Gating of CFTR Cl Channels by ATP Hydrolysis

描述(申请人提供)：CFTR(囊性纤维化跨膜电导调节剂)是一种具有独特性质的氯离子通道。作为ABC(三磷酸腺苷结合盒)转运体超家族的一员，CFTR以三磷酸腺苷的水解酶为能量来源来实现其功能。与这一家族中的其他成员不同，CFTR利用ATP水解的自由能来运输底物，而不是电化学梯度，CFTR在通道的打开和关闭(或门控)期间获取自由能来驱动其构象变化。该家族的蛋白质在多种系统中发挥着重要的生理和病理生理作用，包括上皮氯的分泌、胆固醇的运输、癌症的耐药性、心肌膜的兴奋性和胰岛素的分泌。因此，在分子水平上了解CFTR是如何工作的，将对基础科学和临床医学产生广泛的影响。 最近解决了CFTRN-末端核苷酸结合域(NBD1)的X射线晶体结构，为详细研究NBDS在控制门控转变中的作用打开了大门。目前的提案将使用电生理、分子生物学和结构生物学技术的组合来解决CFTR门控的一些基本问题：单个NBD在调节CFTR门控中扮演什么角色？ATP与其结合口袋之间相互作用的化学本质是什么？通道开放绝对需要两个NBD上的ATP结合吗？由于NBD1缺乏ATP水解所必需的氨基酸，那么NBD1上的ATP结合的作用是什么？我们的具体目标是：目的1.利用结构导向突变技术研究CFTR门控动力学。目的2.利用新的核苷酸类似物确定单个NBD在CFTR门控中的动力学和能量作用。清楚地了解CFTR功能的分子机制将有助于设计治疗囊性纤维化、分泌性腹泻和其他CFTR相关疾病的治疗试剂。

DESCRIPTION (provided by applicant): CFTR (Cystic Fibrosis Transmembrane conductance Regulator) is a chloride channel with unique properties. Being a member of the ABC (ATP Binding Cassette) transporter superfamily, CFTR uses ATP hydrolysis as the energy source to carry out its function. Unlike other members of this family that use the free energy of ATP hydrolysis to transport substrates against the electrochemical gradients, CFTR harvests the free energy to drive its conformational changes during opening and closing (or gating) of the channel. Proteins in this family play numerous physiological and pathophysiological roles in a variety of systems including epithelial chloride secretion, transport of cholesterol, drug resistance in cancers, cardiac membrane excitability and insulin secretion. Thus, understanding how CFTR works at a molecular level will have a broad impact on both basic sciences and clinical medicine. Recent solution of X-ray crystal structure of CFTR's N-terminal nucleotide binding domain (NBD1) has opened the door for detailed studies of the role of NBDs in controlling gating transitions. The current proposal will employ a combination of electrophysiological, molecular biological, and structural biological techniques to address some fundamental questions of CFTR gating: What is the role of individual NBDs in modulating CFTR gating? What is the chemical nature of interactions between ATP and its binding pockets? Is binding of ATP at both NBDs absolutely required for channel opening? Since NBD1 lacks the essential amino acids for ATP hydrolysis, what is the role of ATP binding at NBD1? Our specific aims are: Aim 1. To study CFTR gating kinetics using structure-guided mutagenesis. Aim 2. To determine the kinetic and energetic roles of individual NBDs in CFTR gating using novel nucleotide analogs. A clear understanding of the molecular mechanisms of CFTR function will aid in designs of therapeutical reagents for the treatment of cystic fibrosis, secretory diarrhea, and other CFTR-associated diseases.