Molecular Mechanisms of Mechanosensitive Channel Gating

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着至关重要的作用;当通道 它的功能失调会导致微生物细胞的死亡。因此，它似乎是一种可行的药理学 目标第二，随着纳米技术的进步，生物传感器，特别是MscL的潜力， 用于生物医学纳米机器或药物输送装置正在实现。第三，MscL已经并将 继续作为机械感觉转导研究的分子范例。与 晶体结构的什么似乎是一个'接近关闭'状态的MscL，该通道已经推进了该领域 通过打开结构，遗传和分子分析的途径， 电生理学和通量测定，所有这些都与明确的生理作用有关。MscL继续作为一个 用于确定通道门控的分子机制的易处理的模型以及 蛋白质是如何检测并响应膜张力的然而，要真正利用这个系统， 理解MscL如何感知和响应膜张力的分子机制， 这是我们的目标;这是我们的目标。虽然门控期间的结构转变模型 虽然已经提出，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解决的结构与分子，生物化学， 遗传和电生理学分析，以确定蛋白质区域在 感测和响应膜拉伸，并限定在门控时发生的转变。的 使用的方法包括：嵌合体的产生，以确定与 同源物的功能差异，将直系同源物重建成天然和确定的膜， 利用“取代的半胱氨酸可及性方法”（SCAM）确定在门控中的哪个点 工艺孔残留物暴露于水性环境，二硫化物捕获以确定过渡， 通道的开放状态，以及测试残基在门控时是否相互接近的遗传方法 过渡 公共卫生：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（例如，用于血压和肾脏调节的那些）如何起作用;因此， 我们最终可能会推测出药物是如何调节这些传感器的。这项工作也可以有 抗菌药物设计和生物传感器在未来技术中的应用 比如用于药物输送的纳米装置。

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.