Project 3: Anesthetic Mechanisms on GABAA Receptors

全麻药被广泛应用于外科手术和其他临床程序中，但它们非常 这是危险的，其机制仍然知之甚少。了解他们在分子水平上的行为 这一水平将使开发出更具治疗特异性的新药成为可能。这个项目的总体目标是 是确定有效的全身麻醉药在已知治疗靶点上的机制：突触和 突触外GABAA受体。光标记、结构同源建模、突变研究和 机制分析已经建立了依托咪酯在突触中作用的结构和功能模型 AiP2Y2L受体，为异丙酚和巴比妥酸盐等其他有效麻醉药提供了一种新的范例， 和含有不同亚基的突触外GABA。我们的工作假设是在突触 CCI(32/372和突触外OAPSS GABAA受体)，不同的强效麻醉剂结合到不同的亚区 在a-p亚基交界处形成的膜内口袋，每个亚基构成一个通道衬里。 跨膜M2结构域和另一个：A-M1和P-M3。麻醉剂结合促进重排 这些口袋，稳定开放的通道状态，从而减少神经元的活动。我们建议 电生理研究表达了定义的亚基组成的伽巴尔，开发了详细的 大电流水平麻醉作用的机制和决定大电流水平麻醉作用的结构变化 假想的药物口袋会影响麻醉剂的相互作用和通道门控。具体目标1是调查 Aip2/3y2L(突触)GABA中的麻醉-光标记氨基酸及其在通道门控和信号转导中的作用 依托咪酯、异丙酚和戊巴比妥的亲和力/有效性。诱变，包括半胱氨酸诱变 和状态依赖的修饰，以及基于平衡平衡的电生理分析 将采用激动剂门控模型。具体目标2是建立依托咪酯的变构动力学模型 调节aiP2/3‘y2L GABAARS并建立异丙酚和戊巴比妥的变构共激动剂模型。 大电流动态电生理学将使用“人工突触”进行快速应用 GABA和麻醉剂，以及动力学模型将被评估它们是否能够定量解释 数据。具体目标3是将上述方法扩展到4p35(突触外)GABAARS。具体目标4 是与其他PPG项目合作，评估新的光敏麻醉药并指导时间分辨 光标记实验。 相关性(请参阅说明)：

General anesthetics are widely used during surgery and other clinical procedures, yet they are extremely dangerous and their mechanisms remain poorly understood. Understanding their actions at the molecular level will enable development of new drugs with more therapeutic specificity. The overall aim of this project is to define the mechanisms of potent general anesthetics at known therapeutic targets: synaptic and extrasynaptic GABAA receptors. Photolabeling, structural homology modeling, mutant studies, and mechanistic analysis have established structural and functional models for etomidate actions at synaptic aiP2Y2L receptors, providing a new paradigm for other potent anesthetics such as propofol and barbiturates, and extra-synaptic GABAARS containing different subunits. Our working hypothesis is that at synaptic cci(32/372 and extrasynaptic OAPSS GABAA receptors, different potent anesthetics bind to distinct sub-regions of intra-membrane pockets formed at a-p subunit interfaces, with each subunit contributing a channel-lining transmembrane M2 domain and one other: a-M1 and P-M3. Anesthetic binding facilitates rearrangement of these pockets, stabilizing open channel states, thereby reducing neuronal activity. We propose to electrophysiologically study expressed GABAARS of defined subunit composition, developing a detailed mechanism of anesthetic actions at the macrocurrent level and determining how structural changes in the putative drug pockets affect anesthetic interactions and channel gating. Specific Aim 1 is to investigate anesthetic-photolabeled amino acids in aip2/3y2L (synaptic) GABAARS and their roles in channel gating and the affinity/efficacy of etomidate, propofol, and pentobarbital. Mutagenesis, including cysteine mutagenesis and state-dependent modification, together with electrophysiological analysis based on an equilibrium co¿ agonist gating model will be used. Specific Aim 2 is to build an allosteric-kinetic model for etomidate modulation of aiP2/3'y2L GABAARS and to develop allosteric co-agonist models for propofol and pentobarbital. Macrocurrent kinetic electrophysiology will be performed using an "artificial synapse" for rapid application of GABA and anesthetics, and kinetic models will be assessed for their ability to quantitatively account for the data. Specific Aim 3 is to extend the above approaches to a4p35 (extrasynaptic) GABAARS. Specific Aim 4 is to collaborate with other PPG projects by assessing new photolabel anesthetics and guiding time-resolved photolabeling experiments. RELEVANCE (See instmctions):