FUNCTIONAL CFTR IN ENDOSOMAL COMPARTMENT OF CFTR-EXPRESSING FIBROBLASTS AND T84 CELLS

It was proposed that the cystic fibrosis transmembrane conductance reg ulator (CFTR) functions in the endosomal compartment as a adenosine 3' ,5'-cyclic monophosphate (cAMP)-regulated Cl channel that regulates en dosomal acidification (J. Barasch, B. Kiss, A. Prince, L. Saiman, D. G ruenert, and A. Al-Awqati. Nature Lond. 352: 70-73, 1991). This hypoth esis was tested in stably transfected Swiss 3T3 fibroblasts expressing CFTR or Delta F508 CFTR and in T84 epithelial cells that normally exp ress CFTR. In fibroblasts, the time course of pH in individual endosom es was measured by quantitative image analysis after 1 min pulse label ing with 2 mu M carboxyfluorescein (Cf)-tetramethylrhodamine-transferr in (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) a fter 15 min in the mock-transfected cells with a half time of similar to 3 min; pH was slightly lower (5.97 +/- 0.06) in the CFTR-expressing fibroblasts. The difference did not result from a subpopulation of hi ghly acidic endosomes. Forskolin (10 mu M) increased average pH to 6.6 2 +/- 0.03 and abolished the difference. For determination of Cl condu ctance, endosomes in fibroblasts and T84 cells were labeled with Cf-de xtran (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 mu M). Because the proton flux across the endosomal membrane is limited by the movement of K and Cl, the rat e of alkalinization (dpH/dt) after CCCP addition provided a measure of endosomal Cl conductance. In CFTR-expressing fibroblasts, forskolin ( 10 mu M) increased dpH/dt 1.6 +/- 0.2-fold (n = 14). dpH/dt in mock-tr ansfected and Delta F508 CFTR-expressing fibroblasts was not affected by forskolin and was similar to dpH/dt in CFTR-expressing fibroblasts in the absence of forskolin. In T84 cells, forskolin increased dpH/dt 1.44 +/- 0.09-fold (n = 6). In CFTR-expressing fibroblasts and T84 cel ls, replacement of Cl by the relatively impermeable anion isethionate decreased dpH/dt by 1.4 +/- 0.2- (n = 6; fibroblasts) and 1.6 +/- 0.1- fold (n = 4; T84) and abolished forskolin sensitivity. These studies p rovide evidence for functional cAMP-stimulated CFTR Cl channels in the endocytic compartment of CFTR-expressing 3T3 fibroblasts and T84 epit helial cells.