Molecular Mechanisms of Mechanosensitive Channel Gating

机械敏感通道门控的分子机制

• 批准号: 7142166
• 负责人: PAUL BLOUNT
• 金额: $ 31.7万
• 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 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7142166
• 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

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. Hence, it appears to be a viable pharmacological target. Second, as nanotechnology progresses, the potential for biological sensors, especially MscL, to be used in biomedical nanomachines or drug delivery devices is being realized. Third, MscL has, and will continue to serve as a molecular paradigm for the investigation of mechanosensory transduction. With a crystal structure of what appears to be a 'nearly-closed' state of MscL, the channel has advanced the field considerably by opening the avenues of structural, genetic and molecular analyses coupled with electrophysiology and flux assays, all allied to a well-defined physiological role. MscL continues to serve as a tractable model for determining the molecular mechanisms of channel gating as well as general principles for how a protein detects and responds to membrane tension. To truly exploit this system, however, a better understanding of the molecular mechanisms of how MscL senses and responds to membrane tension must be obtained; this is the objective of this proposal. While models for structural transitions during gating have been proposed, they are not consistent and even many of the fundamental features are not yet resolved. The experiments within this proposal are designed to ally the solved structure with molecular, biochemical, genetic and electrophysiological analyses to determine the functional role that regions of the protein play in sensing and responding to membrane stretch and to define transitions that occur upon gating. The approaches used include: the generation of chimeras to determine the structural elements associated with functional differences of homologues, reconstitution of orthologues into native and defined membranes, utilizing the "Substituted Cysteine Accessibility Method" (SCAM) to determine at what point in the gating process pore residues are exposed to the aqueous environment, disulfide trapping to define transition and open states of the channel, and a genetic approach to test if residues approach each other upon gating transition. PUBLIC HEALTH: Studying how a bacterial sensor detects forces will allow insight into the mechanisms of how human mechano-sensors, e.g. those used in blood pressure and kidney regulation, may function; thus, we may eventually speculate how such sensors can be modulated by drugs. This work could also have implications in anti-bacterial drug design and the utilization of biological sensors for future technological feats, such as nanodevices for drug delivery.

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，可能会导致微生物细胞死亡。因此，它似乎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（尤其是 MscL）在生物医学纳米机器或药物输送设备中的潜力正在被认识到。第三，MscL 已经并将继续作为机械感觉转导研究的分子范例。由于 MscL 的晶体结构似乎处于“接近闭合”状态，该通道通过打开结构、遗传和分子分析以及电生理学和通量测定的途径，极大地推进了该领域的发展，所有这些都与明确的生理作用相关。 MscL 继续作为一个易于处理的模型，用于确定通道门控的分子机制以及蛋白质如何检测和响应膜张力的一般原理。然而，为了真正利用这个系统，必须更好地理解 MscL 如何感知和响应膜张力的分子机制；这是本提案的目标。虽然门控期间的结构转变模型已经被提出，但它们并不一致，甚至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、遗传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方面发挥的功能作用，并定义门控时发生的转变。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，将直系同源物重构为天然和确定的膜，利用“取代半胱氨酸可及性方法”（SCAM）确定门控过程中孔残基在什么点暴露于水性环境，二硫键捕获以确定通道的过渡和开放状态，以及测试的遗传方法 如果残基在门控转换时彼此接近。公共健康：研究细菌传感器如何检测力将有助于深入了解人类机械传感器的机制，例如。用于血压和肾脏调节的药物可能会发挥作用；因此，我们最终可以推测药物如何调节此类传感器。这项工作还可能对抗菌药物设计和利用生物传感器实现未来的技术成就（例如用于药物输送的纳米设备）产生影响。