X-RAY STRUCTURES OF PENTAMERIC ION CHANNELS IN THE ABSENCE AND PRESENCE OF ANEST

不存在和存在 Anest 时五聚体离子通道的 X 射线结构

• 批准号: 8170297
• 负责人: PEI TANG
• 金额: $ 0.03万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170297
• 关键词: Affinity; Anesthetics; Binding; Binding Sites; Breathing; Bromides; Cations; Chloride Ion; Chlorides; Complex; Computer Retrieval of Information on Scientific Projects Database; Data; Dose; Family; Funding; Gated Ion Channel; General Anesthesia; General anesthetic drugs; Grant; Halothane; Homologous Gene; Institution; Intravenous Anesthetics; Ion Channel; Ketamine; Ligands; Molecular; Proteins; Research; Research Personnel; Resolution; Resources; Source; Structure; Sulfur; Thiopental; United States National Institutes of Health; base; beamline; molecular size; novel; protein function

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The molecular mechanism of general anesthesia remains an enigma. Although a superfamily of pentameric ligand gated ion channels (pLGICs) has been identified as putative targets of general anesthetics, the lack of high-resolution structures of these pLGICs hinders the understanding where anesthetic binding sites are in these proteins and how anesthetic bindings impact on protein functions. An exciting platform for getting the answers has recently emerged as the x-ray structures of two bacterial homologs to the pLGIC family, GLIC and ELIC, were solved with resolutions of 2.9 and 3.3 ¿. As cation channels, GLIC and ELIC can be crystallized in the open- and close-channel states, respectively. Similar to mammalian pLGIC cation channels, cation conductance of GLIC could be inhibited by a variety of anesthetics at subclinical doses. Here we propose to co-crystallize GLIC and ELIC with general anesthetics. No anesthetic has been crystallized with any pLGICs in the past. The x-ray structures of GLIC- and ELIC-anesthetics complexes will provide novel structural basis to explain the functional impact of general anesthetics to the pLGICs. Inhaled anesthetic halothane and intravenous anesthetics thiopental and ketamine are chosen for the study. We have crystallized GLIC and ELIC in the absence and presence of anesthetics. Our preliminary data show that the resolutions up to 2.7 ¿ and 2.9 ¿ have been achieved for GLIC in the presence of thiopental and ketamine. Despite tangible protein resolutions, to define precise anesthetic binding sites in the proteins remains challenging because of small anesthetic molecular sizes and relatively low anesthetic binding affinities (~100 ?M). Thus, we request to use specific beamlines that provide high sensitivity and resolution to bromide, sulfur, and possibly chloride atoms. These atoms are contained in the selected anesthetics and can serve as marks for anesthetics in the proteinanesthetic complexes.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以出现在其他 CRISP 条目中 列出的机构是。 对于中心来说，它不一定是研究者的机构。 全身麻醉的分子机制仍然是个谜，尽管五聚体配体门控离子通道 (pLGIC) 超家族已被确定为全身麻醉剂的推定靶标，但这些 pLGIC 缺乏高分辨率结构阻碍了对麻醉剂结合位点的理解。最近出现了一个令人兴奋的平台来获得答案，即 pLGIC 家族的两种细菌同源物 GLIC 和 GLIC 的 X 射线结构。 ELIC，以 2.9 和 3.3 的分辨率解决作为阳离子通道，GLIC 和 ELIC 可以分别在开放通道和封闭通道状态下结晶，与哺乳动物 pLGIC 阳离子通道类似，GLIC 的阳离子电导可以在亚临床剂量下被多种麻醉剂抑制。 -用全身麻醉剂使 GLIC 和 ELIC 结晶。 过去没有任何 pLGIC 使麻醉剂结晶。 GLIC- 和 ELIC 的 X 射线结构。 ELIC-麻醉剂复合物将为解释全身麻醉剂对 pLGIC 的功能影响提供新的结构基础，我们选择吸入麻醉剂氟烷和静脉麻醉剂硫喷妥钠和氯胺酮在不存在和存在麻醉剂的情况下结晶。我们的初步数据显示分辨率高达 2.7 ¿和 2.9 ¿尽管已在硫喷妥钠和氯胺酮存在下实现了 GLIC，但由于麻醉剂分子尺寸较小且麻醉剂结合亲和力相对较低（约 100 μM），因此确定蛋白质中精确的麻醉剂结合位点仍然具有挑战性。我们要求使用特定的光束线，对溴、硫和可能的氯原子提供高灵敏度和分辨率，这些原子包含在选定的麻醉剂中，可以作为麻醉剂的标记。蛋白质麻醉复合物。