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.
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.
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