MOLECULAR MECHANISMS OF CFTR FUNCTION

CFTR 功能的分子机制

• 批准号: 6315441
• 负责人: JOHN R RIORDAN
• 金额: $ 4.81万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-01 至 2000-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6315441
• 关键词: Saccharomyces cerevisiae; adenosine triphosphate; chloride channels; enzyme activity; fungal genetics; intermolecular interaction; microorganism culture; nucleic acid sequence; oligonucleotides; phosphoproteins; protein kinase A; protein kinase C; protein structure function

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.

描述：该项目试图了解监管机制 控制CFTR氯化物通道的活性。 有趣的 过去几年中分子如何起作用的建议 基于分子的想象结构和读出的结构 功能，即氯化物渗透。 但是，有 对这些模型基础的分子事件的测试很少。 CFTR是 在离子频道中独有的独特在仅由事件控制 膜的细胞质侧，而不是通过细胞外结合 膜内电压敏感元件的配体或反应 潜在的变化。 因此，这项研究将注意力集中在 较小的细胞质环（CL）作为界面 形成孔隙和较大细胞质的膜跨膜序列 是监管引擎的域。 重建和 在四个CL中表达了30种与疾病相关的突变的表达表明 他们参与通道的门控和电导。 后者 效应与其中一些（例如Cl3，可能）可能的可能性一致 靠近孔的内嘴。 因为CLS中的突变 主要改变由核苷酸控制的门控动力学 绑定褶皱（NBF），研究人员假设有 NBF和CLS之间的相互作用。 实验证据 相互作用是从酵母2杂交分析中获得的。 持续的 当这些相互作用发生在双层表面附近的观念中，它们 发现改变门控动力学的NBF突变的敏感性是 依赖胆固醇含量，因此脂质的状态 双层。 意识到三维结构信息将是 需要进一步了解机制，研究人员已经产生了 高容量酵母表达系统中的蛋白质。 因此，当前工作的目标是：1）阐明 通过磷酸化控制CFTR。 蛋白激酶A的作用是 NBFS的门控行动的先决条件仍不理解， 蛋白激酶C和酪氨酸的显着影响也没有 磷酸化：2）确定ATP在NBFS中的作用如何控制 CFTR通道门控； 3）确定细胞质环在 主要细胞质结构域调节CFTR通道门控；和 4）在高水平上表达CFTR并从酵母中净化它。