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.

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 全身麻醉的分子机制仍然是个谜。尽管五聚体配基门控离子通道(PLGIC)超家族已被确定为全麻药的靶标，但这些pLGIC缺乏高分辨结构阻碍了对这些蛋白质中麻醉剂结合部位的理解以及麻醉剂结合如何影响蛋白质功能的理解。最近出现了一个获得答案的令人兴奋的平台，pLGIC家族的两个细菌同系物GLIC和ELIC的X射线结构分别以2.9和3.3的分辨率被解决。作为阳离子通道，GLIC和ELIC可以分别在开放和关闭通道状态下结晶。与哺乳动物的pLGIC阳离子通道相似，GLIC的阳离子电导可被多种麻醉剂在亚临床剂量下抑制。在这里，我们建议将GLIC和ELIC与全麻药共结晶。在过去，没有任何麻醉剂与任何pLGICs结晶。GLIC-和ELIC-麻醉药复合体的X射线结构将为解释全麻药对pLGICs的功能影响提供新的结构基础。选择吸入麻醉剂氟烷、静脉麻醉药硫喷妥钠和氯胺酮作为研究对象。我们已经在没有麻醉剂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存在下，GLIC的分离度分别达到2.7°和2.9°。尽管蛋白质具有明显的分辨率，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(~100M)，确定蛋白质中的麻醉剂结合部位仍然具有挑战性。因此，我们要求使用对溴、硫和可能的氯原子提供高灵敏度和高分辨率的特定光束线。这些原子包含在所选择的麻醉剂中，并且可以作为蛋白质麻醉剂复合体中麻醉剂的标志。