GENERAL ANESTHETICS AND LIPID PROTEIN INTERACTIONS

这个项目的广泛的，长期的目标是确定分子 全身麻醉药的作用机制。总的假设是 测试的是，全身麻醉剂改变膜之间的相互作用， 蛋白质和脂质是维持正常蛋白质的关键 功能 这一假说是基于膜蛋白 功能对脂质环境敏感，需要 麻醉剂作用于蛋白质构象转变， 通过脂质-蛋白质相互作用。 本项目的具体目标是：（1）准确识别率 控制烟碱乙酰胆碱的常数和激动剂亲和力 对一般敏感的受体（nAcChoR）脱敏 麻醉药;（2）确定麻醉药敏感速率常数 和激动剂亲和力通过脂质-蛋白质相互作用调节;（3） 确定赋予麻醉剂敏感性的脂质， nAcChoRs和测试的假设，麻醉剂与这些竞争 nAcChoR上疏水位点的脂质;和（4）确定是否 非麻醉化合物改变nAcChoR脱敏动力学，并测试 假设麻醉剂和非麻醉剂化合物竞争 离散的nAcChoR结合位点。 研究设计是为了描述全身麻醉药的作用 在鱼雷的天然膜中的nAcChoRs上，然后改变脂质- 通过将nAcChoR重组成脂质双层来进行蛋白质相互作用， 胆固醇和磷脂酸（PA）含量变化。的假设 麻醉剂与脂质竞争nAcChoR上的疏水位点， 通过评估已被麻醉的nAcChoR的麻醉敏感性进行测试， 重组成胆固醇和PA含量变化的双层。的 假设麻醉和非麻醉化合物竞争结合 将通过确定非麻醉剂是否 降低麻醉剂作用于nAcChoR的效力。 用于表征nAcChoR构象转变的方法将是 停流荧光光谱法这项技术有一个时间 分辨率比放射性配体技术快近1000倍 通常用于表征nAcChoR脱敏动力学。的 nAcChoR将被用作蛋白质模型，因为它是最好的 特征配体门控离子通道，它是敏感的一般 麻醉剂，它是唯一一种可以在数量上纯化的麻醉剂。 和生物物理研究所需的具体活动。

The broad, long term objective of this project is to define the molecular mechanisms by which general anesthetics act. The overall hypothesis to be tested is that general anesthetics alter interactions between membrane proteins and lipids that are critical for maintaining normal protein function. This hypothesis is based on evidence that membrane protein function is sensitive to the lipid environment and requires that anesthetics act on protein conformational transitions that are modulated by lipid-protein interactions. The specific aims of this project are: (1) to precisely identify the rate constants and agonist affinities governing nicotinic acetylcholine receptor (nAcChoR) desensitization that are sensitive to general anesthetics; (2) to determine whether anesthetic-sensitive rate constants and agonist affinities are modulated by lipid-protein interactions; (3) to identify the lipids that confer anesthetic sensitivity to reconstituted nAcChoRs and to test the hypothesis that anesthetics compete with these lipids for hydrophobic sites on nAcChoRs; and (4) to determine whether nonanesthetic compounds alter nAcChoR desensitization kinetics and to test the hypothesis that anesthetics and nonanesthetic compounds compete for discrete nAcChoR binding sites. The research design is to characterize the actions of general anesthetics on nAcChoRs in native membranes from Torpedo and then to alter lipid- protein interactions by reconstituting nAcChoRs into lipid bilayers whose cholesterol and phosphatidic acid (PA) content varies. The hypothesis that anesthetics compete with lipids for hydrophobic sites on nAcChoRs will be tested by assessing the anesthetic sensitivity of nAcChoRs that have been reconstituted into bilayers whose cholesterol and PA content varies. The hypothesis that anesthetic and nonanesthetic compounds compete for binding sites on the nAcChoR will be tested by determining whether nonanesthetics reduce the potencies with which anesthetics act on nAcChoRs. The method used to characterize nAcChoR conformational transitions will be stopped-flow fluorescence spectroscopy. This technique has a time resolution that is nearly 1000 fold faster than radioligand techniques typically used to characterize nAcChoR desensitization kinetics. The nAcChoR will be used as the protein model because it is the best characterized ligand-gated ion channel, it is sensitive to general anesthetics, and it is only one that can be purified in the quantity and specific activity needed for biophysical studies.