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）：细菌机械敏感的MSCL通道的研究具有生物医学意义，原因有几个。首先，该通道在维持微生物的渗透稳态方面起着至关重要的功能。当通道错功能时，它可能导致微生物细胞的死亡，因此可能是可行的药理靶标。其次，随着纳米技术的进展，正在实现生物传感器，尤其是MSCL的潜力，以实现生物医学纳米驱动器。第三，MSCL是研究机械感觉转导的绝佳模型。由于其可牵引性的性质和晶体结构的存在，MSCL是研究机械感知通道中结构功能关系的独特工具。我们一直在利用该系统，以更好地了解机械敏感的通道如何感觉到膜张力的分子机制。这是该提议的长期目标。尽管已经提出了在门控过程中进行结构过渡的模型，但它们并不一致或完整，并且许多基本特征尚未解决。该提案中的实验旨在通过分子，生化和电生理分析来使溶液结构确定，以确定蛋白质区域在感应和响应膜伸展方面起作用的功能作用，以定义在门控的过渡，并确定开放式孔结构的方面。所使用的方法包括：嵌合体的产生，以确定与同源物功能差异相关的结构元素，利用基于硫基水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，以使其是否进入或退出脂质环境，以置于封闭状态，并将其置于通道和开放状态，并将其置于换档状态，并将其定义为跨性别，并将其转变为the频率和开放状态。