GENERAL ANESTHETICS AND LIPID PROTEIN INTERACTIONS

这个项目广泛的、长期的目标是定义分子 全麻药的作用机制。总体假设是 测试表明，全身麻醉剂会改变膜之间的相互作用。 对维持正常蛋白质至关重要的蛋白质和脂类 功能。这一假说是基于膜蛋白 功能对脂质环境敏感，并要求 麻醉剂作用于蛋白质的构象转变，这些转变是被调节的 通过脂质和蛋白质的相互作用。 本项目的具体目标是：(1)准确识别费率 烟碱型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)测定麻醉敏感率常数 激动剂亲和力受脂-蛋白相互作用调节；(3) 确定使重组药物具有麻醉敏感性的脂类 NAcChoRs，并检验麻醉药与这些药物竞争的假设 NAcChoRs上疏水部位的脂类；以及(4)确定 非麻醉性化合物改变nAcChoR脱敏动力学及检测 麻醉剂和非麻醉剂化合物相互竞争的假说 离散的nAcChoR结合位点。 研究设计是为了描述全身麻醉剂的作用。 对鱼雷天然膜中nAcChoRs的影响，进而改变脂质- 通过将nAcChoRs重组为脂双层而实现的蛋白质相互作用 胆固醇和磷脂酸(PA)的含量各不相同。假设 麻醉药与脂类竞争nAcChoRs上的疏水位点 通过评估nAcChoRs的麻醉敏感性进行了测试 重组成胆固醇和PA含量不同的双分子层。这个 麻醉剂和非麻醉剂化合物竞争结合的假说 NAcChoR上的部位将通过确定非麻醉药 降低麻醉剂作用于nAcChoRs的效力。 用于表征nAcChoR构象转变的方法如下 停流荧光光谱分析。这项技术有一段时间 分辨率比放射性配基技术快近1000倍 通常用于表征nAcChoR脱敏动力学。这个 NAcChoR将被用作蛋白质模型，因为它是最好的 表征配体门控离子通道，对一般敏感 麻醉药，而且它是唯一一种可以提纯数量的 以及生物物理研究所需的特定活动。

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.