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：该项目旨在了解监管机制 其控制CFTR氯离子通道的活性。 有趣 在过去的几年里， 根据想象的分子结构和读出的 功能，即产电氯化物渗透。 但一直 对这些模型背后的分子事件几乎没有测试。 CFTR是 在离子通道中是独特的，它完全由细胞上的事件控制。 细胞质侧的膜，而不是通过结合细胞外 配体或膜内电压敏感元件的响应， 潜在的变化。 因此，本研究将注意力集中在 更小的细胞质环（CL）作为细胞间的界面， 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 在四个CL中的30个疾病相关突变的表达揭示， 它们涉及通道的门控和电导。 后者 效果与其中一些的可能性一致，例如，CL 3，可 非常靠近毛孔的内口。 因为CL中的突变 主要改变由核苷酸控制的门控动力学 结合折叠（NBFs），研究人员假设， NBF和CL之间的相互作用。 实验证据表明， 已经从酵母双杂交分析获得了相互作用。 一致 由于这些相互作用发生在双层表面附近， 已经发现对改变门控动力学的NBF突变的敏感性是 这取决于胆固醇含量以及脂质的状态 双层。 意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 通过磷酸化控制CFTR。 蛋白激酶A的作用， NBF的选通动作的先决条件仍然不被理解， 蛋白激酶C和酪氨酸也没有显著影响， 磷酸化：2）确定ATP在NBFs上的作用如何控制 CFTR通道门控; 3）确定细胞质环在CFTR通道门控中的作用。 通过主要胞质结构域调节CFTR通道门控;和 4)以高水平表达CFTR并从酵母中纯化。