FUNCTION OF FETAL LUNG CHLORIDE CHANNELS

University Of Maryland Baltimore, Baltimore MD

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Abstract

The PI of this project is Dr. Carol Murray, a physician who has just finished fellowship training as a pediatric pulmonologist, and is on the faculty at the Johns Hopkins Medical Institutions. The project is designed to study fetal lung chloride channels, because active chloride secretion across the airway epithelium and the obligatory fluid secretion that follows are essential for normal lung morphogenesis to occur. The precise molecular identities of the fetal lung chloride channels responsible for lung development are as yet unknown. One member of the ClC family of chloride channels is abundantly expressed in the fetal lung and is rapidly downregulated in the postnatal lung. The HYPOTHESIS of this grant is that ClC-2 is a fetal lung chloride channel, which conducts a chloride current to drive fluid movement into the developing airways. A secondary hypothesis is it may serve as an alternative channel for conducting chloride, when the cAMP-regulated chloride conductance is non- functional as in the disease, cystic fibrosis (CF). The GOAL of this project is to determine the role of ClC-2 in rat lung development. The SPECIFIC AIMS are as follows: AIM 1: to identify the specific respiratory epithelial cells of the developing lung which express ClC-2 by co- localizing ClC-2 expression with airway epithelial cell markers using immunocytochemistry and in situ hybridization; AIM 2: to determine ClC secretory function in the developing lung by measuring chloride secretion in primary fetal rat lung cells in culture using 36/Cl efflux and Ussing chamber short circuit current and flux experiments after treatment with antisense oligonucleotides; AIM 3: to ascertain the effect of ClC-2 chloride channel activity on fluid secretion in fetal lung explants by knocking out ClC-2 activity with antisense strategies; AIM 4: to identify mechanisms which regulate ClC-2 in the developing lung by treating primary fetal lung cells in vitro, or by in vivo injection in pregnant dams with steroids, and measuring effect on ClC-2 expression and function.