The CFTR protein forms a chloride ion channel in the plasma membranes of many epithelial cells, including cells of the kidney and gut. Mutation of the gene encoding CFTR is the primary defect in Cystic Fibrosis (CF), the most common lethal, autosomal recessive disease among Caucasians, affecting approximately 30,000 Americans. Alteration in CFTR function also plays an important role in the pathophysiology of secretory diarrhea and polycystic kidney disease (PKD). The basic mechanisms of permeation in this channel are not clear. It is not known which portions of the protein contribute to forming the pore, and which amino acids in those domains are involved in the biophysical processes of ion permeation. The long-term objective of this laboratory is to determine the mechanisms of permeation in CFTR. For this proposal, Specific Aim number 1 is to determine the oligometric structure of the functional CFTR channel.
Specific Aim number 2 is to identify transmembrane (TM) helices that line the pore, by localization of binding sites for open-channel blockers.
Specific Aim number 3 is to identify groups of amino acids that serve as determinants of anion selectivity. The proposed approach relies upon the use of molecular biological techniques (site-directed mutagenesis) combined with expression in Xenopus oocytes and quantitative biophysical assays. The working hypothesis is that the pore is lined by TM domains 5, 6, 11, and 12. To achieve these goals, whole-cell and single-channel currents will be measured to determine the kinetics of two structurally-distinct classes of pore-blocking molecules, and to determine whether their binding domains contribute to the permeation pathway. Structural elements that contribute to the architecture of the pore will be defined by comparing the ability of wildtype and mutant channels to interact with open-channel blockers. Previous studies from this laboratory have shown that blocker kinetics are highly sensitive to the structure of the pore. A region within TM6 has been identified that is critical for discrimination between different anions. This region also appears to lie close to the binding sites for pore-blocking molecules. To accurately describe the structure of this region of the channel, it is necessary to consider to contributions made from portions of the channel other than TM6. These studies will be guided by a three- dimensional model of the pore, proposed in the application, which takes into account the experimental data for TM domains 5, 6, 11, and 12. This approach hypothesizes that multiple helical domains contribute both to the binding sites for drugs and to the determinants of selectivity in the channel. A specific subset of residues that may determine the biophysical features of permeation is proposed. Testing the importance of these residues will allow the construction of a detailed map of the conduction pathway. An improved understanding of the function of this channel will aid in the design of novel therapies for Cystic Fibrosis, secretory diarrhea, and polycystic kidney disease.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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General Medicine B Study Section (GMB)
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Mckeon, Catherine T
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Georgia Institute of Technology
Schools of Arts and Sciences
United States
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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
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
Cui, Guiying; Zhang, Zhi-Ren; O'Brien, Andrew R W et al. (2008) Mutations at arginine 352 alter the pore architecture of CFTR. J Membr Biol 222:91-106
Jordan, I King; Kota, Karthik C; Cui, Guiying et al. (2008) Evolutionary and functional divergence between the cystic fibrosis transmembrane conductance regulator and related ATP-binding cassette transporters. Proc Natl Acad Sci U S A 105:18865-70

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