Syntaxin 8 impairs trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) and inhibits its channel activity.
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-dependent chloride channel that mediates electrolyte transport across the luminal surface of epithelial cells. In this paper, we describe the CFTR regulation by syntaxin 8, a t-SNARE protein (target soluble N-ethylmaleimide-sensitive factor attachment protein receptor) involved in the SNARE endosomal complex. Syntaxin family members are key molecules implicated in diverse vesicle docking and membrane fusion events. We found that syntaxin 8 physically interacts with CFTR: recombinant syntaxin 8 binds CFTR in vitro and both proteins co-immunoprecipitate in HT29 cells. Syntaxin 8 regulates CFTR-mediated currents in chinese hamster ovary (CHO) cells stably expressing CFTR and syntaxin 8. Iodide efflux and whole-cell patch-clamp experiments on these cells indicate a strong inhibition of CFTR chloride current by syntaxin 8 overexpression. At the cellular level, we observed that syntaxin 8 overexpression disturbs CFTR trafficking. Confocal microscopy shows a dramatic decrease in green fluorescent protein-tagged CFTR plasma membrane staining, when syntaxin 8 is coexpressed in COS-7 cells. Using antibodies against Lamp-1, TfR or Rab11 we determined by immunofluorescence assays that both proteins are mainly accumulated in recycling endosomes. Our results evidence that syntaxin 8 contributes to the regulation of CFTR trafficking and chloride channel activity by the SNARE machinery.
Bilan
Frédéric
F
Laboratoire de Génétique Cellulaire et Moléculaire, UPRES EA 2622, CHU de Poitiers, BP 577, 86021 Poitiers CEDEX, France.
Thoreau
Vincent
V
Nacfer
Magali
M
Dérand
Renaud
R
Norez
Caroline
C
Cantereau
Anne
A
Garcia
Martine
M
Becq
Frédéric
F
Kitzis
Alain
A
D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D000954:Antigens, Surface / Q000378:metabolism; D016466:CHO Cells; D019556:COS Cells; D002460:Cell Line; D006224:Cricetinae; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D011992:Endosomes / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D007454:Iodides / Q000737:chemistry; D051907:Lysosomal Membrane Proteins; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009419:Nerve Tissue Proteins / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D021381:Protein Transport; D050765:Qa-SNARE Proteins; D011990:Receptors, Transferrin / Q000378:metabolism; D015398:Signal Transduction; D050827:Syntaxin 1; D013997:Time Factors; D014162:Transfection; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D000099193:rab11 GTP-Binding Proteins
D015703:Antigens, CD; D000954:Antigens, Surface; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D018076:DNA, Complementary; D005982:Glutathione Transferase; D007454:Iodides; D051907:Lysosomal Membrane Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050765:Qa-SNARE Proteins; D011990:Receptors, Transferrin; D050827:Syntaxin 1; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C505032:CFTR protein, human