Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR), which is aberrant in patients with cystic fibrosis, normally functions both as a chloride channel and as a pleiotropic regulator of other ion transporters. Here we show, by ratiometric imaging with luminally exposed pH-sensitive green fluorescent protein, that CFTR affects the pH of cellubrevin-labeled endosomal organelles resulting in hyperacidification of these compartments in cystic fibrosis lung epithelial cells. The excessive acidification of intracellular organelles was corrected with low concentrations of weak base. Studies with proton ATPase and sodium channel inhibitors showed that the increased acidification was dependent on proton pump activity and sodium transport. These observations implicate sodium efflux in the pH homeostasis of a subset of endocytic organelles and indicate that a dysfunctional CFTR in cystic fibrosis leads to organellar hyperacidification in lung epithelial cells because of a loss of CFTR inhibitory effects on sodium transport. Furthermore, recycling of transferrin receptor was altered in CFTR mutant cells, suggesting a previously unrecognized cellular defect in cystic fibrosis, which may have functional consequences for the receptors on the plasma membrane or within endosomal compartments.
Highlights
The cystic fibrosis transmembrane conductance regulator (CFTR)1 functions as an apical membrane chloride channel [1]
The endosomal and plasma membrane (GPI)-targeted pHluorin GFP probes were transfected into well characterized human bronchial epithelial cells [21, 22]: IB3-1, C38 (IB3-1 cells corrected with a functional CFTR lacking the first ecto-loop), and S9 (IB3-1 cells corrected with a full size functional CFTR cDNA)
Our observations that the recycling endosomal compartment is hyperacidified in mutant CFTR IB3-1 and CFT1 cells is at variance with the previously published values for CF nasal polyp cells reporting slight alkalinization of the endosome [11]
Summary
Cells and Tissue Culture—CFT1 [19, 20] is a cell line derived from the tracheal epithelium of a CF patient homozygous for the most common CFTR ⌬F508 mutation. Cells were washed after 10 and 60 min, and the ratio of fluorescence emission at 508 nm was determined upon altered excitation at 410 and 470 nm. Fluorescence Microscopy Localization Studies—For localization studies with fluorescently labeled transferrin, IB3-1 cells and derivatives grown on glass slides were transfected with cellubrevin-pHluorin GFP as described above. For localization studies with CFTRGFP and transferrin, IB3-1 cells and derivatives were transfected with CFTR-GFP. For localization studies with dextran-Texas Red, cellubrevin-pHluorin-transfected IB3-1 cells and IB3-1 derivatives were incubated with 10 g/ml dextran-Texas Red followed by three washes. IB3-1 cells and their derivatives were incubated with 125I-labeled transferrin for 45 min in DMEM, 0.2% BSA at 37 °C. Cells were incubated with 5 mg/ml HRP in DMEM, 0.2% BSA for 60 min at 37 °C. Statistics—All statistical analyses were carried out using Fisher’s Protected LSD post hoc test (analysis of variance) (SuperANOVA v1.11, Abacus Concepts, Inc., Berkeley, CA)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
