The long term objective of our research is to understand the chloride conductance pathways that malfunction in Cystic Fibrosis (CF). We have recently identified a CAMP-activated Cl channel in the apical membrane of the T84 cell line. This low-conductance channel closely resembles one reported previously in the pancreatic duct, and which may be responsible for pancreatic dysfunction in CF. We will use the T84 cell line as a model for studying modulation and biophysical properties of this low-conductance channel, then explore its role in cystic fibrosis. We will quantify the effects of CAMP stimulation on open probability and channel density, and test the hypothesis that CAMP causes insertion of new Cl channels into the apical membrane by vesicle fusion. We will also investigate other signalling pathways that impinge on the channel, and we shall determine the relationship between the CAMP- stimulated conductance and cell volume. Several biophysical properties of the channel will be assessed including ion selectivity, gating kinetics, and pharmacology. Efficient methods for incorporating the channel into liposomes will be developed for use in future assays of purified regulatory components. We shall examine CF-affected tissues in order to investigate the role of this channel in CF and investigate its relationship to the defective protein CFTR. To accomplish this we will compare the number of channels and their regulation in normal T84 cells and in antisense T84 cells which have reduced expression of the CFTR protein. Finally, we will examine the effects of antibodies to CFTR on Cl channel function. These approaches should provide new insights into the mechanisms underlying epithelial Cl conductance, and establish the role of this channel in cystic fibrosis.
