Mechanisms, Structure, and Regulation of CFTRs NBDs

描述(申请人提供)：CFTR(囊性纤维化跨膜传导调节蛋白)是48种人类三磷酸腺苷结合盒(ABC)蛋白之一，由CF患者突变的基因编码。CFTR属于ABC-C亚家族，与其他医学上重要的蛋白质MRP(多药耐药相关蛋白)和SUR(磺脲受体)一样。但与任何其他ABC蛋白质不同的是，CFTR是一种离子通道，可以进行高分辨率的功能测试。来自这些测试的证据表明，在两个细胞质核苷酸结合域(NBD)之间的界面上，由ATP结合和水解驱动的相同的构象变化周期，在大多数ABC蛋白中被传递到跨膜域，为底物运输提供动力，在CFTR中，打开和关闭通道门。这种门控调节跨上皮液运动所需的快速下坡阴离子流动。Cftr可以被认为是一种破损的转运蛋白，它是从ABC的祖先进化而来的，因为它的一个门失去了完整性。这项研究的目标仍然是从分子上详细了解cftr的NBD的结构和功能机制，它们之间的相互作用，跨膜区(TMD)中通向通道门的转导途径，以及NBD功能和通道门的调控机制。了解控制CFTRCl-通道开放和关闭的精确机制，可能有助于将来在CF患者中对由于突变的CFTR通道表达而导致离子流不足的病变细胞进行药物救援，包括那些无法以足够的数量到达细胞表面的细胞，那些单通道电导降低的细胞，以及那些开放时间不足的细胞。具体目标是：(1)加强和改进我们目前的CFTR通道选通周期模型；(2)精确定位伴随着通道选通的两个NBD之间以及NBD和TMD中的通道门之间的动态重排的位置；以及(3)确定在其选通周期中CFTR内发生的结构运动的程度。野生型和突变型cftr通道将在卵母细胞中表达，并将使用电生理、生物物理和生化方法分析其结构和功能。单通道浇注动力学的测量将检验浇注周期模型。引入的目标半胱氨酸对单官能和双官能硫醇特异性试剂的可及性的实时门控依赖关系将探索CFTR残基和结构域之间的相互作用。对超嗜热菌的不对称ABC蛋白进行结构分析，具有一个活性和一个残缺的复合催化位点，将有助于阐明CFTR中的分子转导机制。 与公共健康相关：囊性纤维化是美国最常见的致命遗传病，由一种名为CFTR的蛋白质缺陷引起，这种蛋白质通常有助于将盐输送到肺、肠、胰腺和汗管的细胞表面。我们的研究旨在确切地了解CFTR蛋白通常是如何发挥作用的，以帮助医生更好地选择方法来弥补囊性纤维化患者中表现不佳的CFTR蛋白造成的缺陷。

DESCRIPTION (provided by applicant): CFTR (cystic fibrosis transmembrane conductance regulator), encoded by the gene mutated in CF patients, is one of 48 human ATP-binding cassette (ABC) proteins. CFTR belongs to subfamily ABC-C, like other medically important proteins MRP (multidrug resistance related protein) and SUR (sulfonylurea receptor). But unlike any other ABC protein, CFTR is an ion channel, allowing high-resolution tests of function. Evidence from such tests suggests that the same cycle of conformational changes, driven by ATP binding and hydrolysis in the interface between the two cytoplasmic nucleotide-binding domains (NBDs), that in most ABC proteins is transmitted to the transmembrane domains to power substrate transport, in CFTR opens and closes the channel gate. This gating regulates the rapid downhill anion flow needed for transepithelial fluid movement. CFTR can be considered to be a broken transporter that evolved from an ABC ancestor by loss of integrity of one of its gates. The goal of the proposed research remains to understand, in molecular detail, the structure and mechanisms of function of CFTR's NBDs, the interactions between them, the transduction pathway to the channel's gate in the transmembrane domains (TMDs), and the mechanisms by which NBD function and channel gating are regulated. Understanding the precise mechanisms that control opening and closing of CFTR Cl- channels might aid future pharmacological rescue in CF patients of diseased cells with inadequate ion flow due to expression of mutant CFTR channels, including those that fail to reach the cell surface in adequate numbers, those with diminished single-channel conductance, and those that spend an insufficient fraction of the time open. The Specific Aims are: (1) to strengthen and refine our present model of the CFTR channel gating cycle; (2) to pinpoint the locations of the dynamic rearrangements between the two NBDs, and between the NBDs and the channel gates in the TMDs, that accompany channel gating; and (3) to determine the extents of structural motions that occur within CFTR during its gating cycle. Wild-type and mutant CFTR channels will be expressed in oocytes and their structure and function will be analyzed using electrophysiological, biophysical, and biochemical methods. Measurements of single-channel gating kinetics will test gating cycle models. Real-time gating-state dependence of accessibility of introduced target cysteines to monofunctional and bifunctional thiol-specific reagents will probe interactions between residues and domains of CFTR. Structural analysis of asymmetric ABC proteins from hyperthermophiles, with one active and one crippled composite catalytic site, will elucidate molecular transduction mechanisms in CFTR. PUBLIC HEALTH RELEVANCE: Cystic fibrosis, the most common lethal genetic disease in the US, is caused by defects in a single protein (called CFTR) that normally helps move salt across the surfaces of cells in the lungs, intestines, pancreas, and sweat ducts. Our research aims to understand exactly how a CFTR protein usually works, to help doctors better choose ways to make up the deficit caused by poorly performing CFTR proteins in cystic fibrosis patients.