Structural Determinants of GABA-A receptor function

描述(申请人提供)：伽马氨基丁酸(GABA)是哺乳动物中枢神经系统中的主要抑制性神经递质，大脑中最快速的抑制性神经传递是由GABA受体(GABA受体)介导的。有许多家族的GABAR亚基具有多种亚型。α亚基家族是最大的，有六种不同的亚型(alpha1-alpha6)。这些亚型的表达受到区域性和发展性的调节，很可能每个阿尔法亚型都发挥着独特的生理作用。亚型的表达也会受到病理条件的影响，比如癫痫。受体的亚基组成影响GABAR的许多特性，但对阿尔法亚型的特性对通道动力学特性的影响知之甚少。这项工作的长期目标是了解GABAR结构异质性的功能意义，并确定亚基之间的哪些结构差异是其不同功能特性的基础。这项拟议的工作将通过比较含有不同阿尔法亚型的重组受体在单通道、大片和全细胞水平上的特性来研究这个问题。这些结果的结合将允许完整地描述与每个阿尔法亚型相关的动力学特性，并可能预测包含这些亚型的突触受体的行为。将详细研究导致不同动力学性质的结构差异，包括α1和α6亚型，这两个亚型是阿尔法亚单位家族中功能最多样化的。为了分离负责与每个亚型相关的功能特征的一般结构域，将检查六个嵌合的α1-α6亚基。这些嵌合体被设计来分离来自四个跨膜结构域中的每一个的功能贡献。一旦分离出一般的结构域，将通过定点突变识别负责这些特征的特定残基。GABAR在调节神经元活动中起着不可或缺的作用。GABAR是通常用作镇静剂、抗焦虑药和抗癫痫药以及包括巴比妥酸盐和酒精在内的成瘾药物的重要靶标。其中许多调节剂的活性取决于受体的阿尔法亚型组成。所描述的项目将解决阿尔法子类型之间的结构差异如何影响通道的功能属性的问题。这项工作的结果将有助于我们理解天然GABAα亚基组成的变化如何影响神经元的特性及其对GABA的反应。

DESCRIPTION (provided by applicant): Gamma-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the mammalian central nervous system, and most fast inhibitory neurotransmission in the brain is mediated by the GABAA receptors (GABARs). There are many families of GABAR subunits with multiple subtypes. The alpha subunit family is the largest, with six different subtypes (alpha1-alpha6). The expression of the subtypes is regulated regionally and developmentally and it is likely that each alpha subtype performs a unique physiological role. Subtype expression is also altered by pathological conditions, such as epilepsy. The subunit composition of the receptor affects many characteristics of the GABAR, but little is known about the effect of the identity of the alpha subtype on the channel's kinetic properties. The long-term goal of this work is to understand the functional significance of the structural heterogeneity of the GABAR and to determine which structural differences among the subunits underlie their different functional properties. The proposed work will examine this question by comparing the properties of recombinant receptors containing different alpha subtypes at the single channel, macropatch and whole-cell levels. The combination of these results will allow a complete description of the kinetic properties associated with each alpha subtype and may predict the behavior of synaptic receptors containing these subtypes. The structural differences responsible for distinct kinetic properties will be examined in detail for the alpha1and alpha 6 subtypes, which are the most functionally diverse in the alpha subunit family. To isolate the general structural domains responsible for the functional characteristics associated with each subtype, six chimeric alpha1 - alpha6 subunits will be examined. These chimeras were designed to isolate the functional contribution made from each of the four transmembrane domains. Once the general structural domains are isolated, the specific residues responsible for these characteristics will be identified through site-directed mutagenesis. The GABARs play an integral role in regulating neuronal activity. GABARs are important targets for drugs commonly used as sedatives, anxiolytics and anti-epileptics as well as for drugs of addiction, including barbiturates and alcohol. The activity of many of these modulators depends upon the alpha subtype composition of the receptor. The projects described will address the question of how structural differences among the alpha subtypes contribute to the functional properties of the channel. The results of this work will help us to understand how variations in the alpha subunit composition of native GABARs can affect the properties of neurons and their responses to GABA.