。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，因此现在人们对其结构和功能特性有了很好的了解。相反，GABA-B受体的作用仍有待阐明。本课题的目的是：1、研究GAB受体对电压门控性钙通道功能的调节作用；2、建立一个可表达功能性GABA-B受体的模型系统，用于GABA-B受体的电生理分析。关于第一个目的，我用膜片钳技术研究了GABA-B激动剂巴氯芬对高压激活(HVA)-钙通道的作用。实验结果表明，GABA-B受体的激活不仅对钙通道开放有众所周知的抑制作用，而且对HVA-钙通道电流也有一过性的增强作用。提示这种作用可能是通过细胞内Se信使系统的改变来实现的。对于第二个目的，我已经建立了可以作为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。利用荧光GFP(绿色荧光蛋白)结合突变基因的显微注射技术，外源突变基因可以在培养盘中鉴定的细胞中表达。

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.