MOLECULAR MECHANISMS OF MECHANOSENSITIVE CHANNEL GATING

细菌的机械敏感通道正在以分子的形式出现 机械感觉转导的研究范式 生理过程，如触摸、本体感觉、心血管 监管、听证和平衡。大肠杆菌间充质干细胞通道的利用 Coli通过允许分子遗传分析，大大推进了这一领域。 与电生理学和转运分析相结合，用于研究 具有明确生理作用的蛋白质。因此，MSCL是一种很好的 确定机械敏感通道分子机制的模型 以及蛋白质如何检测和响应的一般原理 膜张力。骨髓间充质干细胞通道蛋白的纯化 结构基因的鉴定和最终的3.5埃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，这几乎没有提供关于 沟道选通或到开放沟道的结构。因为一个很大的毛孔 在浇注时产生30到40埃直径，一个大的构象 必须发生变化，几个残基通常嵌入蛋白质或脂肪中 环境必须对开放毛孔的衬里起到作用。残留物 最强烈和最重要的互动 通道关闭，残基和结构域的移动发生在 门控，而构成开口毛孔衬里的残留物是 目前都是未知数。此应用程序中的实验旨在使用 基于已解决的结构的分子分析来确定这些功能 以及通道的结构特性。这些方法包括：1)半胱氨酸 扫描以确定跨膜结构域内的哪些相互作用 在MS通道选通中最重要的。2)利用“取代半胱氨酸” 可及性方法“(SCAM)用于识别进入水中的残留物 环境对渠道开放，因此可能是衬砌的候选人 明渠孔隙。3)分离和鉴定抑癌基因突变 功能增益MSCL突变体识别潜在的残基对 在关闭、过渡或打开状态下进行交互。

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.