MUNC18, A FUNCTIONAL PARTNER OF SYNTAXIN3, REGULATES AQP2 FUSION TO THE APICAL MEMBRANE IN A MOUSE COLLECTING DUCT CELL LINE.

We have identified Syntaxin3 (Stx3) as the Q-SNARE involved in the fusion of Aquaporin2 (AQP2) storage vesicles to the apical membrane in MCD4, a mouse renal collecting duct cell line in response to forskolin. Here we describe the functional involvement of Munc18b, a mem- ber of the Sec/Munc family of proteins, as a negative regulator of the SNARE complex formation catalyzing AQP2 fusion to the apical membrane. It has been reported that Munc18 proteins can sta- bilize syntaxins in their closed conformation thus preventing their interaction with other SNARE motifs and hence the formation of the SNARE complex mediating vesicles to membrane fusion. In MCD4 cells, by RT-PCR, western blotting and immunofluorescence approaches, two isoforms of Munc18 were found expressed: Munc18b, the epithelial cells specific isoform and Munc18c the ubiquitously expressed isoform. As confirmed by confocal xz scanning, both isoforms were mainly associated to the apical plasma membrane where they possibly interact with Stx3. The role of Munc18 in AQP2 exocytosis was investigated in cells in which the expression of both Munc18 isoforms was knocked-down by specific SiRNAs. In control MCD4 cells, surface biotinylation technique indicated a 3 to 4-fold increase of apical AQP2 after treatment with forskolin compared to untreated cells. Interestingly, in cells silenced for Munc18b expression, the amount of AQP2 at the apical membrane was 7-fold higher than in untreated cells even in basal condition and FK pro- moted a small but yet significant increase of AQP2. When Munc18c isoform was knocked-down, the effect on AQP2 exocytosis was less dramatic. This is in line with observation that Munc18b isoform binds preferentially to Stx3. These data, taken together, suggest that Munc18b association to Stx3 prevents AQP2 vesicle fusion to the apical membrane in unstimulated cells. Removal of this negative regulator by SiRNA induced spontaneous fusion of AQP2 vesicles to the apical mem- brane in the absence of FK stimulation. This indicates that Munc18b displacement from Stx3 may be part of the vasopressin regulated signal cascade leading to AQP2 exocytosis in collecting duct renal cells