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: 8362296
• 负责人: PEI TANG
• 金额: $ 0.14万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8362296
• 关键词: Affinity; Anesthetics; Binding; Binding Sites; Breathing; Bromides; Cations; Chloride Ion; Chlorides; Complex; Data; Dose; Family; Funding; Gated Ion Channel; General Anesthesia; General anesthetic drugs; Grant; Halothane; Homologous Gene; Intravenous Anesthetics; Ion Channel; Ketamine; Ligands; Molecular; National Center for Research Resources; Principal Investigator; Proteins; Radiation; Research; Research Infrastructure; Resolution; Resources; Roentgen Rays; Source; Structure; Sulfur; Thiopental; United States National Institutes of Health; base; beamline; cost; molecular size; novel; protein function; structural biology

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 全身麻醉的分子机制仍然是一个谜。虽然五聚体配体门控离子通道（pLGIC）的超家族已被确定为全身麻醉剂的假定目标，这些pLGIC的高分辨率结构的缺乏阻碍了了解麻醉剂结合位点在这些蛋白质中以及麻醉剂结合如何影响蛋白质功能。最近出现了一个令人兴奋的获得答案的平台，因为pLGIC家族的两种细菌同系物GLIC和ELIC的X射线结构以2.9和3.3的分辨率得到解决。作为阳离子通道，GLIC和ELIC可以分别在开放和闭合通道状态下结晶。类似于哺乳动物pLGIC阳离子通道，GLIC的阳离子电导可以被多种麻醉剂在亚临床剂量下抑制。在这里，我们建议与全身麻醉剂共结晶GLIC和ELIC。过去没有麻醉剂与任何pLGIC一起结晶。GLIC-和ELIC-麻醉剂复合物的X射线结构将为解释全身麻醉剂对pLGIC的功能影响提供新的结构基础。选择吸入麻醉剂氟烷和静脉麻醉剂硫喷妥钠和氯胺酮进行研究。我们在没有和存在麻醉剂的情况下结晶了GLIC和ELIC。我们的初步数据表明，在硫喷妥钠和氯胺酮的存在下，GLIC的分辨率达到2.7 <$和2.9 <$。尽管有形的蛋白质分辨率，以确定精确的麻醉剂结合位点的蛋白质仍然具有挑战性，因为小的麻醉剂分子大小和相对较低的麻醉剂结合亲和力（~100？M）。因此，我们要求使用特定的光束线，提供高灵敏度和分辨率的溴，硫，并可能氯原子。这些原子包含在选定的麻醉剂中，可以作为蛋白质麻醉复合物中麻醉剂的标记。