Cystic fibrosis (CF) is characterized as a defect of electrolyte transport of epithelial cells of exocrine tissues. Central to the pathophysiology of CF is the absence of cAMP- stimulated Cl- secretion and an enhanced rate of Na+ absorption. The cloning of the CF gene and characterization of the gene product, the cystic fibrosis transmembrane conductance regulator (CFTR), has revealed that CFTR functions as a cAMP-stimulated Cl- channel. Cloning of the CF gene led to the creation of an animal model for CF, the CFTR (-/-) knockout mouse. Interestingly, the CFTR (-/-) mouse did not exhibit a severe CF phenotype in many epithelial tissues, including the airways. Importantly, the absence of a CF phenotype led to the formal identification of the Ca2+-regulated, or """"""""alternative"""""""" Cl- conduction pathway (Cl-A) that is molecularly distinct from CFTR and plays a protective role in preventing CF pathogenesis in the airways of the CFTR (-/-) mouse. This proposal focuses on Cl-A and hypothesizes that Cl-A is an important airway epithelial ion channel resident in the apical membrane and regulated by elevation of intracellular Ca2+. Cl-A will be investigated at three levels, 1) characterization of transepithelial Cl- currents, 2) regulation of Cl-A by Ca2+-mediated signal transduction pathways, 3) patch clamp identification of the Cl-A single channel properties. Confluent polarized epithelial preparations will be used to characterize the Ca2+-stimulated Cl- current and to identify the agonists and signal transducers that regulate this conductance. Patch Cl-Amp technique will be used to identify the Cl-A single channel properties that correlate with Ca2+-stimulated currents from CF murine airway epithelial cells. Importantly all single channel studies will be performed on cells grown on a permeablized support and only channels resident in the apical membrane will be studied. Finally, comparisons between the endogenous Cl-A and heterologously expressed candidate clones will permit the unequivocal identification of the Ca2+-activated airway Cl- channel. A complete characterization of Cl-A will lead to the genesis of new therapies for CF disease that would circumvent a defective CFTR.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL062564-04
Application #
6638538
Study Section
Medical Biochemistry Study Section (MEDB)
Program Officer
Banks-Schlegel, Susan P
Project Start
2000-04-01
Project End
2005-03-31
Budget Start
2003-04-01
Budget End
2005-03-31
Support Year
4
Fiscal Year
2003
Total Cost
$216,750
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
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