IDENTIFICATION AND FUNCTIONAL ANALYSIS OF A COMPLEMENTARY SET OF SNARE PROTEINS RESPONSIBLE FOR AQP2 EXOCYTOSIS IN RENAL CELLS.

The vasopressin-regulated exocytosis of the water channel Aquaporin 2 (AQP2) at the apical mem- brane of the renal collecting duct principal cells increases water re-absorption from the luminal side of the tubule. Several lines of evidences indicate that the process of AQP2 vesicles fusion into the plasma membrane is mediated by SNARE proteins. We have previously demonstrated the involve- ment of a synaptobrevin/VAMP2 protein in AQP2 exocytosis. In this work we analyzed the pattern of subcellular distribution and the functional role of a set of Q and R-SNARE in a polarized mouse collecting duct cell line, MCD4, stably transfected with human AQP2. Both VAMP2 and VAMP3, were found associated with immunoisolated AQP2 vesicles and local- ized to both plasma membrane and a sub-apical vesicular compartment. As in other cell culture models and in the renal tubules, Syntaxin3 (Stx3) and Syntaxin4 (Stx4) were found associated with the apical ad baso-lateral membrane respectively. A third Q-SNARE, SNAP23, able to form functional complexes with VAMP and Stx in non-neural cells, was mainly localized at the plasma membrane. By using the SiRNA technology we selectively knock-down the expression of the iden- tified SNARE proteins and measured the rate of AQP2 fusion to the apical membrane by apical surface protein biotinylation technique. The obtained results indicated that VAMP2 and VAMP3 knock down reduced by about 70% and 60% respectively the amount of AQP2 at the apical membrane after FK stimulation. This inhibitory effect was additive when both VAMP2 and 3 were knocked down at the same time indicating that both proteins may cooperate to the fusion event. Stx3 but not Stx4 knock-down completely abolished AQP2 fusion to the apical membrane indi- cating that Stx3 is the Qa-SNARE involved in this process. Furthermore, SNAP23 knock-down induced a dramatic reduction of AQP2 fusion at the apical membrane. Immunoprecipitation exper- iments confirmed that in MCD4 cells Stx3 is able to form complexes with VAMP2, VAMP3 and SNAP23. These findings, taken together, propose VAMP2/3, Stx3 and SNAP23 as the complemen- tary set of SNARE proteins responsible for AQP2 storage vesicle fusion into the apical membrane in the renal collecting duct.