CFTR is a member of the ABC Transporter superfamily, but is the only member known to bear ion channel activity. Using a molecular evolution analysis, we have identified residues in CFTR that appear to be critical to the evolutionary transition from transporter to channel. The long-term objective is to understand how the pore of the CFTR channel changes its structure between the open and closed states, how steps in the ATP-dependent gating cycle control pore gating, and how CFTR evolved the capability to interrupt a transporter mechanism in order to gain ion channel function. This proposal will test the hypothesis that chloride channel activity evolved in CFTR by converting the conformational changes in the membrane domain associated with binding and hydrolysis of ATP at the cytoplasmic domains, as are found in true ABC Transporters, into the formation of a stable open state, by means of inter- and intra-domain interactions. Results from the previous funding period show that disruption of intradomain interactions in CFTR, at sites that are also identified as divergent between CFTR and related ABC Transporters, dramatically alter channel behavior in terms of conductance, selectivity, pharmacology, and transitions between multiple conducting states. This renewal application proposes to use a refined evolutionary analysis, coupled to structure/function experiments based upon quantitative electrophysiological assays of CFTR channels expressed in oocytes, and simulations of CFTR homology models, to test the importance of specific residues in CFTR in the switch from transporter to channel.
Aim #1 is to identify residues that underlie the evolution of channel activity in CFTR. At sites that exhibit evolutionary divergence from transporters, the impact of mutations on channel activity will be determined.
Aim #2 is to determine how ATP-dependent gating at the cytoplasmic domains leads to conformational changes in the pore associated with channel function.
This Aim will include both experiment and molecular simulation. At sites predicted to interact to stabilize each of the open conductance states, the rate and state-dependence of crosslinking with bifunctional sulfhydryl-modifying (SH) reagents of various lengths will be determined, allowing us to construct and test a scheme for the movements in the pore associated with both opening and closing. These results will be used to validate molecular dynamics simulations of selected CFTR homology models, to identify which structures most closely reflect the true channel structure.
Aim #3 is to verify the importance of the interacting residues by assessing their role in transporter function, in both CFTR and a related glutathione transporter. It is expected that interactions that promote channel behavior will disrupt transporter behavior, which may impact drug development for CF. These studies will allow us to associate molecular motions in CFTR's pore domain with steps in the ATP- dependent gating cycle, will allow us to identify residues that are critical to ion channel function in CFTR, and may identify sites where drugs can be docked to lock open CFTR channels, leading to increased Cl- secretion.

Public Health Relevance

The CFTR protein is a key element in three devastating diseases: cystic fibrosis (CF), secretory diarrhea, and polycystic kidney disease (PKD). The proposed work is expected to provide information on how the open state of the channel is stabilized, which will aid the rational design of drugs that can lock open CFTR channels in the plasma membranes of CF cells, leading to increased chloride secretion and amelioration of CF disease.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Cellular and Molecular Biology of the Kidney Study Section (CMBK)
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Mckeon, Catherine T
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Emory University
Schools of Medicine
United States
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Simhaev, Luba; McCarty, Nael A; Ford, Robert C et al. (2017) Molecular Dynamics Flexible Fitting Simulations Identify New Models of the Closed State of the Cystic Fibrosis Transmembrane Conductance Regulator Protein. J Chem Inf Model 57:1932-1946
Infield, Daniel T; Cui, Guiying; Kuang, Christopher et al. (2016) Positioning of extracellular loop 1 affects pore gating of the cystic fibrosis transmembrane conductance regulator. Am J Physiol Lung Cell Mol Physiol 310:L403-14
Cui, Guiying; McCarty, Nael A (2015) Murine and human CFTR exhibit different sensitivities to CFTR potentiators. Am J Physiol Lung Cell Mol Physiol 309:L687-99
Hunt, William R; Zughaier, Susu M; Guentert, Dana E et al. (2014) Hyperglycemia impedes lung bacterial clearance in a murine model of cystic fibrosis-related diabetes. Am J Physiol Lung Cell Mol Physiol 306:L43-9
Cui, Guiying; Rahman, Kazi S; Infield, Daniel T et al. (2014) Three charged amino acids in extracellular loop 1 are involved in maintaining the outer pore architecture of CFTR. J Gen Physiol 144:159-79
Rahman, Kazi S; Cui, Guiying; Harvey, Stephen C et al. (2013) Modeling the conformational changes underlying channel opening in CFTR. PLoS One 8:e74574
Cui, Guiying; Freeman, Cody S; Knotts, Taylor et al. (2013) Two salt bridges differentially contribute to the maintenance of cystic fibrosis transmembrane conductance regulator (CFTR) channel function. J Biol Chem 288:20758-67
Sebastian, Aswathy; Rishishwar, Lavanya; Wang, Jianrong et al. (2013) Origin and evolution of the cystic fibrosis transmembrane regulator protein R domain. Gene 523:137-46
Cui, Guiying; Song, Binlin; Turki, Hussein W et al. (2012) Differential contribution of TM6 and TM12 to the pore of CFTR identified by three sulfonylurea-based blockers. Pflugers Arch 463:405-18
Thompson, Christopher H; Olivetti, Pedro R; Fuller, Matthew D et al. (2009) Isolation and characterization of a high affinity peptide inhibitor of ClC-2 chloride channels. J Biol Chem 284:26051-62

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