STRUCTURE OF THE GABA A RECEPTOR BINDING SITES

GABA A 受体结合位点的结构

• 批准号: 2038090
• 负责人: CYNTHIA M CZAJKOWSKI
• 金额: $ 17.02万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-12-01 至 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2038090
• 关键词: GABA receptor; Xenopus; Xenopus oocyte; benzodiazepines; chemical binding; cysteine; gamma aminobutyrate; molecular site; protein structure function; receptor binding; site directed mutagenesis

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.

GABAA受体是脑内主要的抑制性神经递质受体， 哺乳动物中枢神经系统，是苯二氮卓类（BZD）的作用部位。 GABA类似物和BZD用于治疗各种 神经和精神疾病，但分子结构的 GABA和BZD结合位点未知。 我们的长期目标是 研究计划是了解GABAA受体在 从分子结构上来说。 作为第一步，我们建议 在受体结构中识别和定位氨基酸残基 形成GABA和BZD结合位点。 一种创新的方法， 取代的半胱氨酸可及性方法，将用于探测 系统地将GABA和BZD结合位点的整个表面 GABAA受体的口袋。 该方法可以提供详细的 关于结合位点结构的分子信息 通常使用传统诱变或亲和标记获得 技术. 如果GABA和BZD结合位点的结构 在分子细节上已知，有可能整个新类别的 将发现特定位点的化合物，现有的化合物可以 修饰以利用结合位点的物理化学特征 以产生更高亲和力、更有选择性的药物。 取代的 半胱氨酸可及性方法是单一、连续 通过野生型氨基定点突变进行半胱氨酸取代 酸残基，突变体的异源功能表达，和 巯基特异性半胱氨酸的探测 试剂 这些试剂是小的、带电荷的、亲水的， 疏脂的，因此仅在水可接近的表面上反应， 受体的 如果与半胱氨酸取代mutnt的结合不可逆， 被巯基1特异性试剂阻断，如果这种 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与结合线相连 网站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。 具有异常功能和结合的突变体， 与巯基特异性试剂反应的相邻残基 将使我们能够推断出含有 该残留物，从而确定该残留物是否暴露于 结合位点。 因此，在结构上形成所述结构的所有残基都是不饱和的。 结合位点可以被成功地定位，并且三维结构 可以对该网站的特性进行建模。 在没有X光的情况下 晶体结构，这种生物化学方法将提供，在一个分子 水平，GABAA受体迄今为止最详细的结构图 available.