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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK056481-10
Application #
8460504
Study Section
Cellular and Molecular Biology of the Kidney Study Section (CMBK)
Program Officer
Mckeon, Catherine T
Project Start
2001-09-15
Project End
2014-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
10
Fiscal Year
2013
Total Cost
$300,456
Indirect Cost
$87,634
Name
Emory University
Department
Pediatrics
Type
Schools of Medicine
DUNS #
066469933
City
Atlanta
State
GA
Country
United States
Zip Code
30322
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
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
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
Fuller, Matthew D; Zhang, Zhi-Ren; Cui, Guiying et al. (2005) The block of CFTR by scorpion venom is state-dependent. Biophys J 89:3960-75
Zhang, Zhi-Ren; Cui, Guiying; Liu, Xuehong et al. (2005) Determination of the functional unit of the cystic fibrosis transmembrane conductance regulator chloride channel. One polypeptide forms one pore. J Biol Chem 280:458-68
Zhang, Zhi-Ren; Song, Binlin; McCarty, Nael A (2005) State-dependent chemical reactivity of an engineered cysteine reveals conformational changes in the outer vestibule of the cystic fibrosis transmembrane conductance regulator. J Biol Chem 280:41997-2003
Thompson, C H; Fields, D M; Olivetti, P R et al. (2005) Inhibition of ClC-2 chloride channels by a peptide component or components of scorpion venom. J Membr Biol 208:65-76

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