ANESTHETICS AND G-PROTEIN COUPLED CHANNEL REGULATION

鸟嘌呤核苷酸结合蛋白（G蛋白）作为 整合受体激活模式的中间体， 细胞外神经内分泌信号，并将其与适当的 包括离子传导通道在内的效应器正变得越来越重要， 认可. 本项目的总体目标是阐明 麻醉剂改变这些细胞运作的分子机制 信号转导级联，以确定这些分子过程 其改变可以解释全球观察到的 麻醉剂 具体而言，该项目将侧重于一个 选择性麻醉药对鸟嘌呤核苷酸结合功能的影响 蛋白质（G蛋白），其将激素受体的激活与 离子传导通道的打开和关闭。 我们最近 证明了G蛋白对钾通道KACh的开放， GK蛋白可作为一种快速、灵敏、选择性的检测肿瘤生长的指标， 受体激活G蛋白在完整细胞中发挥作用的分子机制 细胞（Szabo和Ostroke，1990）。 该项目将利用各种方法 在主要研究者的实验室中开发，以检查 麻醉剂改变受体中显著动力学步骤的方式 介导的GK激活和失活。 我们会研究 所选麻醉剂对以下方面的不同作用：（a）激动剂-受体 相互作用，（B）受体-G蛋白相互作用，（c）G 蛋白质活化（例如GDP的释放和GTP的摄取），（d）蛋白质活化的速率， G蛋白失活（例如GTP水解）和（e）通道G蛋白 交互. 最初的重点将是GK激活毒蕈碱和 嘌呤能激动剂在心房肌细胞电压钳制中的作用 膜片钳技术的全细胞配置和内部 用适当的鸟嘌呤核苷酸或其类似物透析。 后续 实验将解决腺苷酸环化酶的抑制调节， 毒蕈碱和嘌呤能激动剂以及激活 腺苷酸环化酶，最终目的是 阐明分子机制，通过这些机制， 改变Ca 2+通道的激素调节。 初步实验 表明生理上合理浓度一般 麻醉剂对这些G蛋白偶联调节有显著影响， 流程. 通过选择一组化学性质不同的麻醉剂，我们将 试图建立一个麻醉剂敏感性的独特模式 G蛋白介导的信号转导过程。 通过将这些与 麻醉敏感性的模式的显着的生理过程， 拟议中的实验可能会揭示， 麻醉作用可能是通过改变G蛋白介导的 通道或腺苷酸环化酶功能的调节，如果是，通过什么调节 分子机制

The function of guanine nucleotide binding proteins (G-proteins) as intermediates that integrate patterns of receptor activation by extracellular neuroendocrine signals and couple these to appropriate effectors, including ion conductive channels, is becoming increasingly recognized. The overall objective of this project is to elucidate the molecular mechanisms by which anesthetics alter the operation of these signal transduction cascades in order to identify those molecular processes whose alteration may account for globally observed physiological actions of anesthetics. Specifically, the project will focus on the influence of a selected group of anesthetics on the function of guanine nucleotide binding proteins (G-proteins) that couple the activation of hormone receptors to the opening and closing of ion conductive channels. We have recently demonstrated that the opening of the potassium channel KACh by the G protein GK can be used as a rapid, sensitive and selective indicator of the molecular mechanisms of receptor activated G protein functioning in intact cells (Szabo and Otero, 1990). The project will utilize methodologies developed in the laboratory of the principal investigator to examine the ways in which anesthetics alter the salient kinetic steps in receptor mediated activation and deactivation of GK. We will examine the differential effects of selected anesthetics on: (a) agonist-receptor interactions, (b) receptor-G protein interactions, (c) the rate of G protein activation (e.g. release of GDP and uptake of GTP), (d) the rate of G protein deactivation (e.g. GTP hydrolysis) and (e) channel-G protein interactions. Initial emphasis will be on GK activation by muscarinic and purinergic agonists in cardiac atrial myocytes voltage clamped using the whole-cell configuration of the patch-clamp technique and internally dialyzed with appropriate guanine nucleotides of their analogs. Subsequent experiments will address the inhibitory regulation of adenylate cyclase by muscarinic and purinergic agonists as well as the activation of the adenylate cyclase by beta-adrenergic agonists with the ultimate objective of elucidating the molecular mechanisms by which selected anesthetics may alter the hormonal regulation of Ca2+ channels. Preliminary experiments indicate that physiologically reasonable concentrations of general anesthetics have significant effects on these G-protein coupled regulatory processes. By selecting a group of chemically diverse anesthetics we will attempt to establish a distinctive pattern for the anesthetic sensitivity of G-protein mediated signal transduction processes. By comparing these to the patterns of anesthetic sensitivity of salient physiological processes, the proposed experiments are likely to reveal whether or not significant anesthetic actions may arise through alterations of the G protein mediated regulation of channel or adenylate cyclase function and if so by what molecular mechanism.