CFTR ANION SELECTIVE CHANNEL--STRUCTURE AND FUNCTION

描述(直接从应用程序中获取) 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，一种存在缺陷的蛋白质 囊性纤维化是一种氯离子通道，其活性受 磷酸化和三磷酸腺苷结合。尽管CFTR的职能一直是 经过广泛研究，CFTR的结构就像大多数其他积分一样 膜蛋白，还没有很好地确定。阴离子通道可能是 至少部分地由12个跨膜段的残基构成。 这个项目的目标是系统地识别该线上的残留物 使用扫描-半胱氨酸可及性方法的CFTR通道。在这 接近报告的半胱氨酸被取代，一次一个，变成假定 渠道衬砌管段。每个半胱氨酸替代突变体都在 非洲爪哇卵母细胞和半胱氨酸在水面的暴露 它能与小的、带负电荷和带正电荷的物质反应， 是甲硫磺酸盐的衍生物的巯基专一性试剂。 对于膜跨段中的残留物，我们推断如果工程设计的 半胱氨酸与试剂反应，然后相应的野生型残基 暴露在通道管腔中。通过这种方法，我们已经 在M1和M6膜跨膜中鉴定出18种通道衬里残留物 细分市场。我们将系统地确定其他残留物 Cftr离子通道，并确定其二级结构和位置 栅极、电荷选择性过滤器和尺寸选择性过滤器。 这个项目的成功将使我们能够创建一个低分辨率 CFTR航道的结构模型。渠道模型将提供 对离子传导的分子机制的新见解， 选择性和门控，有助于阐明其机制(S) 疾病引起的突变会改变CFTR通道功能。

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.