title: Proton leak and CFTR in regulation of Golgi pH in respiratory epithelial cells.
abstract: Work addressing whether cystic fibrosis transmembrane conductance regulator (CFTR) plays a role in regulating organelle pH has remained inconclusive. We engineered a pH-sensitive excitation ratiometric green fluorescent protein (pHERP) and targeted it to the Golgi with sialyltransferase (ST). As determined by ratiometric imaging of cells expressing ST-pHERP, Golgi pH (pH(G)) of HeLa cells was 6.4, while pH(G) of mutant (DeltaF508) and wild-type CFTR-expressing (WT-CFTR) respiratory epithelia were 6.7-7.0. Comparison of genetically matched DeltaF508 and WT-CFTR cells showed that the absence of CFTR statistically increased Golgi acidity by 0.2 pH units, though this small difference was unlikely to be physiologically important. Golgi pH was maintained by a H(+) vacuolar (V)-ATPase countered by a H(+) leak, which was unaffected by CFTR. To estimate Golgi proton permeability (P(H(+))), we modeled transient changes in pH(G) induced by inhibiting the V-ATPase and by acidifying the cytosol. This analysis required knowing Golgi buffer capacity, which was pH dependent. Our in vivo estimate is that Golgi P(H(+)) = 7.5 x 10(-4) cm/s when pH(G) = 6.5, and surprisingly, P(H(+)) decreased as pH(G) decreased.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3200, USA.
authors.forename: G
authors.initials: G
authors.lastname: Chandy
authors.forename: M
authors.initials: M
authors.lastname: Grabe
authors.forename: H P
authors.initials: HP
authors.lastname: Moore
authors.forename: T E
authors.initials: TE
authors.lastname: Machen
mesh_terms: D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000502:physiology*; D003600:Cytosol / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017930:Genes, Reporter; D006056:Golgi Apparatus / Q000187:drug effects / Q000502:physiology*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008322:Mammals; D008954:Models, Biological; D009297:Nasal Mucosa / Q000166:cytology / Q000502:physiology*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000032:analysis; D020545:Respiratory Mucosa / Q000502:physiology*; D017384:Sequence Deletion; D014162:Transfection
chemical_list: C505032:CFTR protein, human; D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D049452:Green Fluorescent Proteins; D005576:Colforsin; D003994:Bucladesine