GENETIC DISSECTION OF THE YEAST K+ CHANNEL PROTEIN

酵母 K 通道蛋白的基因解剖

• 批准号: 6386636
• 负责人: YOSHIRO SAIMI
• 金额: $ 23万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386636
• 关键词: Xenopus oocyte; chemical models; fungal genetics; model design /development; molecular cloning; nucleic acid sequence; potassium channel; protein structure function; voltage /patch clamp; yeasts

DESCRIPTION: K+ channels regulate resting potential, spike spacing and many other cellular processes, failure of which leads to diseases found in heritabl syndromes. K+ channels have been found in microbes; the one in yeast has been cloned and analyzed under patch clamp. While many aspects of channel molecular biology have been elucidated through site-directed mutagenesis, further structure-function studies can be advanced through the use of microbial genetics. Mutant selection and biophysical analysis have recently been married in the study of the yeast K+ channel, YKC1. This union yielded thus far, among others, mutations in regions that destabilize close-state conformations as deduced from the gating kinetics of K+ currents. The continued application of power of yeast genetics and biophysical analysis will be pursued. The approach allows specific alteration of the YKC1 gene in plasmid without modification of the yeast genome. As in progress, more gain-of-function mutants will be isolated to exhaustively map the sites involved in gating between closed and open conformations. The window of selection will be altered to select mutants of different phenotypes. Other YKC1-channel mutants with specific ion sensitivity or over-tolerance and those that rescue a K+-uptake deficiency will be isolated to further dissect the ion filter and the rectification mechanism. In all instances, the sequence defects will be correlated with the electrophysiological defects detected by patch-clamping native yeast cells as well as Xenopus oocytes expressing the YKC1 genes. Molecular structure-and -function correlations gathered will be used to address existing models or to construct new models of this K+ channel.

描述：K+通道调节静息潜力，尖峰间距和许多 其他细胞过程，导致在 遗传性综合征。 在微生物中发现了K+通道。一个In 酵母已被克隆并在斑块夹下进行分析。 而许多方面 通道分子生物学已通过现场定向阐明 诱变，进一步的结构功能研究可以通过 使用微生物遗传学。 突变选择和生物物理分析具有 最近在研究酵母K+通道YKC1的研究中结婚。 这 迄今为止，工会在不稳定的地区产生了突变 从K+的门控动力学推论出的封闭状态构象 电流。 酵母遗传学和生物物理的能力的继续应用 将进行分析。 该方法允许对 质粒中的YKC1基因无修饰酵母基因组。 如 进度，将隔离更多功能突变体以详尽地绘制 封闭和开放构象之间涉及门控的站点。 这 选择窗口将更改以选择不同的突变体 表型。 其他具有特定离子敏感性的YKC1通道突变体或 耐受性和挽救K+摄取缺陷的人将被隔离 进一步剖析离子滤波器和整流机制。 总的来说 实例，序列缺陷将与 通过贴片夹具的天然酵母细胞检测到的电生理缺陷 以及表达YKC1基因的爪蟾卵母细胞。 分子 收集的结构和功能相关将用于解决 现有模型或构建此K+通道的新模型。