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

γ-氨基丁酸A型受体（GABA（A）Rs）家族是主要的抑制性神经元受体， 人类大脑中的受体。大约二十几种同种型，每种由五个亚基组成，选自 19个已建立的亚基，介导与意识相关的许多不同的生理途径， 镇静、焦虑、癫痫、抑郁和产后抑郁。GABA（A）R亚基突变 与癫痫和精神分裂症等疾病有关。他们也是许多药物的目标 例如可逆地暂停意识或试图控制癫痫或焦虑而不诱发 镇静剂我们的总体长期目标是建立突触结构与 突触外GABA（A）R及其功能。在最近的两篇论文中确立的基本战略是， 使用单颗粒cryo-EM测定全长、糖基化人GABA（A）Rs的结构， 并在保持构象变化的状态下重建，并使用相同的受体进行研究 结构和功能并行。这是可能的，因为发展诱导，高产 稳定的细胞系在激活和失活过程中，GABA（A）Rs发生构象变化， 和接近的变构位点，其中一些被内源性配体（例如神经类固醇）使用，另一些被内源性配体（例如神经类固醇）使用 药物（如全身麻醉药、苯二氮卓类）。这些偶然发现的变构配体， 提供了这些网站存在的线索。我们的目标是提供一个基本的了解， 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，使其他人能够开发控制GABA（A）R功能的方法。我们的工作 假设是位于TMD亚基之间的变构位点可以容纳阳性和 负变构调节剂（分别为PAM和NAM）以及无效变构配体（NAL）， 就像苯二氮卓类药物在ECD中的α+/γ-界面一样。我们的第一个具体目标涉及突触外 受体，不含苯二氮卓类位点。在这里，化学计量的基本信息和 缺乏亚单位排列，必须首先解决这个问题。两种配体DS 2和氯胺酮 两者都对含有δ-亚基的突触外受体具有选择性，为存在独特的 变构位点，可能与δ亚基接触，在活化过程中打开。第二特定 目的是检验这一假设，即在突触受体的TMD中结合的试剂可以充当PAM、NAM和 NALS。政策和行动措施已经确立，这一目标侧重于不适用市场机制和不适用市场机制。根据现有结构， 几种构象，我们将设计和合成NAM和NAL的目标是获得配体 适用于结构测定，这将提高对GABA（A）R功能的机械理解。 意义在于，这可能导致麻醉拮抗剂（NALS）和镇静剂的发展。 （部分NAMs），以及对GABA（A）R功能机制的更深入了解。

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.