EVIDENCE FOR LOCATION OF THE CFTR IN HUMAN PLACENTAL APICAL MEMBRANE-VESICLES

Ion transport (Cl-36 uptake) and immunochemical studies were undertaken to detect the cystic fibrosis transmembrane conductance regulator (CFTR) in apical membrane vesicles prepared from human placenta. Cl-36 uptake into membrane vesicles was studied in the absence and presence of inwardly directed potassium gradients and valinomycin (K-o = K-i and K-o > K-i, where K-o is potassium concentration outside and K-i is potassium concentration inside the vesicles). The sensitivities of Cl-36 uptake to the inhibitors 4,4'-diisothiocyanostilbene-2,2' -disulfonic acid (DIDS), bumetanide, and diphenylamine-2-carboxylate were investigated. Each compound significantly inhibited uptake under both sets of conditions. Additional inhibition of Cl-36 uptake was found when the compounds were added together, indicating that they were acting at least partly on different components of the Cl-36 uptake. The DIDS- and bumetanide-insensitive component of transport was more selective for Cl than I. These findings suggested that this component may, at least in part, represent Cl transport via CFTR. Addition of adenosine 5'-O-(3-thiotriphosphate) (0.8 mM) led to a decrease in total Cl-36 uptake but masked in the overall decrease was an increase in the DIDS- and bumetanide-insensitive component of Cl-36 uptake. Western blot analysis of the apical membrane proteins with an antibody specific for a region of human CFTR detected a protein band of similar to 190 kDa. These ion transport and immunochemical studies provide evidence that CFTR is located in human placental apical membrane vesicles.