Molecular Mechanisms of Mechanosensitive Channel Gating

描述(申请人提供)：由于几个原因，对细菌机械敏感的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的渗透平衡方面起着至关重要的作用；当该通道发生故障时，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装置的潜力正在实现。第三，骨髓间充质干细胞已经并将继续作为研究机械感觉转导的分子范例。该通道的晶体结构看起来像是一种“近乎封闭”的MSCL状态，它开辟了结构、遗传和分子分析与电生理学和通量分析相结合的途径，所有这些都与明确的生理作用相结合。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，为了真正利用这一系统，必须更好地了解MSCL如何感知和响应膜张力的分子机制；这就是本提案的目标。虽然已经提出了门控过程中结构转变的模型，但这些模型并不一致，甚至许多基本特征还没有解决。这一方案中的实验旨在将已解决的结构与分子、生化、遗传和电生理分析结合起来，以确定蛋白质区域在感知和响应膜拉伸方面所起的功能作用，并定义在门控时发生的转变。所使用的方法包括：产生嵌合体以确定与同系物的功能差异相关的结构元件，将直系物重组为天然和定义的膜，利用“替代半胱氨酸可及性方法”(SCAM)来确定在门控过程中孔残基暴露在水环境中的哪个点，二硫化物捕获来定义通道的过渡和开放状态，以及遗传方法来测试在门控过渡时残基是否彼此接近。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如何发挥作用的机制，例如用于血压和肾脏调节的那些传感器；因此，我们最终可能会推测这些传感器如何受到药物的调节。这项工作还可能对抗菌药物设计和生物传感器的未来技术壮举的利用产生影响，例如用于药物输送的纳米设备。

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.