Molecular Mechanisms of Mechanosensitive Channel Gating

描述（由申请人提供）：细菌机械敏感性MscL通道的研究具有生物医学意义，原因有几个。首先，通道在维持微生物的渗透稳态中起重要作用;当通道功能失调时，它可以导致微生物细胞的死亡，因此可能是可行的药理学靶标。其次，随着纳米技术的进步，生物传感器，特别是MscL，用于生物医学纳米器件的潜力正在实现。第三，MscL是一个很好的模型，用于研究机械感觉转导。由于其易处理的性质和晶体结构的存在，MscL是一种独特的工具，以调查结构功能的关系，在mechanosensory通道。我们一直在利用这个系统，以更好地了解机械敏感通道如何感知和响应膜张力的分子机制;这是本提案的长期目标。虽然已经提出了门控期间的结构转变模型，但它们并不一致或完整，并且许多基本特征尚未解决。本提案中的实验旨在将解决的结构与分子，生物化学和电生理学分析结合起来，以确定蛋白质区域在传感和响应膜拉伸中发挥的功能作用，定义门控时发生的转换，并确定开孔结构的各个方面。所采用的方法包括：产生嵌合体以确定与同源物功能差异相关的结构元件，利用基于巯基的翻译后扫描的结果来确定应通过诱变进一步测试的残基，以确定它们是否在门控时进入或退出脂质环境，二硫键捕获以定义通道的闭合、过渡和开放状态，并尝试使通道在这些多种状态下结晶。

DESCRIPTION (provided by applicant): The study of the bacterial mechanosensitive MscL channel has biomedical significance for several reasons. First, the channel serves a vital function in maintaining osmotic homeostasis of microbes; when the channel misfunctions it can lead to the death of the microbial cell, and thus may be a viable pharmacological target. Second, as nanotechnology progresses, the potential for biological sensors, especially MscL, to be used in biomedical nano-devices is being realized. Third, MscL is an excellent model for the study of mechanosensory transduction. Because of its tractable nature and the existence of a crystal structure, MscL is a unique tool to investigate structure-function relationships in a mechanosensory channel. We have been exploiting this system to obtain a better understanding of the molecular mechanisms of how a mechanosensitive channel senses and responds to membrane tension; this is the long-term objective of this proposal. While models for structural transitions during gating have been proposed, they are not consistent or complete, and many of the fundamental features are not yet resolved. The experiments within this proposal are designed to ally the solved structure with molecular, biochemical and electrophysiological analyses to determine the functional role that regions of the protein play in sensing and responding to membrane stretch, to define transitions that occur upon gating, and to determine aspects of the open-pore structure. The approaches used include: the generation of chimeras to determine the structural elements associated with functional differences of homologues, utilizing the results from a sulfhydryl-based post-translational scan to determine residues that should be further tested by mutagenesis for whether they enter or exit a lipid environment upon gating, disulfide trapping to define closed, transition and open states of the channel, and attempts to crystallize the channel in these multiple states.