MOLECULAR MECHANISMS OF CFTR FUNCTION

描述：本项目旨在了解监管机制 它们控制着CFTR氯离子通道的活性。有意思的 在过去的几年里，已经出现了关于该分子如何工作的建议 基于分子的想象结构并读出其 功能，即电生氯离子渗透。然而，已经有了 几乎没有对这些模型背后的分子事件进行测试。CFTR是 在离子通道中是独一无二的，它仅由 细胞膜的细胞质一侧，而不是细胞外的结合 膜内电压敏感元件的配体或响应 潜在的变化。因此，本研究将注意力集中在 更小的细胞质环(CLS)作为 形成孔和较大细胞质的跨膜序列 这些领域是监管的引擎。重建和 对4种CL中30个疾病相关突变的表达表明 它们同时参与通道的选通和导通。后者 效果与它们中的一些，例如Cl3，可能 非常靠近毛孔的内口。因为CLS中的突变 主要改变由核苷酸控制的门控动力学 结合折叠(NBF)，研究人员假设有 NBF和CLS之间的相互作用。这方面的实验证据 从酵母双杂交分析中获得了相互作用。一致 根据这些相互作用发生在双层表面附近的概念，他们 已经发现，对改变门控动力学的NBF突变的敏感性是 取决于胆固醇的含量，因此取决于脂质的状态 双层的。认识到三维结构信息将被 为了进一步了解机制，调查人员制作了 在高容量酵母表达系统中的蛋白质。 因此，本工作的目标是：1)阐明心绞痛的发病机制 通过磷酸化调控cftr。蛋白激酶A的作用是 NBF的选通动作的先决条件仍然不被理解， 蛋白激酶C和酪氨酸的影响也不显著 磷酸化：2)确定ATP在NBF的作用是如何控制的 Cftr通道门控；3)确定细胞质环在 主要胞质结构域对cftr通道门控的调控； 4)在酵母中高效表达并纯化CFTR。

DESCRIPTION: This project seeks to understand the regulatory mechanisms which control the activity of the CFTR chloride channel. Interesting proposals of how the molecule may work have arisen in the past several years based on the imagined structure of the molecule and read-out of its function, i.e. electrogenic chloride permeation. However, there has been little testing of the molecular events underlying these models. CFTR is unique among ion channels in being controlled exclusively by events on the cytoplasmic side of the membrane rather than by binding of extracellular ligands or responses of voltage sensitive elements within the membrane to potential changes. Therefore, this study focuses its attention on the smaller cytoplasmic loops (CLs) as the interface between the membrane-spanning sequences which form the pore and the larger cytoplasmic domains that are the engines of the regulation. Reconstruction and expression of 30 disease-associated mutations in the four CLs revealed that they are involved in both gating and conductance of the channel. The latter effect is consistent with the possibility that some of them, e.g., CL3, may be very near the inner mouth of the pore. Because mutations in CLs primarily alter gating kinetics which are controlled by the nucleotide binding folds (NBFs), the investigators postulate that there are interactions between the NBFs and the CLs. Experimental evidence for such interactions has been obtained from yeast 2-hybrid analysis. Consistent with the notion that these interactions occur near the bilayer surface, they have found that sensitivity to NBF mutations which change gating kinetics is dependent on the cholesterol content and hence the state of the lipid bilayer. Realizing that 3-dimensional structure information will be required to further understand mechanism, the investigators have produced the protein in a high capacity yeast expression system. Hence, the goals of the current work are: 1) to elucidate the mechanism of control of CFTR by phosphorylation. The action of protein kinase A which is a prerequisite to the gating actions of the NBFs is still not understood, nor are the significant influences of protein kinase C and tyrosine phosphorylation: 2) to determine how the action of ATP at the NBFs controls CFTR channel gating; 3) to determine the role of the cytoplasmic loops in the regulation of CFTR channel gating by the major cytoplasmic domains; and 4) to express CFTR at high levels and purify it from yeast.