title: Targeting of carbonic anhydrase IV to plasma membranes is altered in cultured human pancreatic duct cells expressing a mutated (deltaF508) CFTR.
abstract: Human pancreatic duct cells secrete HCO3- ions mediated by a Cl-/HCO3- exchanger and a HCO3- channel that may be a carbonic anhydrase IV (CA IV) in a channel-like conformation. This secretion is regulated by CFTR (Cystic Fibrosis Transmembrane conductance Regulator). In CF cells homozygous for the deltaF508 mutation, the defect in targeting of CFTR to plasma membranes leads to a disruption in the secretion of Cl- and HCO3 ions along with a defective targeting of other proteins. In this study, we analyzed the targeting of membrane CA IV in the human pancreatic duct cell line CFPAC-1, which expresses a deltaF508 CFTR, and in the same cells transfected with the wild-type CFTR (CFPAC-PLJ-CFTR6) or with the vector alone (CFPAC-PLJ6). The experiments were conducted on cells in the stationary phase the polarized state of which was checked by the distribution of occludin and actin. We show that both cell lines express a 35-kDa CA IV at comparable levels. Analysis of fractions of plasma membranes purified on a Percoll gradient evidenced lower levels of CA IV (8-fold) in the CFPAC-1 than in the CFPAC-PLJ-CFTR6 cells. Quantitative analyses showed that 6- to 10-fold fewer cells in the CFPAC-1 cell line exhibited membrane CA IV-immunoreactivity than in the CFPAC-PLJ-CFTR6 cell line. Taken together, these results suggest that the targeting of CA IV to apical plasma membranes is impaired in CFPAC-1 cells. CA IV/gamma-adaptin double labeling demonstrated the presence of CA IV in the trans-Golgi network (TGN) of numerous CFPAC-1 cells, indicating that trafficking was disrupted on the exit face of the TGN. The retargeting of CA IV observed in CFPAC-PLJ-CFTR6 cells points to a relationship between the traffic of CFTR and CA IV. On the basis of these observations, we propose that the absence of CA IV in apical plasma membranes due to the impairment in targeting in cells expressing a deltaAF508 CFTR largely contributes to the disruption in HCO3- secretion in CF epithelia.
authors.affiliation: Laboratoire de Biologie Cellulaire et Moléculaire des Epithéliums, Université Paul Sabatier, Toulouse, France.
authors.forename: Marjorie
authors.initials: M
authors.lastname: Fanjul
authors.forename: Christel
authors.initials: C
authors.lastname: Salvador
authors.forename: Laetitia
authors.initials: L
authors.lastname: Alvarez
authors.forename: Sylvie
authors.initials: S
authors.lastname: Cantet
authors.forename: Etienne
authors.initials: E
authors.lastname: Hollande
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D000328:Adult; D001639:Bicarbonates / Q000378:metabolism*; D030741:Carbonic Anhydrase IV / Q000235:genetics / Q000378:metabolism*; D002277:Carcinoma / Q000201:enzymology / Q000235:genetics / Q000503:physiopathology; D002455:Cell Division / Q000235:genetics; D002462:Cell Membrane / Q000201:enzymology*; D016764:Cell Polarity / Q000235:genetics; D003550:Cystic Fibrosis / Q000201:enzymology / Q000235:genetics / Q000503:physiopathology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000172:deficiency* / Q000235:genetics; D003594:Cytoplasmic Granules / Q000201:enzymology / Q000235:genetics / Q000648:ultrastructure; D004847:Epithelial Cells / Q000166:cytology / Q000201:enzymology*; D005455:Fluorescent Antibody Technique; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D009154:Mutation / Q000502:physiology; D062793:Occludin; D010183:Pancreatic Ducts / Q000166:cytology / Q000201:enzymology*; D010190:Pancreatic Neoplasms / Q000201:enzymology / Q000235:genetics / Q000503:physiopathology; D021381:Protein Transport / Q000235:genetics; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000201:enzymology*; D021601:trans-Golgi Network / Q000201:enzymology / Q000235:genetics / Q000648:ultrastructure
chemical_list: D000199:Actins; D001639:Bicarbonates; C505032:CFTR protein, human; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D030741:Carbonic Anhydrase IV