Using Microbial Genetics to Study Eukaryotic Channels

利用微生物遗传学研究真核通道

• 批准号: 6524649
• 负责人: PAUL BLOUNT
• 金额: $ 15.6万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-30 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6524649
• 关键词: Escherichia coli; Schizosaccharomyces pombe; bacterial genetics; behavioral /social science research tag; complementary DNA; eukaryote; fungal genetics; mechanoreceptors; membrane channels; osmosis; phenotype; protein structure function; sensory feedback; sensory signal detection; thermoreceptors; voltage /patch clamp; voltage gated channel

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).

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功能 和热调节。此外，机械感觉是必要的主要 听觉、平衡、触觉和疼痛感的事件。然而，很少的是 了解这些感觉背后的分子机制。最好的 基于哺乳动物系统中这些现象的传感器候选者是 最近才发现的离子通道。 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蛋白和通道。鉴于这些 无数的例子，我们预计一些新发现的候选者 机械和热敏感通道可以在功能上表达 微生物系统。微生物异源表达具有独特优势 表达的基因可以随机突变并且罕见的突变事件 快速筛选或选择。通过这种方式，可以将结构变化关联起来 具有功能差异，从而深入了解分子机制 的蛋白质。 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与功能关系被认为是 门响应机械和/或热刺激。此前，它是 证明缺乏机械敏感通道的大肠杆菌菌株 渗透压依赖性细胞死亡表型。候选哺乳动物传感器将是 在这些菌株之一中异源表达及其抑制的能力 测定了该表型。将审查几名候选人，并且 将积极追求给出最有希望的结果。突变 引起抑制改善、生长减慢或通量依赖性表型 决定。所有产生的突变体将进行渗透依赖性离子分析 通量和通道活动，以允许结构与 功能变化。最后，我们将致力于开发一个系统 将允许对同源和异源表达进行类似的研究 酵母（S. pombe）中的机械和热敏感真核通道。