ACTIVATION OF CFTR MUTANTS IN VIVO FOR CF THERAPY

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DESCRIPTION (Taken directly from the application) lt is generally accepted that the symptoms of cystic fibrosis are in part, if not wholly, due to defective electrolyte transport mediated by CFTR, although the contribution of CFTR as a chloride channel or as a regulator of other ion channels in pathogenesis is unclear. The hypothesis to be examined in this proposal is that increasing the activity of CFTR mutants is likely to have therapeutic potential for CF patients. As a measure of predicted efficacy, we will measure electrogenic chloride transport across CF tissues in response to various drugs which activate CFTR. First, as a model for human CF airway tissues, the inferior nasal turbinate of CF patients will be exposed to beta-adrenergic agonists and class-specific phosphodiesterase inhibitors, the combination of which has been found to induce CFTR-mediated chloride efflux from primary CF nasal epithelial cells. The ability of these compounds, in the presence of amiloride, to increase nasal potential difference will be measured as an indicator of chloride secretion. Patients of various genotypes will be studied. As a more versatile model system for CF, transepithelial chloride transport will be measured across tissues from mice carrying CF mutations. Nasal potential difference measurements will be made to examine transport in the airway and short circuit current across the jejunum measured in response to beta-adrenergic agonists and class-specific phosphodiesterase inhibitors. The second focus of this application is to examine the effects of chronic exposure to these potential therapeutic compounds on electrolyte transport. Cells expressing CFTR will be cultured in the presence of compounds found to induce transepithelial chloride transport and the effects on CFTR mRNA and protein will be measured, as well as short circuit current. The mechanism by which these compounds increase chloride transport will also be investigated. To discriminate between the mechanisms of increasing channel activity and increasing channel number, the quantity of CFTR in the plasma membrane will be measured by cell-surface biotinylation before and after stimulation. The level of CFTR phosphorylation will also be measured by metabolic labeling of CFTR with 32P in the presence and absence of stimulation. Comparison of the amount in the membrane with the amount phosphorylated should indicate the contribution of CFTR activity and quantity. If beta-adrenergic agonists and class-specific phospho-diesterase inhibitors can significantly increase chloride transport, it is important to carry out studies on mechanism to determine if the process can be further improved.