Cystic fibrosis is an autosomal recessive disease. Affected tissues release less fluid resulting in congestion of the lungs, airway passages, pancreas and intestine. This pathology is due to reduced secretion of chloride ions from epithelial cells. Normally, chloride channels open in response to the second messengers, cyclic AMP or calcium. Epithelia from CF patients fail to secrete chloride in response to cyclic AMP. In 1989 the gene linked with cystic fibrosis (CFTR) was identified and sequenced. For the past several years this project has been directed toward elucidating the mediation of intracellular calcium through the investigation of high affinity calcium-binding proteins. Several calcium- dependent phospholipid binding proteins were identified and immunohistochemical techniques were used to localize these annexins in transporting epithelia. Annexin IV is associated with the apical membrane of epithelial cells. Anti-annexin IV antibody and oligonucleotides anti- sense to annexin IV suppress the functional levels of annexin IV and, in turn, alter Ca2+-activated Cl- conductance in perfused cells. Use of protein kinases and their peptide inhibitors suggests that phosphorylation events are associated with the annexin IV-modulated chloride secretory pathways. Phosphorylation of annexin IV will be correlated with chloride conductance. Photoaffinity cross-linking studies and selection of peptides from a library of random sequences will be performed to identify annexin IV 'nearest neighbor' target proteins. Their partial sequences will be compared with known proteins. Should the target proteins be previously uncharacterized, antibodies will be raised and used by immunoblot and indirect immunofluorescence to determine cellular expression and localization. Additional studies have been designed to obtain and test peptides as specific modifiers of cellular chloride conductance in CF cells. Peptide(s) reflecting the active site of annexin IV will also be purified by screening random peptide libraries with the anti-annexin IV antibodies. The effect of the peptides on chloride currents will be tested in patch-clamp studies. These findings elucidating the role annexin IV in Ca2+-dependent chloride and fluid secretion may provide insight into the development of pharmacologic strategies in the treatment of cystic fibrosis. Activation of a functional Cl- conductance pathway which is independent of CFTR could potentially ameliorate the chronic pathological condition associated with cystic fibrosis.
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