Structure/function study of the CFTR (ABCC7) chloride channel

CFTR (ABCC7) 氯离子通道的结构/功能研究

• 批准号: RGPIN-2017-05528
• 负责人: Chappe, Valerie
• 金额: $ 1.89万
• 依托单位: Dalhousie University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2017
• 资助国家: 加拿大
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=629568
• 关键词: Structure; function; study; CFTR; ABCC7

ABC transporters form one of the largest superfamily of membrane proteins which are found in all organisms from bacteria to plants and human. They use the energy generated by ATP binding and hydrolysis to translocate a wide variety of substrates across membranes. Human ABC proteins are mostly exporters involved in secretion, drug detoxification and antigen presentation. Despite the evolutionary divergence and heterogeneity of transported substrates, the basic architecture of ABC transporters is highly conserved. Many questions remain unanswered on ABCs mechanism of transport, especially for eukaryotic transporters for which high resolution 3D structures are limited. Biochemical and functional studies to evaluate the physiological significance of structural prediction based on bacterial transporters are thus needed. ABCC7 or CFTR (Cystic Fibrosis Transmembrane conductance Regulator) is an ATP-dependent, phosphorylation-activated Cl- channel mainly expressed in epithelial cells of the lung, intestine and pancreas. It uses the fundamental mechanism shared by all asymmetric ABC proteins driven by the formation/dissociation of their Nucleotide Binding Domains (NBD1/NBD2). CFTR contains additional domains with physiological significance: a regulatory extension (RE) in NBD1 and a regulatory domain (RD) which plays a key role in the channel activation by Protein kinases A and C. Despite this divergence, CFTR still retains important structural characteristics of asymmetric exporters. The mechanism by which the deferentially phosphorylated RD controls CFTR function remains an important issue to explore. Our main objective is to elucidate the mechanism by which phosphorylation promotes or restrains domains' interactions to regulate chloride secretion. More specifically, we will: 1) Map important interaction sites between the phosphorylated RD and other CFTR domains and identify phosphorylation sites involved. We propose that different phosphorylation profiles of the RD are correlated to changes in binding strength and sites with other domains; 2) Determine the functional significance of phosphorylation-dependent changes in domains interaction. We will study the impact of changes in domains interaction by phosphorylation on CFTR membrane stability and abundance at the cell surface. 3) Study the functional relevance of the current hypothesis that the phosphorylated RD regulates CFTR gating by restraining large movements of NBDs, favoring the dimer stability. The RD is unique to CFTR, and much remains to be elucidated on its role in CFTR gating.

ABC转运蛋白是最大的膜蛋白超家族之一，广泛存在于从细菌到植物和人类的所有生物体中。它们利用ATP结合和水解产生的能量将各种底物跨膜转运。人类ABC蛋白主要是参与分泌、药物解毒和抗原呈递的输出者。尽管转运底物的进化差异和异质性，ABC转运蛋白的基本结构是高度保守的。ABCs的转运机制仍有许多问题没有得到解答，特别是对于高分辨率三维结构有限的真核转运蛋白。因此，需要进行生物化学和功能研究，以评估基于细菌转运蛋白的结构预测的生理意义。ABCC 7或CFTR（囊性纤维化跨膜传导调节器）是ATP依赖性、磷酸化激活的Cl-通道，主要在肺、肠和胰腺的上皮细胞中表达。它使用所有不对称ABC蛋白共有的基本机制，由其核苷酸结合结构域（NBD 1/NBD 2）的形成/解离驱动。CFTR包含具有生理意义的其他结构域：NBD 1中的调节延伸（RE）和在蛋白激酶A和C的通道激活中起关键作用的调节结构域（RD）。尽管存在这种差异，CFTR仍然保留了非对称出口国的重要结构特征。去磷酸化的RD控制CFTR功能的机制仍然是一个重要的问题来探索。我们的主要目的是阐明磷酸化促进或抑制结构域的相互作用来调节氯分泌的机制。具体而言，我们将：1）定位磷酸化RD和其他CFTR结构域之间的重要相互作用位点，并鉴定所涉及的磷酸化位点。我们提出RD的不同磷酸化特征与其与其他结构域的结合强度和位点的变化相关; 2）确定结构域相互作用中磷酸化依赖性变化的功能意义。我们将研究结构域相互作用的磷酸化对CFTR膜稳定性和细胞表面丰度的影响。3)研究当前假设的功能相关性，即磷酸化RD通过抑制NBD的大幅度移动来调节CFTR门控，有利于二聚体稳定性。RD是CFTR所独有的，其在CFTR门控中的作用仍有待阐明。