The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent protein kinase (PKA)- and ATP-regulated chloride channel whose activity determines the rate of electrolyte and fluid transport in a variety of epithelial tissues. Dysfunctional CFTR activity is involved in the pathogenesis of diseases including cystic fibrosis (CF), secretory diarrhea and pancreatitis. CFTR is expressed in the apical membrane of epithelial cells in tissues affected in these diseases, where its activity contributes to the transport of transepithelial salt and fluid. CFTR is thus directly involved in the pathology of at least two major diseases that cause morbidity and mortality in millions of individuals throughout the world. However, the molecular mechanisms that modulate CFTR activity in epithelial tissues are poorly understood. The development of effective treatments for these diseases would be facilitated by detailed knowledge of various cellular factors that regulate the activity of CFTR. In addition, recent evidences suggest that CFTR may exist in macromolecular complexes, in which protein-protein interactions may influence its Cl- channel activity.? ? Identification of proteins that interact with CFTR might provide important insights into various cellular mechanisms involving CFTR. Our laboratory has isolated a number of proteins that bind to CFTR. One such protein NHERF causes a pronounced increase in CFTR channel activity. Since CF is caused by insufficient CFTR activity at the plasma membrane, a detailed understanding of CFTR?NHERF interaction could yield important clinical benefits, including possible therapeutic strategies for CF. Using RNAi techniques, we have generated a cell line inwhich NHERF expression is reduced. In these cell lines, CFTR channel actvity is drastically affected. Currently, dissecting the molecular mechanisms involved in the regulation of CFTR channel activity with a view to provide crucial insights leading to a more effective treatment for cystic fibrosis.? ? Cystic fibrosis transmembrane conductance regulator (CFTR) is unique among ABC transporters in that it functions as a Cl- ion channel. In CFTR channels, the opening and closing of the channel is regulated by phosphorylation of residues in the R domain by cAMP dependent protein kinase. Once phosphorylated, binding and hydrolysis of ATP to the nucleotide binding domains is allosterically coupled to the opening and closing of the pore. Although residues in transmembrane 6 are thought to form the lining of the pore, little is known about how opening and closing of the pore is accomplished. To learn more about the structure of TM6 and its role in gating, we have used site-directed mutagenesis to substitute cysteines at amino acid positions 325-353 of CFTR, and then determined their accessibility to thiol-reactive MTS reagents and Cd2+ applied to extra-cellular side of the membrane in the open and closed states of the channel. We found a series of residues whose modifications were state dependent. Modifications of these residues were 10-1000 folds slower in the open state. In contrast modification rates of R334C and T338C, residues that are involved in Cl- binding and permeation were state independent. At three of these sites Cd2+ binding holds the channel in the closed state. Our results suggest that the transmembrane helix undergoes a conformations change associated with ATP binding and is coupled to the gating mechanism that restricts ion conduction.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Intramural Research (Z01)
Project #
1Z01HL001286-03
Application #
7321640
Study Section
(LKEM)
Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2006
Total Cost
Indirect Cost
Name
U.S. National Heart Lung and Blood Inst
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Beck, Edward J; Yang, Yu; Yaemsiri, Sirin et al. (2008) Conformational changes in a pore-lining helix coupled to cystic fibrosis transmembrane conductance regulator channel gating. J Biol Chem 283:4957-66
Liedtke, Carole M; Raghuram, Viswanathan; Yun, C Chris et al. (2004) Role of a PDZ1 domain of NHERF1 in the binding of airway epithelial RACK1 to NHERF1. Am J Physiol Cell Physiol 286:C1037-44
Yoo, Dana; Flagg, Thomas P; Olsen, Olav et al. (2004) Assembly and trafficking of a multiprotein ROMK (Kir 1.1) channel complex by PDZ interactions. J Biol Chem 279:6863-73