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First published online 23 March 2004
doi: 10.1242/jcs.01070

Journal of Cell Science 117, 1923-1935 (2004)
Published by The Company of Biologists 2004
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Research Article

Syntaxin 8 impairs trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) and inhibits its channel activity

Frédéric Bilan1,*, Vincent Thoreau1,*, Magali Nacfer1, Renaud Dérand2, Caroline Norez2, Anne Cantereau2, Martine Garcia3, Frédéric Becq2 and Alain Kitzis1,{ddagger}

1 Laboratoire de Génétique Cellulaire et Moléculaire, UPRES EA 2622, CHU de Poitiers, BP 577, 86021 Poitiers CEDEX, France
2 Laboratoire de Biomembranes et Signalisation Cellulaire, CNRS UMR 6558, Université de Poitiers, France
3 Laboratoire d'Immunologie et Interactions Moléculaires, UPRES EA 2224, Université de Poitiers, France

{ddagger} Author for correspondence (e-mail: a.kitzis{at}chu-poitiers.fr)

Accepted 23 December 2003

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.

Key words: CFTR, Syntaxin 8, SNARE, Recycling endosome, Trafficking


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