CFTR ANION SELECTIVE CHANNEL--STRUCTURE AND FUNCTION

CFTR阴离子选择性通道--结构和功能

• 批准号: 2856794
• 负责人: Myles H. Akabas
• 金额: $ 15.6万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 2000-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2856794
• 关键词: Xenopus oocyte; adenosine triphosphate; anions; chloride channels; cysteine; ion transport; membrane proteins; methane sulfonate; mutant; protein engineering; protein structure function; site directed mutagenesis; thiols

DESCRIPTION (Taken directly from the application) The long term goal of this research is to elucidate the structural basis of ion conduction, selectivity and gating in the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR, the protein which is defective in cystic fibrosis, is a chloride channel whose activity is regulated by phosphorylation and ATP binding. Although the functions of CFTR have been extensively studied, the structure of CFTR, like most other integral membrane proteins, is not well determined. The anion channel is presumably lined, at least in part, by residues from the 12 membrane-spanning segments. The goal of this project is to identify systematically the residue that line the CFTR channel using the scanning-cysteine accessibility method. In this approach reporter cysteines are substituted, one at a time, into putative channel-lining segments. Each cysteine-substitution mutant is expressed in Xenopus oocytes and the water-surface exposure of the cysteine is determined by its ability to react with small, negatively and positively charged, sulfhydryl-specific reagents which are derivatives of methanethiosulfonate. For residues in membrane- spanning segments, we infer that if an engineered cysteine reacts with the reagents then the corresponding wild-type residue is exposed in the channel lumen. By this approach we have already identified 18 channel-lining residues in the M1 and M6 membrane-spanning segments. We will systematically identify the other residues that line the CFTR ion channel, and determine their secondary structure, and the position of the gate, the charge-selectivity filter and the size- selectivity filter. The success of this project will allow us to create a low resolution structural model of the CFTR channel. A model of the channel will provide new insights into the molecular mechanisms underlying ion conduction, selectivity and gating and help to elucidate the mechanism(s) by which disease causing mutations alter CFTR channel function.

描述（直接取自应用程序） 这项研究的长期目标是阐明 囊性纤维化跨膜中的离子传导、选择性和门控 电导调节器（CFTR）。 CFTR，一种有缺陷的蛋白质 囊性纤维化，是一种氯离子通道，其活性受以下因素调节 磷酸化和 ATP 结合。 尽管 CFTR 的功能已被 与大多数其他积分一样，CFTR 的结构得到了广泛研究 膜蛋白，尚未确定。 阴离子通道可能是 至少部分由 12 个跨膜片段的残基排列。 该项目的目标是系统地识别该线的残留物 使用扫描半胱氨酸可及性方法的 CFTR 通道。 在这个 方法报告者半胱氨酸被一次一个替换为假定的 渠道内衬段。 每个半胱氨酸取代突变体均表达于 非洲爪蟾卵母细胞和半胱氨酸的水面暴露量被测定 通过其与小电荷、负电荷和正电荷发生反应的能力， 硫氢基特异性试剂，是甲硫代磺酸盐的衍生物。 对于跨膜片段中的残留物，我们推断，如果工程化 半胱氨酸与试剂反应，然后产生相应的野生型残基 暴露在通道内腔中。 通过这种方法我们已经 鉴定出 M1 和 M6 跨膜中的 18 个通道衬里残基 段。 我们将系统地识别排列在 CFTR离子通道，并确定其二级结构，以及位置 栅极、电荷选择性滤波器和尺寸选择性滤波器。 这个项目的成功将使我们能够创建一个低分辨率 CFTR通道的结构模型。 通道模型将提供 对离子传导分子机制的新见解， 选择性和门控，并有助于阐明其机制 引起疾病的突变会改变 CFTR 通道功能。