Toward a 3-Dimensional View of Permeation at CFTR

离子通道是一种膜蛋白，负责离子和其他底物在细胞膜上的被动运动。通道在各种细胞类型中起作用，包括上皮细胞，它们调节离子在分离体内主要隔室的膜上的流动，以及可兴奋细胞，它们在细胞膜上传递电信号。离子通道通常是信号转导途径的终点或效应器。典型离子通道的整体结构可以分为两个主要区域——通过在膜上形成一个“孔”来形成离子渗透途径的部分，以及（通常是分开的）通过响应适当的刺激来调节孔的开/闭结构的部分。这一建议涉及一种通道——一种在上皮细胞中对氯化物分泌和重吸收过程至关重要的通道。这个通道，CFTR蛋白，是遗传性疾病囊性纤维化中基因缺陷的产物。CFTR的一种变体也参与心室肌细胞膜兴奋性的调节。该项目的长期目标是了解离子通道和转运体的传导、特异性和门控机制，重点是阴离子通道。与阳离子通道相比，阴离子通道的结构结构尚不清楚。本项目的总体目标是确定CFTR中控制渗透的机制。目标1是通过定位开放通道阻滞剂的结合位点来鉴定排列在孔内的跨膜螺旋。目标2是确定作为阴离子选择性决定因素的氨基酸群。提出的方法依赖于使用分子生物学技术（定点诱变），结合爪蟾卵母细胞的表达和定量生物物理测定。工作假设是孔隙由TM结构域5、6、11和12排列。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，并确定它们的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滞剂相互作用的能力，将定义有助于孔结构的结构元素。先前来自首席研究员实验室的研究表明，阻断剂动力学对孔隙结构高度敏感。TM6中的一个区域也被确定为区分不同阴离子的关键区域。这个区域似乎也靠近孔阻断分子的结合位点。为了准确地描述孔隙的结构，有必要考虑除TM6以外的部分通道的贡献。本项目将以应用程序中提出的孔隙三维模型为指导，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既有助于药物的结合位点，也有助于通道的选择性结构域。残基的一个特定子集可能决定渗透的生物物理特征被提出。TM6和TM12中的残基将首先被处理。测试这些残基的重要性将允许构建CFTR传导途径的详细地图。用于渗透的基本机制在CFTR和其他阴离子通道之间可能是共同的。因此，从这个分子模型的研究中得出的结论很可能与理解其他阴离子通道的渗透有关。

Ion channels are membrane proteins responsible for the passive movement of ions, and sometimes other substrates, across cell membranes. Channels function in various cell types, including epithelial cells where they regulate the flow of ions across membranes that separate major compartments in the body, and excitable cells where they transduce electrical signals across cell membranes. Ion channels are often the endpoint, or effector, of signal transduction pathways. The overall structure of a typical ion channel can be broken into two major domains -- portions that form the pathway for ion permeation by creating a "pore" through the membrane, and (usually separate) portions that serve to regulate the open/closed configuration of the pore by gating in response to an appropriate stimulus. This proposal concerns one type of channel -- one crucial to the processes of chloride secretion and reabsorption in epithelial cells. This channel, the CFTR protein, is the product of the gene defective in the inherited disease, cystic fibrosis. A variant of CFTR is also involved in modulation of membrane excitability in cardiac ventricular myocytes. The long-term goal of this project is to understand the mechanisms of conduction, specificity, and gating in ion channels and transporters, with an emphasis on anion channels. Compared to cation channels, the structural architecture of anion channels is poorly understood. For this project, the overall objective is to determine the mechanisms controlling permeation in CFTR. Goal #1 is to identify transmembrane (TM) helices that line the pore, by localization of binding sites for open-channel blockers. Goal #2 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 the principal investigator's laboratory have shown that blocker kinetics are highly sensitive to the structure of the pore. A region within TM6 has also 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 the pore, it is necessary to consider the contributions made from portions of the channel other than TM6. This project 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 selectivity domains of the channel. A specific subset of residues that may determine the biophysical features of permeation is proposed. Residues in TM6 and TM12 will be addressed initially. Testing the importance of these residues will allow the construction of a detailed map of the conduction pathway in CFTR. Basic mechanisms used for permeation are likely to be common between CFTR and other anion channels. Hence, it is likely that conclusions drawn from the study of this molecular model will be relevant to the understanding of permeation in other anion channels.