Abstract

CFTR (Cystic Fibrosis Transmembrane conductance Regulator) is a chloride channel with unique properties. Being a member of the ABC (ATP Binding Cassette) transporter superfamily, CFTR uses ATP hydrolysis as the energy source to carry out its function. Unlike other members of this family that use the free energy of ATP hydrolysis to transport substrates against the electrochemical gradients, CFTR harvests the free energy to drive its conformational changes during opening and closing (or gating) of the channel. Proteins in this family play numerous physiological and pathophysiological roles in a variety of systems including epithelial chloride secretion, transport of cholesterol, drug resistance in cancers, cardiac membrane excitability and insulin secretion. Thus, understanding how CFTR works at a molecular level will have a broad impact on both basic sciences and clinical medicine. Recent solution of X-ray crystal structure of CFTR's N-terminal nucleotide binding domain (NBD1) has opened the door for detailed studies of the role of NBDs in controlling gating transitions. The current proposal will employ a combination of electrophysiological, molecular biological, and structural biological techniques to address some fundamental questions of CFTR gating: What is the role of individual NBDs in modulating CFTR gating? What is the chemical nature of interactions between ATP and its binding pockets? Is binding of ATP at both NBDs absolutely required for channel opening? Since NBD1 lacks the essential amino acids for ATP hydrolysis, what is the role of ATP binding at NBD1? Our specific aims are:
Aim 1. To study CFTR gating kinetics using structure-guided mutagenesis.
Aim 2. To determine the kinetic and energetic roles of individual NBDs in CFTR gating using novel nucleotide analogs. A clear understanding of the molecular mechanisms of CFTR function will aid in designs of therapeutical reagents for the treatment of cystic fibrosis, secretory diarrhea, and other CFTR-associated diseases.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL053445-12
Application #
7644974
Study Section
Special Emphasis Panel (ZRG1-MDCN-C (02))
Program Officer
Banks-Schlegel, Susan P
Project Start
1996-04-19
Project End
2011-07-31
Budget Start
2009-08-01
Budget End
2011-07-31
Support Year
12
Fiscal Year
2009
Total Cost
$329,972
Indirect Cost

  Institution

Name
University of Missouri-Columbia
Department
Type
Schools of Medicine
DUNS #
153890272
City
Columbia
State
MO
Country
United States
Zip Code
65211

  Publications

Hwang, Tzyh-Chang; Kirk, Kevin L (2013) The CFTR ion channel: gating, regulation, and anion permeation. Cold Spring Harb Perspect Med 3:a009498
Sohma, Yoshiro; Yu, Ying-Chun; Hwang, Tzyh-Chang (2013) Curcumin and genistein: the combined effects on disease-associated CFTR mutants and their clinical implications. Curr Pharm Des 19:3521-8
Jih, Kang-Yang; Hwang, Tzyh-Chang (2012) Nonequilibrium gating of CFTR on an equilibrium theme. Physiology (Bethesda) 27:351-61
Jih, Kang-Yang; Sohma, Yoshiro; Li, Min et al. (2012) Identification of a novel post-hydrolytic state in CFTR gating. J Gen Physiol 139:359-70
Bai, Yonghong; Li, Min; Hwang, Tzyh-Chang (2011) Structural basis for the channel function of a degraded ABC transporter, CFTR (ABCC7). J Gen Physiol 138:495-507
Cai, Zhiwei; Sohma, Yoshiro; Bompadre, Silvia G et al. (2011) Application of high-resolution single-channel recording to functional studies of cystic fibrosis mutants. Methods Mol Biol 741:419-41
Tsai, Ming-Feng; Jih, Kang-Yang; Shimizu, Hiroyasu et al. (2010) Optimization of the degenerated interfacial ATP binding site improves the function of disease-related mutant cystic fibrosis transmembrane conductance regulator (CFTR) channels. J Biol Chem 285:37663-71
Tsai, Ming-Feng; Li, Min; Hwang, Tzyh-Chang (2010) Stable ATP binding mediated by a partial NBD dimer of the CFTR chloride channel. J Gen Physiol 135:399-414
Shimizu, Hiroyasu; Yu, Ying-Chun; Kono, Koichi et al. (2010) A stable ATP binding to the nucleotide binding domain is important for reliable gating cycle in an ABC transporter CFTR. J Physiol Sci 60:353-62
Huang, Sheng-You; Bolser, Diana; Liu, Hao-Yang et al. (2009) Molecular modeling of the heterodimer of human CFTR's nucleotide-binding domains using a protein-protein docking approach. J Mol Graph Model 27:822-8

Showing the most recent 10 out of 19 publications