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，通过 细胞外神经内分泌信号并将这些信号耦合到适当的 效应器，包括离子传导通道，正变得越来越多 被认可了。这个项目的总体目标是阐明 麻醉剂改变这些脑区操作的分子机制 为了识别这些分子过程，信号转导级联 其改变可能解释了全球观察到的 麻醉剂。具体地说，该项目将侧重于 部分麻醉药对鸟嘌呤核苷酸结合功能的影响 将激素受体的激活偶联到 离子传导通道的打开和关闭。我们最近做了 证明了G对钾通道Kach的开放 蛋白GK可作为一种快速、灵敏和选择性的指标 受体激活的G蛋白在正常人体发挥作用的分子机制 细胞(Szabo和Otero，1990)。该项目将利用方法论 在首席调查员的实验室中开发，以检查 麻醉药改变受体显著运动步骤的方式 介导GK的激活和失活。我们将研究 部分麻醉药对：(A)激动剂-受体的不同作用 相互作用，(B)受体-G蛋白相互作用，(C)G 蛋白质活化(例如国内生产总值的释放和GTP的摄取)，(D) G蛋白失活(如GTP水解)和(E)通道G蛋白 互动。最初的重点将是毒扁豆碱和 心脏心房肌细胞嘌呤能激动剂的电压钳制 膜片钳技术的全细胞构型和内部 用其类似物的适当鸟嘌呤核苷酸进行透析。后续 实验将通过以下方式解决腺苷酸环化酶的抑制调节 毒扁豆碱和嘌呤能激动剂以及激活 以β-肾上腺素能激动剂为终极目标的腺苷环化酶 阐明所选麻醉药可能通过的分子机制 改变钙离子通道的激素调节。初步实验 表明生理上合理的一般浓度 麻醉药对这些G蛋白偶联调节有显著影响 流程。通过选择一组化学成分不同的麻醉剂，我们将 尝试建立一种独特的麻醉敏感度模式 G蛋白介导的信号转导过程。通过将这些与 显著生理过程的麻醉敏感性模式， 拟议中的实验可能会揭示出是否有重要意义 麻醉作用可能是通过改变G蛋白介导的 通道或腺苷环化酶功能的调节，如果是，是通过什么来调节的？ 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.