Project 2 Subunit and State Dependence of Anesthetic Action on GABAA Receptors

项目2《S总结/摘要》。 项目2的总体假设是，全身麻醉药与GABA上的几个不同部位结合 而且每个不同的部位都有自己的药理作用。，我们的目标是设计有选择性地与每种药物结合的试剂 截然不同的地点。此外，每个位点的亲和力取决于：(1)受体的亚基组成。我们的目标是 开发针对GABAARs特定亚群的麻醉药，如含有？亚基的 突触外与含有突触受体的亚基；(2)其构象状态。最好的将军 麻醉剂与开放状态的亲和力比与静息状态的亲和力更高，亲和力的比率定义了 麻醉剂的功效。在极端情况下，休眠状态的亲和力可能非常低，以至于结合位点可能仅 在GABAAR的打开状态下可检测到。该项目得到以下支持：(1)合成化学核心 (核心B)，我们将与他一起设计新的麻醉剂和麻醉剂光标签，以支持所有目标； 蛋白质化学核心(核心C)，提供目标1中要求的高效液相分析和目标1中要求的测序 目标2和3，以及(3)提供大量GABAARs的蛋白质生产核心(核心D) 所有目标所需的各种亚基组成。目的1：PPG发现[~3H]R-阿齐-依托咪酯 巴比妥酸盐[~3H]R-mTFD-MPAB结合在跨膜区亚基之间的不同位置。 这些位点在二级结构上是同源的，但在序列上略有不同。这两个特工为我们提供了 使用工具来研究控制选择性结合到其单独位点的结构活性关系。在……里面 此外，PPG已经确定在胞外区域中有一个单独的非同源位点 抽搐的巴比妥酸盐与。因为全身麻醉药通常会引起兴奋和相似之处 通常是抽搐或前抽搐的，重要的是发现与此结合的结构规则 在设计新的通用方案时，可以消除不必要的兴奋性副作用 麻醉剂，从而降低毒性并改善病人护理。AIM 2在GABAAR上寻找新的选址。(1) 吸入麻醉药可以装进比PPG迄今检测到的更小的捆绑袋中 静脉麻醉剂，所以我们将设计更小的光标签来验证这一假设。(2)结构是什么？ GABAARs代表亚基附近跨膜结构域结合部位的活性关系 突触外感受器？目标3使用时间分辨光标记来检验以下假设：(1)存在 跨膜结构域中四个螺旋束中的亚基内位点 以及(2)变构模型的预测，即一个分子的相对亲和力 开放状态的麻醉剂与静息状态的麻醉剂对增强GABA诱发电流的效果成正比。 这一跨学科项目旨在通过提供新的信息和概念来提高患者的安全性 将指导更具选择性的全身麻醉药的开发。

Project 2's Summary/Abstract. The overall hypothesis of Project 2 is that general anesthetics bind to several distinct sites on the GABAAR and that each distinct site has its own pharmacology. ,We aim to devise agents that bind selectively to each distinct site. Furthermore, the affinity for each site depends on: (1) the receptor's subunit composition. We aim to develop anesthetics that target specific subpopulations of GABAARs, such as ¿-subunit containing extrasynaptic vs. ¿-subunit containing synaptic receptors; (2) its conformational state. The best general anesthetics will bind with higher affinity to the open than to the resting state, the ratio of affinities defining the anesthetic's efficacy. In extreme cases, affinity for the resting state may be so low that a binding site may only be detectable in the open state of a GABAAR. The project is supported by: (1) the Synthetic Chemistry Core (Core B), with whom we will devise new anesthetics and anesthetic photolabels to support all aims; (2) the Protein Chemistry Core (Core C) who provide HPLC assays required in Aim 1 and the sequencing required in Aims 2 & 3, and (3) the Protein Production Core (Core D) who provide the large amounts of GABAARs of various subunit compositions required in all aims. Aim 1: The PPG has discovered that [3H]R-azi-etomidate and a barbiturate, [3H]R-mTFD-MPAB, bind at different sites between subunits in the transmembrane domain. These sites are homologous in secondary structure, but differ subtly in sequence. These two agents provide us with tools to study the structure activity relationships that govern selective binding to their separate sites. In addition, the PPG has determined that there is a separate nonhomologous site in the extracellular domain that a convulsant barbiturate interacts with. Because general anesthetics often cause excitation and close analogs are often convulsant or pro-convulsant, it is important to discover the structural rules governing binding to this convulsant site so that undesirable excitatory side effects can be eliminated when designing new general anesthetics, thus lowering toxicity and improving patient care. Aim 2 seeks new sites on the GABAAR. (1) Inhalational anesthetics can fit into smaller binding pockets than those the PPG has detected to date for intravenous anesthetics, so we will devise smaller photolabels to test this hypothesis. (2) What are the structure activity relationships of the transmembrane domain sites neighboring ¿-subunits in GABAARs representative of extrasynaptic receptors? Aim 3 uses time resolved photolabeling to test the hypotheses that: (1) There are intrasubunit sites within the bundle of four helices in the transmembrane domain that only become occupied upon activation, and (2) the prediction of the allosteric model that the relative affinity of an anesthetic for the open vs. the resting state is proportional to efficacy for enhancing GABA-induced currents. This interdisciplinary project aims to improve patient safety by providing new information and concepts that will guide the development of more selective general anesthetics.