Using Small Compounds as Probes for Studying Mechanosensitive Channel Gating

项目总结/摘要 研究细菌大电导机械敏感通道MscL的主要原因之一是， 已经并将继续作为机械感觉转导研究的分子范式。 其晶体结构似乎是M的“近闭合”状态。结核病MscL，该通道具有 通过允许研究人员叠加遗传和分子分析， 用电生理学的方法，在一个结构模型上因此，从E.大肠杆菌（Ec-MscL），这是第一个定义， 确定的机械敏感通道，继续作为确定原则的易处理模型， 蛋白质是如何感知和响应膜张力的然而，研究MscL的另一个原因是， 通道在维持微生物的渗透稳态中起着重要作用。它通常作为一种生物- 紧急释放阀;在渗透性下行冲击时，它打开一个巨大的30毫米孔，允许快速释放。 释放许多积累的细胞质成分，包括钾和谷氨酸，从而阻止细胞 溶解当通道门控不适当时，它可以导致微生物细胞的死亡;因此，它是一种活的微生物细胞。 潜在抗生素的药理学靶点。从历史上看，MscL功能研究的局限之一是 而药理学则完全缺乏结合和调节通道的小分子探针。从 一个高通量筛选（HTS）设施在校园里，我们已经确定了18种新的化合物， 抑制E.大肠杆菌中以MscL依赖的方式;令人惊讶的是，另外的命中是链霉素， 其似乎直接结合到MscL通道并增加打开MscL通道的可能性。我们已经用这种 系统开发和完善测定化合物功效的试验。在96孔板中，我们可以测定细胞 生长，以确定化合物在存在或不存在以下物质的情况下的最小抑制浓度（MIC）： 表达的Ec-MscL、MscL直向同源物或不相关的细菌机械敏感通道MscS作为负- 主动控制此外，我们有钾和谷氨酸通量测定来测量MscL门控的结果， 在体内，通过电生理学确定通道功能的能力，并且已经开发了测定 化合物的结合位点。我们将使用这一系列的分析来确定新化合物是否以及如何 在HTS中鉴定的结合和调节MscL活性。这些研究将深入了解机械敏感性 通道门控机制;此外，MscL与一种或多种这些化合物的共结晶可能 为MscL产生一个开放状态结构，这些发现最终可能导致新一代抗生素的产生。

Project Summary/Abstract One of the main reasons to study the bacterial mechanosensitive channel of large conductance, MscL, is that it 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 M. tuberculosis MscL, the channel has advanced the field considerably by allowing researchers to overlay genetic and molecular analyses, coupled with electrophysiology, onto a structural model. Thus, MscL from E. coli (Ec-MscL), which was the first defini- tive mechanosensitive channel identified, continues to serve as a tractable model for determining principles for how a protein senses and responds to membrane tension. However, another reason to study MscL is that the channel serves a vital function in maintaining osmotic homeostasis of microbes. It normally serves as a biolog- ical emergency release valve; upon osmotic downshock it opens a huge 30Å pore that allows for the rapid re- lease of many accumulated cytoplasmic components, including potassium and glutamate, thus preventing cell lysis. When the channel gates inappropriately it can lead to the death of the microbial cell; it thus is a viable pharmacological target for potential antibiotics. Historically, one of the limitations in the study of MscL function and pharmacology has been the total lack of small molecular probes that bind and modulate the channel. From a High Throughput Screening (HTS) facility on campus, we have identified 18 novel chemical compounds that inhibit the growth of E. coli in a MscL-dependent manner; surprisingly, an additional hit was streptomycin, which appears to directly bind to and increase the probability of opening the MscL channel. We have used this system to develop and refine assays for determining compound efficacy. In 96 well plates we can assay cell growth to determine the minimal inhibitory concentrations (MIC) of compounds in the presence or absence of expressed Ec-MscL, MscL orthologues, or the unrelated bacterial mechanosensitive channel MscS as a nega- tive control. In addition, we have potassium and glutamate flux assays to measure the results of MscL gating in vivo, the ability to determine channel function by electrophysiology, and have developed assays to determine the binding sites of compounds. We will use this array of assays to determine if and how the novel compounds identified in the HTS bind and modulate MscL activity. These studies will yield insight into mechanosensitive channel gating mechanisms; in addition, co-crystallization of MscL with one or more of these compounds may yield an open state structure for MscL, and the findings could eventually lead to a new generation of antibiotics.