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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK051767-16
Application #
8241015
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Mckeon, Catherine T
Project Start
1996-09-30
Project End
2015-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
16
Fiscal Year
2012
Total Cost
$344,041
Indirect Cost
$123,694
Name
Rockefeller University
Department
Physiology
Type
Other Domestic Higher Education
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065
Csanády, László; Töröcsik, Beáta (2014) Catalyst-like modulation of transition states for CFTR channel opening and closing: new stimulation strategy exploits nonequilibrium gating. J Gen Physiol 143:269-87
Csanády, László; Töröcsik, Beáta (2014) Structure-activity analysis of a CFTR channel potentiator: Distinct molecular parts underlie dual gating effects. J Gen Physiol 144:321-36
Csanady, Laszlo; Mihalyi, Csaba; Szollosi, Andras et al. (2013) Conformational changes in the catalytically inactive nucleotide-binding site of CFTR. J Gen Physiol 142:61-73
Csanady, Laszlo; Vergani, Paola; Gadsby, David C (2010) Strict coupling between CFTR's catalytic cycle and gating of its Cl- ion pore revealed by distributions of open channel burst durations. Proc Natl Acad Sci U S A 107:1241-6
Muallem, Daniella; Vergani, Paola (2009) Review. ATP hydrolysis-driven gating in cystic fibrosis transmembrane conductance regulator. Philos Trans R Soc Lond B Biol Sci 364:247-55
Csanady, Laszlo (2009) Application of rate-equilibrium free energy relationship analysis to nonequilibrium ion channel gating mechanisms. J Gen Physiol 134:129-36
Gadsby, David C (2009) Ion channels versus ion pumps: the principal difference, in principle. Nat Rev Mol Cell Biol 10:344-52
Mense, Martin; Vergani, Paola; White, Dennis M et al. (2006) In vivo phosphorylation of CFTR promotes formation of a nucleotide-binding domain heterodimer. EMBO J 25:4728-39
Csanady, Laszlo; Nairn, Angus C; Gadsby, David C (2006) Thermodynamics of CFTR channel gating: a spreading conformational change initiates an irreversible gating cycle. J Gen Physiol 128:523-33
Artigas, Pablo; Al'aref, Subhi J; Hobart, E Ashley et al. (2006) 2,3-butanedione monoxime affects cystic fibrosis transmembrane conductance regulator channel function through phosphorylation-dependent and phosphorylation-independent mechanisms: the role of bilayer material properties. Mol Pharmacol 70:2015-26

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