Promising strategies for the treatment of CF are under investigation in many laboratories but it remains uncertain whether any single approach will restore enough Cl conductance to the appropriate cells to eliminate symptoms. The goal of this project is to study practical aspects of CFTR function that could be used to improve the effectiveness of new therapies as they become available. The first and most general approach would involve manipulating pathways that normally regulate CFTR activity.
Thus Specific Aim I focuses on characterizing one of these pathways, a membrane-associated phosphatase activity which normally downregulates CFTR. Evidence that this may succeed is provided by our finding that phosphatase inhibitors are potent activators of wild type CFTR channels and channels with the disease -causing mutations R117H, G551D, or deltaF508 after exposure to low temperature to reduce biosynthetic arrest. An alternative approach is feasible with CFTR gene and protein therapies, since these begin with cDNA which can be manipulated in vitro.
Specific Aim II will investigate mutations in CFTR designed to make it a more effective therapeutic agent. Mutagenesis and functional analysis will identify regions of CFTR that line the pore and residues that increase single channel conductance, Cl affinity and open probability. CFTR channels in multi-channel patches open and close in 'waves' that suggest cooperative interactions between the conducting units. The lack of such interactions may lower open probability when channels are sparse; for example, when biosynthetic arrest is only partially overcome or therapeutic delivery of the gene or protein is inadequate. Cooperativity and the relationship between channel number and open probability will be investigated. These studies should provide practical insights in CFTR channel function that will be useful in improving the effectiveness of newly-developed CF therapies.
