Endosomal SNARE proteins regulate CFTR activity and trafficking in epithelial cells

Exp Cell Res. 2008 Jul 1;314(11-12):2199-211. doi: 10.1016/j.yexcr.2008.04.012. Epub 2008 May 4.

Abstract

The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) protein is a chloride channel localized at the apical plasma membrane of epithelial cells. We previously described that syntaxin 8, an endosomal SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) protein, interacts with CFTR and regulates its trafficking to the plasma membrane and hence its channel activity. Syntaxin 8 belongs to the endosomal SNARE complex which also contains syntaxin 7, vti1b and VAMP8. Here, we report that these four endosomal SNARE proteins physically and functionally interact with CFTR. In LLC-PK1 cells transfected with CFTR and in Caco-2 cells endogenously expressing CFTR, we demonstrated that endosomal SNARE protein overexpression inhibits CFTR activity but not swelling- or calcium-activated iodide efflux, indicating a specific effect upon CFTR activity. Moreover, co-immunoprecipitation experiments in LLC-PK1-CFTR cells showed that CFTR and SNARE proteins belong to a same complex and pull-down assays showed that VAMP8 and vti1b preferentially interact with CFTR N-terminus tail. By cell surface biotinylation and immunofluorescence experiments, we evidenced that endosomal SNARE overexpression disturbs CFTR apical targeting. Finally, we found a colocalization of CFTR and endosomal SNARE proteins in Rab11-positive recycling endosomes, suggesting a new role for endosomal SNARE proteins in CFTR trafficking in epithelial cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Cystic Fibrosis Transmembrane Conductance Regulator / genetics
  • Cystic Fibrosis Transmembrane Conductance Regulator / metabolism*
  • Endosomes / metabolism
  • Epithelial Cells / cytology
  • Epithelial Cells / metabolism*
  • Humans
  • Iodides / metabolism
  • Protein Transport / physiology
  • Qa-SNARE Proteins / genetics
  • Qa-SNARE Proteins / metabolism*
  • Qb-SNARE Proteins / genetics
  • Qb-SNARE Proteins / metabolism*
  • R-SNARE Proteins / genetics
  • R-SNARE Proteins / metabolism*
  • RNA Interference
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • SNARE Proteins / genetics
  • SNARE Proteins / metabolism*
  • rab GTP-Binding Proteins / genetics
  • rab GTP-Binding Proteins / metabolism

Substances

  • Iodides
  • Qa-SNARE Proteins
  • Qb-SNARE Proteins
  • R-SNARE Proteins
  • Recombinant Fusion Proteins
  • SNARE Proteins
  • VAMP8 protein, human
  • VTI1B protein, human
  • Cystic Fibrosis Transmembrane Conductance Regulator
  • rab11 protein
  • rab GTP-Binding Proteins