Molecular Pharmacology of the Synaptic and Extrasynaptic GABA(A) Receptors

γ-氨基丁酸A型受体家族(GABA(A)Rs)是一类主要的抑制性神经元。 人脑中的受体。大约24个亚型，每个亚基由5个亚基组成 19个已建立的亚基，调节与意识相关的许多不同的生理通路， 镇静、焦虑、癫痫、抑郁和产后抑郁症。GABA(A)R亚基的突变 与癫痫和精神分裂症等疾病有关。他们也是许多药物的靶子。 例如，可逆地暂停意识或寻求在不诱导的情况下控制癫痫或焦虑 镇静剂。我们的总体长期目标是建立突触的结构和 突触外GABA(A)受体及其功能。在最近的两篇论文中确立的基本战略是 用单颗粒冷冻-EM测定纯化的全长糖基化人GABA(A)受体的结构 并在保持构象变化的状态下重组，并使用相同的受体进行研究 结构和功能并行。这是可能的，因为发展了可诱导的、高产的 稳定的细胞系。在激活和失活过程中，GABA(A)受体经历开放的构象变化 和封闭的变构部位，其中一些由内源配体(例如神经类固醇)使用，另一些由 药物(如全身麻醉药、苯二氮卓类药物)。这些变构配体是偶然发现的， 为此类网站的存在提供线索。我们的目标是提供一个基本的了解如何 这些变构口袋的结构取决于受体的构象。这将提供基本的 机械知识，这将使其他人能够开发方法来控制GABA(A)R功能。我们的工作 假设位于TMD亚基之间的变构位点可以容纳正的和 负变构调节剂(分别为PAM和NAM)以及零变构配体(NAL)，仅 就像苯二氮类药物可以在电子捕获中的α/γ-界面上一样。我们的第一个具体目标是突触外 受体，不包含苯二氮卓类部位。以下是化学计量学和化学计量学的基本信息 缺乏亚单位的安排，这个问题必须首先解决。两个配体，DS2和氯胺酮， 两者都选择性地为δ亚单位所含的突触外受体，提供了唯一存在的线索 变构位点，可能与δ亚单位接触，在激活过程中打开。第二个具体问题 Aim验证了以下假设：结合在突触受体TMD中的药物可以作为PAM，NAMS和 纳尔。PAM建立得很好，这一目标侧重于NAM和NAL。基于中的现有结构 几种构象，我们将设计和合成NAMS和NAL，目标是获得配体 适用于结构测定，这将提高对GABA(A)R功能的机理理解。 其意义在于，这可能会导致麻醉剂拮抗剂(NAL)和镇静剂的发展 (部分NAMS)，以及对GABA(A)R功能机制的深入了解。

The family of γ-aminobutyric acid type A receptors (GABA(A)Rs) are the major inhibitory neuro– receptors in the human brain. Some two dozen isoforms, each made up of five subunits selected from the 19 established subunits, mediate many different physiological pathways associated with consciousness, sedation, anxiety, epilepsy, depression and postpartum depression. Mutations in the GABA(A)R subunits are associated with diseases such as epilepsy and schizophrenia. They are also the target of many drugs that for example reversibly suspend conciousness or seek to control epilepsy or anxiety without inducing sedation. Our overall long-term goal is to establish the relationship between the structure of synaptic and extrasynaptic GABA(A)R and how they function. The basic strategy, established in two recent papers, is to use single particle cryo-EM to determine structures of full-length, glycosylated human GABA(A)Rs, purified and reconstituted in a state that preserves conformation changes and to use the same receptors to study structure and function in parallel. This is possible because of the development of inducible, high yielding stable cell lines. During activation and deactivation, GABA(A)Rs undergo conformation changes that open and close allosteric sites some of which are used by endogenous ligands (e.g. neurosteroids) and other by drugs (e.g. general anesthetics, benzodiazepines). These allosteric ligands, discovered serendipitously, provide clues to the existence of such sites. We aim to provide a basic understanding of how the structure of these allosteric pockets depends on a receptor’s conformation. This will provide the basic mechanistic knowledge that will enable others to develop ways to control GABA(A)R function. Our working hypothesis is that allosteric sites located between subunits in the TMD can accommodate positive and negative allosteric modulators (PAMs and NAMs respectively) as well as null allosteric ligands (NALs), just as benzodiazepines can in the α+/γ– interface in the ECD. Our first specific aim concerns extrasynaptic receptors, which do not contain a benzodiazepine site. Here the basic information on stoichiometry and subunit arrangement is lacking, and this problem must be addressed first. Two ligands, DS2 and ketamine, both selective for δ-subunit–containing extrasynaptic receptors, provide clues to the existence of unique allosteric sites, perhaps in contact with the δ-subunit, that open up during activation. The second specific aim tests the hypothesis that agents binding in the TMD of synaptic receptors can act as PAMs, NAMs and NALs. PAMs are well established, and this aim focuses on NAMs and NALs. Based on existing structures in several conformations, we will design and synthesize NAMs and NALs with the goal of obtaining ligands suitable for structural determination that will improve the mechanistic understanding of GABA(A)R function. The significance is that this may lead to the development of anesthetic antagonists (NALs) and sedative (partial NAMs), as well as a deeper understanding of the mechanisms of GABA(A)R function.