MODULATORY SITES ON GABA A RECEPTORS

GABA-A受体受许多静脉全身麻醉药（IV）的调节 GAs），以临床相关剂量。低浓度的IV GA，例如 麻醉类固醇、巴比妥类药物和异丙酚可增强GABA 神经元的反应。较高的浓度，存在于血液中， 静脉麻醉，直接激活GABA-A受体开放Cl-通道， 从而降低神经元的兴奋性。这代表了一种机制， 有些静脉输液可以达到麻醉效果。 GABA-A受体是异质性的，由五个亚基组成。每个 亚基可能是迄今为止克隆的15个品种之一。使用贴片- 用钳夹技术记录单细胞GABA反应 已经发现具有不同亚基组合的受体具有 独特的药理学特性。初步数据显示，GABA-A 受体组合，由神经元GT 1 -7细胞系表达， 直接被高浓度的静脉内GA激活。为了研究亚基 GABA-A受体调节和IV GAs激活的特异性，GABA-A 受体亚基cDNA将转染到缺乏 内源性受体细胞将首先用同源重组质粒转染。 受体。这些将使用膜片钳技术进行测试， 确定同源受体是否是功能性的（特异性目标1）。 在确定了受体对GABA做出反应所需的亚基后， IV GAs增强GABA反应所需的亚基将是 具体目标（2）。如果需要额外的子单元， 功能性受体，或通过IV GAs调节GABA-A受体，这些 将被共转染到细胞中。测试的子单元将是那些 已经使用分子生物学方法在GT 1 -7细胞中鉴定了哪些mRNA 技术.最后，重组GABA-A受体将被测试，以确定 IV GA直接激活所需的亚基（特异性目标3）。 在未来，已经建立了GABA-A的亚基特异性， GABA和IV GAs直接激活受体，IV GAs调节， 这些将与类似作用的亚基特异性进行比较 在这个受体上的挥发性物质。对亚基的理解 GAs对GABA-A受体调节的特异性和GABA-A受体的分布 大脑中的这些亚单位可能暗示与麻醉有关的区域。一旦 确定了GA的靶位点，可能会开发更多 更安全有效的药物。

GABA-A receptors are modulated by many intravenous general anesthetics (IV GAs), at clinically relevant doses. Low concentrations of IV GAs, such as the anesthetic steroids, barbiturates and propofol, potentiate GABA responses in neurons. Higher concentrations, present in blood during total IV anesthesia, directly activate GABA-A receptors opening Cl- channels and thus reducing neuronal excitability. This represents a mechanism by which some IV GAs may achieve anesthesia. GABA-A receptors are heterogeneous consisting of five subunits. Each subunit could be one of fifteen varieties cloned so far. Using the patch- clamp technique to record GABA responses from single cells, GABA-A receptors with different subunit combinations have been found to possess distinct pharmacological properties. Preliminary data indicate that GABA-A receptor combinations, expressed by the neuronal GT1-7 cell line are not directly activated by high concentrations of IV GAs. To study the subunit specificity of GABA-A receptor modulation and activation by IV GAs, GABA-A receptor subunit cDNAs will be transfected into cell lines lacking endogenous receptors. The cells will first be transfected with homomeric receptors. These will be tested using the patch-clamp technique to determine whether homomeric receptors are functional (Specific Aim l). Having determined the subunits required for a receptor to respond to GABA, the subunits needed for potentiation of GABA-responses by IV GAs will be investigated (Specific Aim 2). If additional subunits are required for functional receptors, or modulation of GABA-A receptors by IV GAs, these will be co-transfected into cells. The subunits tested will be those for which mRNAs have been identified in GT1-7 cells using molecular techniques. Finally, recombinant GABA-A receptors will be tested to define the subunits required for direct activation by IV GAs (Specific Aim 3). In future, having established the subunit specificities for GABA-A receptor direct activation by GABA and IV GAs, and modulation by IV GAs, these will be compared to the subunit specificity for the similar actions of volatile agents at this receptor. An understanding of the subunit specificity of GABA-A receptor modulation by GAs and the distribution of these subunits in the brain may suggest areas involved in anesthesia. Once the target site(s) for GAs are defined it may be possible to develop more efficacious and safer agents.