Evidence against the acidification hypothesis in cystic fibrosis.
The pleiotropic effects of cystic fibrosis (CF) result from the mislocalization or inactivity of an apical membrane chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR may also modulate intracellular chloride conductances and thus affect organelle pH. To test the role of CFTR in organelle pH regulation, we developed a model system to selectively perturb the pH of a subset of acidified compartments in polarized cells and determined the effects on various protein trafficking steps. We then tested whether these effects were observed in cells lacking wild-type CFTR and whether reintroduction of CFTR affected trafficking in these cells. Our model system involves adenovirus-mediated expression of the influenza virus M2 protein, an acid-activated ion channel. M2 expression selectively slows traffic through the trans-Golgi network (TGN) and apical endocytic compartments in polarized Madin-Darby canine kidney (MDCK) cells. Expression of M2 or treatment with other pH perturbants also slowed protein traffic in the CF cell line CFPAC, suggesting that the TGN in this cell line is normally acidified. Expression of functional CFTR had no effect on traffic and failed to rescue the effect of M2. Our results argue against a role for CFTR in the regulation of organelle pH and protein trafficking in epithelial cells.
Gibson
G A
GA
Laboratory of Epithelial Cell Biology, Renal-Electrolyte Division, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
Hill
W G
WG
Weisz
O A
OA
D000143:Acids / Q000378:metabolism*; D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001692:Biological Transport / Q000235:genetics; D002451:Cell Compartmentation / Q000187:drug effects; D002460:Cell Line; D016764:Cell Polarity; D002738:Chloroquine / Q000494:pharmacology; D003550:Cystic Fibrosis / Q000235:genetics / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000096:biosynthesis / Q000235:genetics; D004285:Dogs; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D006056:Golgi Apparatus / Q000378:metabolism; D006387:Hemagglutination, Viral / Q000235:genetics; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007070:Immunoglobulin A / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D010190:Pancreatic Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D014162:Transfection; D014407:Tumor Cells, Cultured; D014763:Viral Matrix Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D000086082:Viroporin Proteins
D000143:Acids; D002738:Chloroquine; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004791:Enzyme Inhibitors; D007070:Immunoglobulin A; D014763:Viral Matrix Proteins; C031540:M-protein, influenza virus; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins