Molecular Mechanisms of Mechanosensitive Channel Gating

机械敏感通道门控的分子机制

• 批准号: 8726991
• 负责人: PAUL BLOUNT
• 金额: $ 33.39万
• 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 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8726991
• 关键词: Address; Ally; Anti-Bacterial Agents; Biochemical; Biosensor; Blood Pressure; Cardiovascular system; Cells; Cellular Mechanotransduction; Cessation of life; Chimera organism; Complex; Crystallization; Cysteine; Data; Detergents; Disulfides; Drug Delivery Systems; Drug Design; Elements; Emergency Situation; Environment; Equilibrium; Escherichia coli; Event; Foundations; Future; Generations; Goals; Hearing; Homeostasis; Homologous Gene; Human; Kidney; Lead; Libraries; Life; Lipids; Mechanics; Membrane; Microbe; Molecular; Mutagenesis; Mutate; Mycobacterium tuberculosis; Nanotechnology; Nature; Pain; Pharmaceutical Preparations; Physiological; Play; Process; Property; Protein Region; Proteins; Reagent; Regulation; Resolution; Role; Scanning; Site-Directed Mutagenesis; Stretching; Structural Models; Structure; Structure-Activity Relationship; Study models; System; Tertiary Protein Structure; Testing; Touch sensation; Transmembrane Domain; Work; X-Ray Crystallography; base; design; in vivo; insight; microbial; mutant; nanodevice; potassium channel protein TREK-1; reconstitution; research study; sensor; tool

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.

描述（由申请人提供）：出于多种原因，细菌机械敏感性 MscL 通道的研究具有生物医学意义。首先，该通道在维持微生物渗透压稳态方面发挥着重要作用；当通道功能失调时，会导致微生物细胞死亡，因此可能是一个可行的药理学靶点。其次，随着纳米技术的进步，生物传感器，特别是MscL，在生物医学纳米设备中的应用潜力正在被认识到。第三，MscL 是研究机械感觉转导的优秀模型。由于其易于处理的性质和晶体结构的存在，MscL 是研究机械感觉通道中结构-功能关系的独特工具。我们一直在利用这个系统来更好地理解机械敏感通道如何感知和响应膜张力的分子机制；这是该提案的长期目标。虽然门控过程中的结构转变模型已经被提出，但它们并不一致或不完整，并且许多基本特征尚未解决。该提案中的实验旨在将已解决的结构与分子、生化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所发挥的功能作用，定义门控时发生的转变，并确定各个方面的开孔结构。使用的方法包括：生成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，以确定它们是否进入或退出门控、二硫键捕获等脂质环境来定义通道的关闭、过渡和开放状态，并尝试使通道在这些多种状态下结晶。