Building a 3-Dimensional Model of the Pore of CFTR

CFTR蛋白在许多上皮细胞的质膜中形成氯离子通道，包括肾和肠的细胞。 编码CFTR的基因突变是囊性纤维化（CF）的主要缺陷，囊性纤维化是高加索人中最常见的致死性常染色体隐性遗传疾病，影响约30，000名美国人。 CFTR功能的改变也在分泌性腹泻和多囊肾病（PKD）的病理生理学中起重要作用。 该通道中渗透的基本机制尚不清楚。 目前尚不清楚蛋白质的哪些部分有助于形成孔，以及这些结构域中的哪些氨基酸参与离子渗透的生物物理过程。 该实验室的长期目标是确定CFTR中的渗透机制。 对于该提议，具体目标1是确定功能性CFTR通道的寡聚结构。 具体目标2是通过定位开放通道阻断剂的结合位点来鉴定排列在孔中的跨膜（TM）螺旋。 具体目标3是确定作为阴离子选择性决定因素的氨基酸基团。 所提出的方法依赖于使用分子生物学技术（定点诱变）结合表达在非洲爪蟾卵母细胞和定量生物物理测定。 工作假设是，孔内衬TM域5，6，11和12。 为了实现这些目标，将测量全细胞和单通道电流，以确定两种结构不同的孔阻断分子的动力学，并确定其结合域是否有助于渗透途径。 通过比较野生型和突变型通道与开放通道阻断剂相互作用的能力，定义有助于孔结构的结构元件。 该实验室以前的研究表明，阻滞剂动力学对孔的结构高度敏感。 TM6内的一个区域已被确定，这是关键的不同阴离子之间的歧视。 该区域似乎也位于孔封闭分子的结合位点附近。 为了准确地描述沟道的该区域的结构，有必要考虑除TM6之外的沟道部分的贡献。 这些研究将由本申请中提出的孔隙的三维模型指导，该模型考虑了TM域5、6、11和12的实验数据。 这种方法假设多个螺旋结构域有助于药物的结合位点和通道中选择性的决定因素。 提出了一个特定的子集的残留物，可以确定渗透的生物物理特征。 测试这些残基的重要性将允许构建传导通路的详细地图。 对该通道功能的进一步了解将有助于设计囊性纤维化，分泌性腹泻和多囊肾病的新疗法。

The CFTR protein forms a chloride ion channel in the plasma membranes of many epithelial cells, including cells of the kidney and gut. Mutation of the gene encoding CFTR is the primary defect in Cystic Fibrosis (CF), the most common lethal, autosomal recessive disease among Caucasians, affecting approximately 30,000 Americans. Alteration in CFTR function also plays an important role in the pathophysiology of secretory diarrhea and polycystic kidney disease (PKD). The basic mechanisms of permeation in this channel are not clear. It is not known which portions of the protein contribute to forming the pore, and which amino acids in those domains are involved in the biophysical processes of ion permeation. The long-term objective of this laboratory is to determine the mechanisms of permeation in CFTR. For this proposal, Specific Aim number 1 is to determine the oligometric structure of the functional CFTR channel. Specific Aim number 2 is to identify transmembrane (TM) helices that line the pore, by localization of binding sites for open-channel blockers. Specific Aim number 3 is to identify groups of amino acids that serve as determinants of anion selectivity. The proposed approach relies upon the use of molecular biological techniques (site-directed mutagenesis) combined with expression in Xenopus oocytes and quantitative biophysical assays. The working hypothesis is that the pore is lined by TM domains 5, 6, 11, and 12. To achieve these goals, whole-cell and single-channel currents will be measured to determine the kinetics of two structurally-distinct classes of pore-blocking molecules, and to determine whether their binding domains contribute to the permeation pathway. Structural elements that contribute to the architecture of the pore will be defined by comparing the ability of wildtype and mutant channels to interact with open-channel blockers. Previous studies from this laboratory have shown that blocker kinetics are highly sensitive to the structure of the pore. A region within TM6 has been identified that is critical for discrimination between different anions. This region also appears to lie close to the binding sites for pore-blocking molecules. To accurately describe the structure of this region of the channel, it is necessary to consider to contributions made from portions of the channel other than TM6. These studies will be guided by a three- dimensional model of the pore, proposed in the application, which takes into account the experimental data for TM domains 5, 6, 11, and 12. This approach hypothesizes that multiple helical domains contribute both to the binding sites for drugs and to the determinants of selectivity in the channel. A specific subset of residues that may determine the biophysical features of permeation is proposed. Testing the importance of these residues will allow the construction of a detailed map of the conduction pathway. An improved understanding of the function of this channel will aid in the design of novel therapies for Cystic Fibrosis, secretory diarrhea, and polycystic kidney disease.