MAPPING BENZODIAZEPINE BINDING SITES ON GABAA RECEPTORS

γ-氨基丁酸A型受体(GABA/A)是哺乳动物中枢神经系统神经元抑制的主要效应物，负责传递苯二氮类药物的神经活性。BZDS是一类临床上重要的药物，具有强大的抗焦虑、镇静催眠、肌松和抗癫痫作用。虽然阿尔法和伽马亚基都被认为有助于BZD结合口袋的形成，但结合位点的详细结构图并不存在。利用定点突变和取代半胱氨酸可及性方法，我将鉴定对BZD结合重要的新残基，并绘制α(α1P96-S106)和伽马亚基(Gamma2 Y72-783)中两个潜在结合部位结构域的二级结构图。此外，比较各种巯基特定试剂和残基诱变的修饰率，将得到有关BZD结合的局部化学环境的信息。最后，我将通过检测GABAA受体激活剂存在下取代半胱氨酸可及性的变化来确定BZD调节受体的结合部位的结构变化。BZD位点的变构变化也将通过使用非天然氨基酸取代来破坏构象运动中涉及的残基的主干结构来检查。这些研究代表着朝着理解BZD调节GABA/Z受体的结构决定因素迈出的一步，并将提供迄今为止BZD结合结构域的最详细图。

Gamma-aminobutyric acid type A (GABA/A) receptors are the main effectors of neuronal inhibition in the mammalian central nervous system and are responsible for transducing the neuroactive properties of benzodiazepines (BZDs). BZDs are a clinically important class of drugs with potent anxiolytic, sedative-hypnotic, myorelaxant, and anti-epileptic effects. Although both alpha and gamma subunits are thought to contribute to the formation of the BZD binding pocket, a detailed structural map of the binding site does not exist.. Using site-directed mutagenesis and the substituted cysteine accessibility method, I will identify novel residues important for BZD binding and will map the secondary structure of two potential binding site domains in the alpha (alpha1P96-S106) and gamma subunits (gamma2 Y72-783). Additionally, comparisons of the rates of modification for a variety of sulfhydryl-specific reagents and residue mutagenesis will yield information about the local chemical environment in which BZDs bind. Lastly, I will identify the structural changes in the binding site that underlie BZD modulation of the receptor by examining changes in the accessibility of substituted cysteines in the presence of GABAA receptor activators. Allosteric changes in the BZD site will also be examined by destabilizing backbone structure of residues implicated in conformation movements using unnatural amino acid substitution. These studies represent a step towards understanding the structural determinants of BZD modulation of the GABA/Z receptor, and will provide the most detailed map of the BZD binding domain to date.