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First published online 27 May 2008
doi: 10.1242/jcs.022210

Journal of Cell Science 121, 2097-2106 (2008)
Published by The Company of Biologists 2008
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Research Article

AQP2 exocytosis in the renal collecting duct – involvement of SNARE isoforms and the regulatory role of Munc18b

Giuseppe Procino1, Claudia Barbieri1, Grazia Tamma1, Leonarda De Benedictis2, Jeffrey E. Pessin3, Maria Svelto1 and Giovanna Valenti1,*

1 Department of General and Environmental Physiology, University of Bari, Via Amendola 165/A, Bari 70126, Italy
2 Department of Pharmacology and Human Physiology, University of Bari Medical School, Bari 70124, Italy
3 Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461, USA

* Author for correspondence (e-mail: g.valenti{at}biologia.uniba.it)

Accepted 31 March 2008

Vasopressin regulates the fusion of the water channel aquaporin 2 (AQP2) to the apical membrane of the renal collecting-duct principal cells and several lines of evidence indicate that SNARE proteins mediate this process. In this work MCD4 renal cells were used to investigate the functional role of a set of Q- and R-SNAREs, together with that of Munc18b as a negative regulator of the formation of the SNARE complex. Both VAMP2 and VAMP3 were associated with immunoisolated AQP2 vesicles, whereas syntaxin 3 (Stx3), SNAP23 and Munc18 were associated with the apical plasma membrane. Co-immunoprecipitation experiments indicated that Stx3 forms complexes with VAMP2, VAMP3, SNAP23 and Munc18b. Protein knockdown coupled to apical surface biotinylation demonstrated that reduced levels of the R-SNAREs VAMP2 and VAMP3, and the Q-SNAREs Stx3 and SNAP23 strongly inhibited AQP2 fusion at the apical membrane. In addition, knockdown of Munc18b promoted a sevenfold increase of AQP2 fused at the plasma membrane without forskolin stimulation.

Taken together these findings propose VAMP2, VAMP3, Stx3 and SNAP23 as the complementary set of SNAREs responsible for AQP2-vesicle fusion into the apical membrane, and Munc18b as a negative regulator of SNARE-complex formation in renal collecting-duct principal cells.

Key words: Aquaporin2, Vasopressin, VAMP, SNARE, Syntaxin, Munc18, Exocytosis


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