Propofol Interactions with GABA-A Receptors

描述(申请人提供)：每年数以百万计的人接受全身麻醉，以便外科医生进行挽救生命的手术。对于大多数患者来说，全身麻醉是安全有效的，然而，副作用，如恶心和呕吐，以及并发症，如心血管不稳定或认知功能障碍，增加了麻醉相关的发病率。了解全身麻醉作用的分子基础可能会导致新的和更安全的全身麻醉药的识别。该项目的长期目标是阐明全身麻醉的分子基础。异丙酚是一种广泛使用的静脉全麻药，是本项目的主要研究重点。在临床使用的浓度下，异丙酚与许多蛋白质相互作用，然而，其麻醉作用的主要靶点是GABA-A受体。该项目将重点研究异丙酚与GABA-A受体的相互作用。为了了解异丙酚作用的分子基础，我们必须确定形成其结合部位的氨基酸，阐明其诱导的构象变化，并确定这些构象变化如何调节受体功能。随着最近同源乙酰胆碱结合蛋白(AChBP)和烟碱型乙酰胆碱(ACh)受体结构的公布，我们实现这些目标的能力向前迈进了一大步。这些结构为麻醉剂与GABA-A受体相互作用的结构研究提供了坚实的基础。这些结构信息将指导我们的实验设计和对结果的解释。我们提出了以下具体目标：1)鉴定靠近异丙酚结合位点的残基，并利用异丙酚的巯基反应性异丙酚衍生物来确定异丙酚在结合部位的定位。2)探讨异丙酚浓度升高对离子通道形成通道门的M2段构象变化的影响。这些实验将起到双重作用：验证ACh衍生的结构信息对GABA-A受体的适用性，并将麻醉剂与离子通道相互作用的结构-功能研究建立在合理的结构基础上。该项目的成功完成将提供丙泊酚与GABA-A受体相互作用的动态信息，并对全身麻醉作用的分子基础提供新的见解。这可能为设计新的、更安全的全身麻醉药提供合理的依据。

DESCRIPTION (provided by applicant): Each year millions of people undergo general anesthesia so that surgeons can perform life-saving operations. For most patients general anesthesia is safe and effective, however, side effects, such as nausea and vomiting, and complications, such as cardiovascular instability or cognitive dysfunction, increase anesthesia-associated morbidity. Understanding the molecular basis of general anesthetic action may lead to the identification of newer and safer general anesthetics. The long term goal of this project is to elucidate the molecular bases of general anesthesia. Propofol, a widely used intravenous general anesthetic, is the major focus of this project. At concentrations used clinically, propofol interacts with many proteins, however, a major target for its anesthetic effects is the GABA-A receptors. This project will focus on propofol's interactions with GABA-A receptors. In order to understand the molecular basis of propofol's action we must identify the amino acids that form its binding sites, elucidate the conformational changes that it induces, and determine how these conformational changes modulate receptor function. Our ability to accomplish these goals took a quantum leap forward with the recent publication of the homologous acetylcholine binding protein (AChBP) and nicotinic acetylcholine (ACh) receptor structures. These structures provide a solid foundation for structure-based studies of anesthetic interactions with GABA-A receptors. This structural information will guide our experimental design and the interpretation of our results. We propose the following Specific Aims: 1) To identify the residues that lie in close proximity to the propofol binding site near the beta subunit Met286 position and to determine the orientation of propofol in the binding site using sulfhydryl- reactive propofol derivatives. 2) To probe the extent of conformational changes induced by potentiating concentrations of propofol in the M2 segment that lines the ion channel and forms the channel gate. These experiments will serve the dual purposes of verifying the applicability of the ACh-derived structural information to GABA-A receptors and placing structure-function studies of anesthetic interactions with ion channels on a sound structural basis. The successful completion of this project will provide dynamic information on propofol's interactions with GABA-A receptors and new insights into the molecular basis of general anesthetic action. They may provide a rational basis for design of new, safer general anesthetics.