STRUCTURE OF THE GABA A RECEPTOR BINDING SITES

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，也是苯二氮类药物的作用部位。GABA类似物和BZD被用于治疗各种神经和精神疾病，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从分子结构的角度了解GABA/A受体的功能。作为第一步，我们建议在受体结构中鉴定和定位形成GABA和BZD结合位点的氨基酸残基，并确定有助于形成这些结合位点的二级蛋白质结构。我们正在利用取代半胱氨酸可及性方法，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，可以提供关于这些结合部位的分子结构的细节，而这些结合部位的分子结构不是单靠诱变和光亲和标记技术所能获得的。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，并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生产更高亲和力、更具选择性的药物。取代半胱氨酸可及性方法是通过野生型氨基酸残基的定点突变、突变体的异源功能表达和用SH-试剂探测取代半胱氨酸的组合。如果半胱氨酸取代的残基是结合位点的一部分，与SH试剂的反应将不可逆转地改变结合，而位点选择性配体将保护工程半胱氨酸免受共价修饰。通过使用SH试剂MTSEA-生物素，结合亲和素珠沉淀和Western blotting技术，可以实现对修饰半胱氨酸的直接检测。通过检查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，α-螺旋或β-链。这种方法的详细阐述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，可以获得详细的GABA和BZD结合部位的结构图，并且可以潜在地模拟这些部位的三维结构。在没有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可用的GABA/A受体最详细的结构图。

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.