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

ABC转运蛋白是最大的膜蛋白超家族之一，存在于从细菌到植物和人类的所有生物体中。它们利用三磷酸腺苷结合和水解产生的能量在膜上转移各种各样的底物。人的ABC蛋白主要是参与分泌、药物解毒和抗原提呈的出口蛋白。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。关于ABC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3D结构有限的真核转运蛋白。因此，需要进行生化和功能研究，以评估基于细菌转运蛋白的结构预测的生理学意义。ABCC7或CFTR(囊性纤维化跨膜电导调节因子)是一种ATP依赖的、磷酸化激活的氯离子通道，主要表达于肺、肠和胰腺的上皮细胞。它使用所有不对称ABC蛋白共有的基本机制，由它们的核苷酸结合域(NBd1/Nbd2)的形成/解离驱动。CFTR含有更多具有生理意义的结构域：NBD1中的一个调节延伸(RE)和一个调节结构域(RD)，它在蛋白激酶A和C的通道激活中发挥关键作用。尽管存在这种差异，CFTR仍然保留了不对称输出体的重要结构特征。顺式磷酸化的RD控制CFTR功能的机制仍然是一个重要的问题。我们的主要目标是阐明磷酸化促进或抑制结构域之间相互作用以调节氯离子分泌的机制。更具体地说，我们将：1)定位磷酸化的RD和其他CFTR结构域之间的重要相互作用位点，并确定涉及的磷酸化位点。我们认为，不同的RD磷酸化谱与结合强度和与其他结构域的结合位点的变化相关；2)确定结构域相互作用中磷酸化依赖的变化的功能意义。我们将研究通过磷酸化引起的结构域相互作用的变化对CFTR膜的稳定性和细胞表面丰度的影响。3)研究当前的假设，即磷酸化的RD通过抑制NBD的大运动来调节CFTR门控，有利于二聚体的稳定性。RD是CFTR所特有的，其在CFTR门控中的作用仍有许多有待阐明。

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.