CFTR ANION SELECTIVE CHANNEL--STRUCTURE AND FUNCTION

描述（直接取自应用程序） 本研究的长期目标是阐明 囊性纤维化跨膜离子传导、选择性和门控 电导调节器（CFTR）。 CFTR是一种蛋白质， 囊性纤维化，是一种氯离子通道，其活性受 磷酸化和ATP结合。 虽然CFTR的功能已经被 广泛研究，CFTR的结构，像大多数其他积分 膜蛋白，没有很好地确定。 负离子通道大概是 内衬，至少部分，由残基从12个跨膜节。 这个项目的目标是系统地识别残留物， 使用扫描半胱氨酸可及性方法的CFTR通道。 在这 将接近报告半胱氨酸一次一个地取代成推定的半胱氨酸。 渠道衬砌段。 每种半胱氨酸取代突变体均表达于 非洲爪蟾卵母细胞和水表面暴露的半胱氨酸是确定的 通过它与小的，带负电荷和正电荷的， 巯基特异性试剂，其是甲硫基磺酸盐的衍生物。 对于跨膜片段中的残基，我们推断， 半胱氨酸与试剂反应， 暴露在通道内腔中。 通过这种方法，我们已经 在M1和M6跨膜蛋白中鉴定了18个通道衬里残基， 片段 我们将系统地确定其他残留物， CFTR离子通道，并确定它们的二级结构和位置 门、电荷选择性过滤器和尺寸选择性过滤器的。 这个项目的成功将使我们能够创造一个低分辨率的 CFTR渠道的结构模型。 该通道的模型将提供 对离子传导的分子机制有了新的认识， 选择性和门控，有助于阐明 致病突变改变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.