Functional CFTR in endosomal compartment of CFTR-expressing fibroblasts and T84 cells

Abstract

It was proposed that the cystic fibrosis transmembrane conductance regulator (CFTR) functions in the endosomal compartment as a adenosine 3',5'-cyclic monophosphate (cAMP)-regulated Cl channel that regulates endosomal acidification (J. Barasch, B. Kiss, A. Prince, L. Saiman, D. Gruenert, and A. Al-Awqati, Nature Lond. 352: 70-73, 1991). This hypothesis was tested in stably transfected Swiss 3T3 fibroblasts expressing CFTR or delta F508 CFTR and in T84 epithelial cells that normally express CFTR. In fibroblasts, the time course of pH in individual endosomes was measured by quantitative image analysis after 1 min pulse labeling with 2 microM carboxyfluorescein (Cf)-tetramethylrhodamine-transferrin (K. Zen, J. Biwersi, N. Periasamy, and A. S. Verkman. J. Cell Biol. 119: 99-110, 1992). Average endosomal pH reached 6.20 +/- 0.07 (SE) after 15 min in the mock-transfected cells with a half time of approximately 3 min; pH was slightly lower (5.97 +/- 0.06) in the CFTR-expressing fibroblasts. The difference did not result from a subpopulation of highly acidic endosomes. Forskolin (10 microM) increased average pH to 6.62 +/- 0.03 and abolished the difference. For determination of Cl conductance, endosomes in fibroblasts and T84 cells were labeled with Cf-dextran (5 mg/ml); dissipation of the endosomal pH gradient was measured in response to rapid addition of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP; 20 microM). Because the proton flux across the endosomal membrane is limited by the movement of K and Cl, the rate of alkalinization (dpH/dt) after CCCP addition provided a measure of endosomal Cl conductance. In CFTR-expressing fibroblasts, forskolin (10 microM) increased dpH/dt 1.6 +/- 0.2-fold (n = 14).(ABSTRACT TRUNCATED AT 250 WORDS)