STRUCTURE OF THE GABA A RECEPTOR BINDING SITES

GABAA受体是脑内主要的抑制性神经递质受体 哺乳动物的中枢神经系统，是苯二氮类药物(BZD)的作用部位。 GABA类似物和BZD用于治疗各种 神经和精神障碍，但分子结构 GABA和BZD结合位点尚不清楚。我们的长期目标是 研究计划是为了了解GABAA受体在 关于它的分子结构。作为第一步，我们建议 识别和定位受体结构中的氨基酸残基 它们形成了GABA和BZD结合位点。一种创新的方法， 取代半胱氨酸可及性方法，将用于探查 系统地将GABA和BZD结合部位的整个表面 GABAA受体的口袋。该方法可以提供详细的 关于该区域以外结合部位结构的分子信息 通常使用传统突变或亲和标记获得 技巧。如果GABA和BZD结合位点的结构是 在分子细节上已知，有可能整个新类别的 特定部位的化合物将被发现，现有的化合物可能 被修改以利用结合部位的物理化学特征 以产生更高的亲和力，更具选择性的药物。被取代的人 半胱氨酸可及性方法是一种单次、连续 野生型氨基酸的定点突变半胱氨酸取代 酸残基，突变体的异源功能表达，以及 巯基专一性取代半胱氨酸的探测 试剂。这些试剂很小，带电，亲水，而且 疏水性，因此只在可接触水的表面发生反应 受体。如果与半胱氨酸取代变种的结合是不可逆转的 被巯基1特异性试剂阻止，如果这种阻止 结合可以被位点特异性配体阻止，我们可以推断 相应野生型残基的侧链排列结合 工地口袋。连续工程的可达性模式 半胱氨酸与巯基1-特异性试剂反应反映了 这些残基的二级结构；例如α螺旋或β折叠。 功能和结合异常的突变体，其可及性 邻近残基与1-硫基-1-特异试剂反应 将使我们能够推断包含该区域的二级结构 该残留物，从而确定残留物是否暴露在 结合部位。因此，在结构上形成的所有残基 结合位点可以成功作图，并且三维结构 可以潜在地进行建模。在没有X光的情况下 晶体结构，这一生化方法将在分子水平上提供 水平，迄今为止最详细的GABAA受体结构图 可用。

GABAA 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 the molecular structures of the GABA and BZD binding sites are unknown. The long-term goal of our researach program is to understand the function of the GABAA 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. An innovative approach, the substituted cysteine accessibility method, will be used to probe systematically the entire surface of the GABA and BZD binding site pockets of the GABAA receptor. This method can provide detailed molecular information about the structure of binding sites beyond that usually obtained using traitional mutagenesis or affinity labeling techniques. If the structure of the GABA and BZD binding sites were known in molecular detail, it is possible that whole neew 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-substitution by site directed mutagenesis of wild-type amino acid residues, heterologous functional expression of the mutants, and the probing of the substituted cysteines with sulfhydry1-specific reagents. These reagents are small, charged, hydrophilic, and lipophobic, and thus reactonly at the water-accessible surface of the receptor. If binding to a cysteine substitution mutnt is irreversibly blocked by the sulfhydry1-specific reagents, and if this blockade of binding can be prevented by site-specific ligands, we would infer that the side chain of the corresponding wild-type residue lines the binding site pocket. The pattern of accessibility of consecutive engineered cysteines to reaction with the sulfhydry1-specific reagents reflects the secondary structure of these residues;e.g. alpha helix or beta sheet. Inmutants with abnormal function and binding, the accessibility of neighboring residues to reaction with the sulfhydry1-specific reagents will allow us to deduce the secondary structure of the region containing this residue,and thus determine whether or not the residue is exposedin the binding site. Thus, all residues which structurally form the binding site can be successfully mapped, and the 3-dimensional structure of the site can be potentially modeled. In the absence of an X-ray crystal structure, this biochemical aproach will provide, at a molecular level, the most detailed structural picture of the GABAA receptor yet available.