STRUCTURE OF THE GABA A RECEPTOR BINDING SITES

GABA A 受体结合位点的结构

• 批准号: 6321998
• 负责人: CYNTHIA M CZAJKOWSKI
• 金额: $ 5万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2003-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6321998
• 关键词: GABA receptor; Xenopus; benzodiazepines; binding sites; chemical binding; cysteine; electrophysiology; molecular site; protein structure function; receptor binding; site directed mutagenesis; western blottings

GABA/A receptors are the major inhibitory neurotransmitter receptors in the mammalian CNS and are the site of action of benzodiazepines (BZDs). GABA analogues and BZDs are used in the treatment of a variety of neurological and psychiatric disorders, but detailed molecular structures of the GABA and BZD binding sites are unknown. The long-term goal of our research program is to understand the function of the GABA/A receptor in terms of its molecular structure. As a first step, we propose to identify and locate in the receptor structure the amino acid residues that form the GABA and BZD binding sites and determine the secondary protein structures which contribute to forming these sites. We are utilizing the substituted cysteine accessibility method in combination with mutagenesis, affinity labeling and cross-linking experiments to achieve these aims. The combination of these approaches is quite powerful and can provide details about the molecular structure of these binding sites which are not obtainable by mutagenesis and photoaffinity labeling techniques alone. If the structure of the GABA and BZD binding sites were known in detail, it is possible that whole new classes of site-specific compounds would be discovered and existing compounds could be modified to exploit the physicochemical features of the binding site to yield higher affinity, more selective drugs. The substituted cysteine accessibility method is a combination of single, consecutive cysteine- substitutions by site-directed mutagenesis of wild-type amino acid residues, heterologous functional expression of the mutants, and the probing of the substituted cysteines with sulfhydryl-specific (SH-) reagents. If a cysteine-substituted residue is part of a binding site, reaction with a SH-reagent will alter binding irreversibly, and site-selective ligands will protect the engineered cysteine from covalent modification. Direct detection of a modified cysteine can be accomplished by using the SH- reagent, MTSEA-Biotin, in combination with avidin-bead precipitation and Western blotting techniques. By examining the pattern of accessibility of consecutively engineered cysteines to reaction with SH-reagents, the secondary structure of these residues can be determined; e.g. alpha-helix or beta-strand. Elaborations of this approach allow us to determine the charge-selectivity of the binding sites and to determine the distance between pairs of engineered cysteines. Thus, detailed structural maps of the GABA and BZD binding sites can be obtained, and the 3-dimensional structure of the sites can be potentially modeled. In the absence of an X- ray crystal structure, this biochemical approach will provide, at a molecular level, the most detailed structural picture of the GABA/A receptor available.

GABA/A受体是哺乳动物中枢神经系统中主要的抑制性神经递质受体，是苯二氮卓类药物（BZDs）的作用部位。GABA类似物和BZD被用于治疗多种神经和精神疾病，但GABA和BZD结合位点的详细分子结构尚不清楚。我们研究计划的长期目标是了解GABA/A受体在分子结构方面的功能。作为第一步，我们建议在受体结构中识别和定位形成GABA和BZD结合位点的氨基酸残基，并确定有助于形成这些位点的二级蛋白质结构。我们正在利用替代半胱氨酸接近性方法结合诱变、亲和标记和交联实验来实现这些目标。这些方法的结合是非常强大的，可以提供这些结合位点的分子结构的细节，这些细节是单独通过诱变和光亲和标记技术无法获得的。如果详细了解GABA和BZD结合位点的结构，就有可能发现全新的位点特异性化合物类别，并且可以对现有化合物进行修饰，以利用结合位点的物理化学特征来产生更高亲和力，更具选择性的药物。取代半胱氨酸可及性方法是通过对野生型氨基酸残基进行定点诱变，将单个连续的半胱氨酸取代，突变体的异源功能表达，以及用巯基特异性（SH-）试剂探测取代的半胱氨酸。如果半胱氨酸取代残基是结合位点的一部分，与sh试剂的反应将不可逆地改变结合，位点选择性配体将保护工程半胱氨酸免受共价修饰。使用SH-试剂MTSEA-Biotin，结合亲和素珠沉淀和Western blotting技术，可以直接检测修饰的半胱氨酸。通过检测连续工程半胱氨酸与sh试剂反应的可及性模式，可以确定这些残基的二级结构；例如-螺旋或-链。这种方法的细化使我们能够确定结合位点的电荷选择性，并确定工程半胱氨酸对之间的距离。因此，可以获得GABA和BZD结合位点的详细结构图，并且可以潜在地建模这些位点的三维结构。在缺乏X射线晶体结构的情况下，这种生化方法将在分子水平上提供GABA/ a受体最详细的结构图像。