MOLECULAR MECHANISMS OF MECHANOSENSITIVE CHANNEL GATING

机械敏感通道门控的分子机制

• 批准号: 6086569
• 负责人: PAUL BLOUNT
• 金额: $ 27.14万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6086569
• 关键词: bacterial proteins; biological signal transduction; cysteine; electrophysiology; intermolecular interaction; membrane channels; molecular genetics; site directed mutagenesis; structural biology; voltage /patch clamp; voltage gated channel

Bacterial mechanosensitive channels are emerging as molecular paradigms for investigation of the mechanosensory transduction that occurs in physiological processes such as touch, proprioception, cardiovascular regulation, hearing and balance. The use of the MscL channel of Escherichia coli has advanced the field considerably by allowing molecular genetic analysis to be combined with electrophysiology and transport assays in the study of a protein with a well-defined physiological role. Hence, MscL serves as a good model for determining the molecular mechanisms of mechanosensitive channel gating as well as the general principles of how proteins detect and respond to membrane tension. Purification of the MscL channel protein led to the identification of the structural gene and ultimately to a 3.5 angstrom X-ray crystallographic structure of the Mycobacterium tuberculosis MscL. This is a closed structure, which unfortunately provides few clues to the process of channel gating or to the structure of the open channel. Because a large pore of 30 to 40 angstrom diameter is generated upon gating, a large conformational change must occur and several residues normally embedded in protein or lipid environments must contribute to the lining of the open pore. The residue interations that are strongest and of greatest importance for keeping the channel closed, the movements of residues and domains that take place upon gating, and the residues that contribute to the lining of the open pore are currently all unknown. The experiments in this application are designed to use molecular analyses based on the solved structure to determine these functional and structural properties of the channel. The approaches include: 1) Cysteine scanning to determine which interactions within the transmembrane domains are of most importance in MS channel gating. 2) Utilizing the "Substituted Cysteine Accessibility Method" (SCAM) to identify the residues that move into an aqueous environment upon channel opening, and thus are likely candidates for lining the open-channel pore. 3) Isolating and characterizing suppressor mutations of gain-of-function MscL mutants to identify pairs of residues that potentially interact in the closed, transition or open states.

细菌机械敏感通道正在作为分子 研究发生在中的机械感觉转导的范例 生理过程，如触觉、本体感觉、心血管 调节、听力和平衡。埃希氏菌MscL通道的利用 大肠杆菌通过分子遗传分析极大地推进了该领域的发展 与电生理学和转运测定相结合来研究 具有明确生理作用的蛋白质。因此，MscL 是一个很好的选择 确定机械敏感通道分子机制的模型 门控以及蛋白质如何检测和响应的一般原理 膜张力。 MscL 通道蛋白的纯化导致 结构基因的鉴定并最终获得 3.5 埃的 X 射线 结核分枝杆菌 MscL 的晶体结构。这是一个 封闭的结构，不幸的是它提供的过程线索很少 通道门控或开放通道的结构。因为毛孔粗大 门控时会产生 30 至 40 埃的直径，这是一种大的构象 必须发生变化，并且通常嵌入蛋白质或脂质中的几个残留物 环境必须有助于形成开孔的内衬。残留物 对于保持最强大和最重要的相互作用 通道关闭，残基和结构域的移动发生在 门控，以及有助于形成开孔内衬的残留物是 目前一切未知。本应用中的实验旨在使用 基于已解析结构的分子分析以确定这些功能 和通道的结构特性。这些方法包括：1）半胱氨酸 扫描以确定跨膜域内的相互作用 在 MS 通道门控中最重要。 2) 利用“取代半胱氨酸 可及性方法”（SCAM）来识别进入水溶液的残留物 渠道开放时的环境，因此可能是衬里的候选者 开放通道孔隙。 3) 分离和表征抑制突变 功能获得性 MscL 突变体可识别可能具有潜在作用的残基对 在关闭、过渡或开放状态下交互。