MECHANISMS OF ACTION OF GENERAL ANESTHETICS ON LIGAND GATED ION CHANNELS

在美国，每年约有2500万名患者接受全身麻醉。 使用治疗指数非常低的药物。如果没有更好的 对全身麻醉的作用机制的理解将是 不可能设计出更安全的特工。目前的证据表明 麻醉药影响突触传递，在超家族中起作用 配体门控通道包括GABA/A(GABAAR)和 烟碱型乙酰胆碱受体(NAcChoR)。后者拥有独特的 可从电极板获得毫克量的优点 鱼雷(以与其哺乳动物高度同源的形式 相应的)，使拟议的实验可行。整体而言 假设一般有几个不同的位置 NAcChoR上的麻醉剂，并且每个位置的亲和力随 NAcChoR的构象状态。我们已经提供了强有力的证据 全身麻醉药作用于经络附近的部位以及脂质- NAcChoRs的蛋白质界面。我们将特别关注麻醉剂 在休眠和开放状态下与这些部位的相互作用。衍生品 可以被光激活的脂肪族和芳香族全身麻醉药 将被用来确定哪些站点负责通过 比较这些药物抑制静息和开放的速度 国家与他们的照片标签网站在频道和 脂-蛋白界面。将测定光标记的动力学。 使用快速混合设备和新型快速(1&lt；1ms)冷冻- 停止反应的夹持装置，允许后续的 在冷冻的、非反应的、 州政府。氨基酸序列中的光掺入部位为 下定决心。此外，胆固醇与受体结合的假说 脂-蛋白界面上的位点调节异构性变构 全麻药和激动剂之间的相互作用将在 重组的nAcChoR和GABAAR。NAcChoR的工作与以下内容密切相关 在项目II和III中，并使用合成蛋白质 化学核心。GABAAR的工作与项目IV合作。

Some 25 million patients are given general anesthesia each year in the USA using agents which have a very low therapeutic index. Without a better understanding of the mechanism of action of general anesthesia will be impossible to design safer agents. Current evidence indicates that anesthetics affect synaptic transmission and are active on a super-family of ligand gated channels which include the GABA/A (GABAAR) and the nicotinic acetylcholine receptors (nAcChoR). The latter has the unique advantage of being obtainable in milligram quantities from the electroplax of Torpedo (in a form which is highly homologous with its mammalian counterpart) making the proposed experiments feasible. The overall hypothesis is that there are several different sites for general anesthetics on the nAcChoR, and that each site's affinity varies with the nAcChoR's conformational state. We have provided strong evidence that general anesthetics act at a site near the channel as well as the lipid- protein interface of nAcChoRs. We will specifically focus on anesthetic interactions with these sites in the resting and open states. Derivatives of aliphatic and aromatic general anesthetics which can be photo-activated will be used to establish which sites are responsible for inhibition by comparing the rate at which these agents inhibit the resting and open states with the rate that they photo-label sites in the channel and the lipid-protein interface. The kinetics of photo-labeling will be determined using a rapid mixing device coupled with a novel rapid (1<1 ms) freeze- clamping apparatus which stops the reaction, allowing a subsequent prolonged, high-yield photo-labeling step in the frozen, non-reactive, state. The sites of photo-incorporation in the amino acid sequence will be determined. In addition, the hypothesis that cholesterol-receptor binding sites at the lipid-protein interface modulate heterotropic allosteric interactions between general anesthetics and agonists will be tested in reconstituted nAcChoR and GABAAR. The nAcChoR work is closely related to that in Project II & III, and makes use of the Synthetic & Protein Chemistry Cores. The GABAAR work collaborates with Project IV.