Functional cystic fibrosis transmembrane conductance regulator tagged with an epitope of the vesicular stomatis virus glycoprotein can be addressed to the apical domain of polarized cells

Eur J Cell Biol. 2000 Nov;79(11):795-802. doi: 10.1078/0171-9335-00116.

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-activated chloride channel apically localized in epithelial cells. In cystic fibrosis patients, the gene encoding this N-linked glycoprotein is mutated. About 70% of CF patients express a mutated form of CFTR, deleted at the phenylalanine residue at position 508 (deltaF508). CFTR-deltaF508 fails to exit the endoplasmic reticulum; it remains incompletely glycosylated and is rapidly degraded. To optimize CFTR detection for membrane localization studies and biochemical studies, we tagged wild-type and deltaF508 CFTR with the VSV-G epitope at their carboxy-terminal ends. We have generated pig kidney epithelial cell clones (LLCPK1) expressing VSV-G-tagged human wild-type and deltaF508-CFTR. In CFTR-expressing cells, the transfected protein is maturated and transported to the apical membrane where it is concentrated. The cells exhibit a strong anion channel activity after stimulation by cAMP, as demonstrated by a halide sensitive fluorescent dye assay (6-methoxy-N-ethylquinominium, SPQ), and whole-cell patch-clamp approach. This activity of CFTR-VSV-G is indistinguishable from the wild-type CFTR. In contrast, in cells expressing tagged deltaF508-CFTR or in non-transfected cells, no anion channel activity could be detected after stimulation by cAMP. In deltaF508-CFTR-VSV-G-expressing cells, the mutated CFTR remained in the incompletely glycosylated form and was localized in the endoplasmic reticulum. These cell lines reproduce the cellular fate of wild-type and mutated CFTR-deltaF508. To our knowledge, they are the first differentiated epithelial cell lines stably expressing tagged CFTR and CFTR-deltaF508 in which cellular processing and functional activity of these two proteins are reproduced. Thus the addition of the VSV-G epitope does not impair the localization and function of CFTR, and these cell lines can be used to examine CFTR function in vitro.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Cell Membrane / metabolism*
  • Cell Polarity
  • Cyclic AMP / pharmacology
  • Cystic Fibrosis Transmembrane Conductance Regulator / genetics*
  • Cystic Fibrosis Transmembrane Conductance Regulator / metabolism*
  • Endoplasmic Reticulum / metabolism
  • Epitopes
  • Fluorescent Antibody Technique
  • Fluorescent Dyes / metabolism
  • Humans
  • Membrane Glycoproteins*
  • Mutation
  • Patch-Clamp Techniques
  • Protein Processing, Post-Translational
  • Protein Transport*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Transfection
  • Vesicular stomatitis Indiana virus
  • Viral Envelope Proteins / immunology
  • Viral Envelope Proteins / metabolism*

Substances

  • CFTR protein, human
  • Epitopes
  • Fluorescent Dyes
  • G protein, vesicular stomatitis virus
  • Membrane Glycoproteins
  • Recombinant Fusion Proteins
  • Viral Envelope Proteins
  • Cystic Fibrosis Transmembrane Conductance Regulator
  • Cyclic AMP