Polarization-dependent apical membrane CFTR targeting underlies cAMP-stimulated Cl- secretion in epithelial cells

The relationship between adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion and the cellular location of the cystic fibrosis transmembrane conductance regulator (CFTR) was determined in both polarized (Cl.19A) and unpolarized (parental) HT-29 colonocytes expressing similar levels of CFTR mRNA and protein. CFTR immunolocalized to the apical membrane domain of polarized colonocytes exhibiting cAMP-responsive Cl- secretion. In contrast, CFTR staining was perinuclear in unpolarized colonocytes, which gave little or no cAMP-stimulated Cl- conductance responses. Thus cAMP-stimulated Cl- secretion coincided with an apical localization of CFTR. Brefeldin A (BFA) was used to perturb glycoprotein targeting in these cells. In polarized colonocytes, BFA caused a reversible, time-dependent decrease in the Cl-conductance response to cAMP but not Ca2+. Apical CFTR redistributed into large coalesced intracellular vesicles, located within the same plane as the microtubule organizing center, a marker for th...