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方面起着至关重要的作用;当通道功能失调时，它可能导致微生物细胞的死亡。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，特别是MscL，用于生物医学纳米机器或药物输送设备的潜力正在实现。第三，MscL已经并将继续作为机械感觉转导研究的分子范式。由于MscL的晶体结构似乎是“接近闭合”状态，该通道通过打开结构、遗传和分子分析的途径，加上电生理学和通量测定，大大推进了该领域，所有这些都与明确的生理作用有关。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则。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;这就是本提案的目的。虽然已经提出了门控过程中的结构转变模型，但它们并不一致，甚至许多基本特征也没有得到解决。本提案中的实验旨在将解决的结构与分子，生物化学，遗传和电生理分析结合起来，以确定蛋白质区域在感知和响应膜拉伸中发挥的功能作用，并定义门控时发生的转换。所采用的方法包括：产生嵌合体以确定与同源物的功能差异相关的结构元件，将直向同源物重建成天然的和限定的膜，利用“取代的半胱氨酸可及性方法”（SCAM）以确定在门控过程中孔残基暴露于水性环境的点，二硫化物捕获以限定通道的过渡和开放状态，以及测试残基在门控转换时是否彼此接近的遗传方法。公共卫生：研究细菌传感器如何检测力将使我们能够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机械传感器;因此，我们最终可能会推测这些传感器如何被药物调节。这项工作也可能对抗菌药物设计和生物传感器在未来技术壮举中的应用产生影响，例如用于药物输送的纳米器件。

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.