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ATP结合和水解产生的能量将多种底物跨膜转运。人类ABC蛋白主要是参与分泌、药物解毒和抗原呈递的输出蛋白。尽管转运底物在进化上存在差异和异质性，但ABC转运体的基本结构是高度保守的。关于abc转运机制，特别是真核转运体的高分辨率三维结构尚不清楚，目前仍有许多问题有待解决。因此，需要进行生化和功能研究，以评估基于细菌转运体的结构预测的生理意义。ABCC7或CFTR（囊性纤维化跨膜传导调节因子）是一种atp依赖性、磷酸化激活的Cl-通道，主要表达于肺、肠和胰腺上皮细胞。它使用了所有不对称ABC蛋白共享的基本机制，由其核苷酸结合域（NBD1/NBD2）的形成/解离驱动。CFTR包含其他具有生理意义的结构域：NBD1中的调控延伸域（RE）和在蛋白激酶a和c激活通道中起关键作用的调控结构域（RD）。尽管存在这种差异，CFTR仍然保留了不对称出口蛋白的重要结构特征。特异性磷酸化的RD调控CFTR功能的机制仍然是一个重要的研究课题。我们的主要目的是阐明磷酸化促进或抑制结构域相互作用以调节氯化物分泌的机制。更具体地说，我们将：1)绘制磷酸化的RD与其他CFTR结构域之间的重要相互作用位点，并确定所涉及的磷酸化位点。我们认为，RD的不同磷酸化谱与其他结构域的结合强度和位点的变化有关；2)确定结构域相互作用中磷酸化依赖性变化的功能意义。我们将研究通过磷酸化改变结构域相互作用对CFTR膜稳定性和细胞表面丰度的影响。3)研究目前关于磷酸化的RD通过抑制nbd的大运动来调节CFTR门控，有利于二聚体稳定性的假设的功能相关性。RD是CFTR独有的，其在CFTR控制中的作用仍有待阐明。***我们的研究将有助于阐明CFTR监管的基本方面，这些方面是针对不对称运输方式的。此外，我们将获得CFTR结构-功能关系的生理学相关模型，以补充当前的结构研究。**

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. ***Our research will help elucidate fundamental aspects of CFTR regulation which are specific to the asymmetric mode of transport. Moreover, we will obtain physiologically relevant models on CFTR structure-function relationship that will complement current structural studies. **