Using Microbial Genetics to Study Eukaryotic Channels

描述(由申请人提供)： 检测机械和热刺激的能力是 许多维持生命的系统，包括肾功能和心血管系统 和温度调节。此外，机械传感是必要的首要任务 听觉、平衡感、触觉和痛觉的活动。然而，几乎没有什么是 了解这些感官背后的分子机制。最好的 作为哺乳动物系统中这些现象基础的传感器的候选对象是 最近才被发现的离子通道。 此前，研究人员已经证明了异源功能表达 细菌和酵母中的许多真核转运体和通道。考虑到这些 无数的例子，我们预计一些新发现的候选人 机械和热敏通道可以在功能上表示为 微生物系统。微生物异源表达具有独特的优势 表达的基因可以随机突变和罕见的突变事件 迅速筛选或挑选的。通过这种方式，结构性变化可以相互关联 功能差异，从而深入了解分子机制 蛋白质的含量。 在这里，我们建议利用微生物遗传学的力量来研究 真核细胞通道的结构-功能关系 闸门对机械和/或热刺激的反应。此前，它是 证明缺乏机械敏感通道的大肠杆菌菌株有 一种依赖渗透压的细胞死亡表型。候选的哺乳动物传感器将是 在其中一株菌株中异源表达及其抑制能力 这一表型分析。将有几名候选人参加考试，而那个人(S) 给出最有希望的成果将被积极追求。突变 引起更好的抑制，减慢的生长或依赖于通量的表型 下定决心。将对所有产生的突变体进行渗透压依赖性离子检测。 通量和通道活性，以允许结构与 功能变化。最后，我们将致力于开发一种 将允许对同源和异源表达的 酵母(S.pombe)中对机械和温度敏感的真核细胞通道。

DESCRIPTION(provided by applicant): The ability to detect mechanical and thermal stimuli is at the foundation of many life-sustaining systems including renal function as well as cardiovascular and thermal regulation. In addition, mechanosensation is the necessary primary event for the senses of hearing, balance, touch and pain. However, little is known about the molecular mechanisms underlying these senses. The best candidates for sensors at the base of these phenomena in mammalian systems are ion channels that have only recently been discovered. Previously, researchers have demonstrated heterologous functional expression of many eukaryotic transporters and channels in bacteria and yeast. Given these numerous examples, we anticipate that some of the newly discovered candidate mechano- and thermo-sensitive channels may be functionally expressed in microbial systems. Microbial heterologous expression has the unique advantage that the expressed gene can be randomly mutated and rare mutational events rapidly screened or selected. In this way, structural changes can be correlated with functional differences, thus giving insight into the molecular mechanisms of the protein. Here we propose to utilize the power of microbial genetics to study the structure-function relationships of eukaryotic channels which are thought to gate in response to mechanical and/or thermal stimuli. Previously, it was demonstrated that E. coli strains deficient in mechanosensitive channels have an osmotic-dependent cell death phenotype. Candidate mammalian sensors will be heterologously expressed in one of these strains and their ability to suppress this phenotype assayed. Several candidates will be examined and the one(s) that give the most promising results will be aggressively pursued. Mutations that evoke improved suppression, slowed growth or flux-dependent phenotypes will be determined. All resulting mutants will be assayed for osmotic-dependent ion fluxes and channel activity to allow for the correlation of structural with functional changes. Finally, we will pursue the development of a system that will allow for a similar study of homologously and heterologously expressed mechano- and thermo-sensitive eukaryotic channels in yeast (S. pombe).