Project 2: Action of general anesthetics on transient states of ligand-gated ion

说明）： 总体目标是更好地了解全身麻醉药与 半胱氨酸环超家族的配体门控离子通道的激活状态。总的假设是 全身麻醉剂在变构位点与这些通道相互作用，这些变构位点的性质随 通道的构象状态。给定的结合位点的大小和对麻醉剂的亲和力随着时间而变化， 受体在加入激动剂后改变其构象。该方法的结构是为了鼓励 动力学和结构方法之间的紧密结合，动力学研究产生关于 麻醉剂对特定瞬时构象状态的相对亲和力和时间分辨冻结 猝灭光标记用于确定光掺入程度和定位麻醉剂 网站.目的1和2探讨受体激动剂的麻醉增强作用的机制和结合位点。 感应离子流目标1针对家族的阳离子成员解决了这个问题，其中仅 最小的麻醉剂增强电流。待检验的具体假设是， 随着受体从封闭状态到开放状态再到脱敏状态，其大小增加。的 这一动力学最容易在超家族中最慢的成员--人类神经元5--上进行剖析。 HT3AR。目标2将与项目3合作定位人类神经元GABAAR的增强位点 并使用来自依托咪酯、丙泊酚、巴比妥酸盐和酒精家族的光活化麻醉剂。的 这个目标的第二部分是询问依托咪酯本身激活GABA电流的位点是否相同 与增强激动剂诱导电流的相同。目的3探讨亚基结合位点的作用 界面与离子通道中的界面相比，使用专门设计用于 任务。这项工作将使用电鳐乙酰胆碱受体，因为它的已知结构将促进 解释。在每个目标中，所选择的受体都是最适合回答问题的受体。光标记 和纯化的异源表达受体将由合成化学和蛋白质 分别生产核心，并通过蛋白质化学核心测序。 相关性（参见说明）：

instnjctions): The overall aim is to gain a better understanding of the manner in which general anesthetics interact with the activated states of the ligand-gated ion channels of the Cys-loop superfamily. The overall hypothesis is that general anesthetics interact with these channels at allosteric sites whose properties vary with the channel's conformational state. A given binding site's size and affinity for anesthetics varies with time as the receptor changes its conformation following addition of agonist. The approach is structured to encourage a tight integration between kinetic and structural approaches with kinetic studies generating hypotheses about the relative affinity of anesthetics for specific transient conformational states and time-resolved freeze quenched photolabeling being used to detemriine the degree of photoincorporation and to locate anesthetic sites. Aims 1 and 2 probe the mechanisms and binding sites involved in anesthetic enhancement of agonist- induced ion currents. Aim 1 addresses this issue for the cationic members of the family where only the smallest anesthetics enhance currents. The specific hypothesis to be tested is that the enhancing site increases in size as the receptor passes from the closed to the open and then desensitized states. The kinetics of this are most easily dissected on the slowest member of the superfamily, the human neuronal 5 HT3AR. Aim 2 will locate the enhancing site in human neuronal GABAARs in collaboration with Project 3 and using photoactivable anesthetics from the etomidate, propofol, barbiturate and alcohol families. The second part of this aim asks whether the site at which etomidate itself activates GABA currents is the same as that which enhances agonist-induced currents. Aim 3 explores the role of binding sites at the subunit interface vs. those in the ion channel in inhibition and enhancement using probes specifically designed for the task. This work will use the Torpedo acetylcholine receptor because its known structure will facilitate interpretation. In each aim, the receptor chosen is that best suited to answering the question. Photolabels and purified, heterologously expressed receptors will be provided by the Synthetic Chemistry and Protein Production Cores respectively, and sequencing by the Protein Chemistry Core. RELEVANCE (See instructions):