Aim: The expression and the functional involvement of SNARE proteins and SNARE-regulating proteins (Munc18b and Munc18c) in catalyzing aquaporin 2 vesicles fusion at the apical membrane is investigated in a mouse cortical collecting duct cell line. Methods: Localization of SNARE and SNARE-regulating proteins was performed using a combination of confocal microscopy and cell fractionation. SNARE complex formation was demonstrated by co-immunoprecipitation. Functional involvement of each protein was demonstrated coupling RNA interference to apical surface biotinylation in order to quantify the effect of protein knockdown on AQP2 exocytosis in response to FK stimulation. Results: Both VAMP2 and VAMP3 were found associated with AQP2 immunoisolated vesicle. Syntaxin 3 was found associated with the apical plasma membrane along with its functional partner Munc18b. Upon FK stimulation, Synataxin 3 is able to dissociate from Munc18b and to form a stable complex with VAMP2, VAMP3 and SNAP23. siRNA experiments indicate that Syntaxin 3, VAMP2, VAMP3 and SNAP23 are required for AQP2 vesicle fusion at the apical membrane. Moreover, Munc18 knockdown induced accumulation of AQP2 at the apical plasma membrane. Conclusion: These findings, taken together, propose a complementary set of SNARE proteins and SNARE-regulators responsible for AQP2 storage vesicle fusion into the apical membrane in the renal collecting duct.
SNARE Proteins And SNARE Regulators Mediate Aquaporin2 Exocytosis In Renal Cells
PROCINO, Giuseppe;TAMMA, GRAZIA;SVELTO, Maria;VALENTI, Giovanna
2008-01-01
Abstract
Aim: The expression and the functional involvement of SNARE proteins and SNARE-regulating proteins (Munc18b and Munc18c) in catalyzing aquaporin 2 vesicles fusion at the apical membrane is investigated in a mouse cortical collecting duct cell line. Methods: Localization of SNARE and SNARE-regulating proteins was performed using a combination of confocal microscopy and cell fractionation. SNARE complex formation was demonstrated by co-immunoprecipitation. Functional involvement of each protein was demonstrated coupling RNA interference to apical surface biotinylation in order to quantify the effect of protein knockdown on AQP2 exocytosis in response to FK stimulation. Results: Both VAMP2 and VAMP3 were found associated with AQP2 immunoisolated vesicle. Syntaxin 3 was found associated with the apical plasma membrane along with its functional partner Munc18b. Upon FK stimulation, Synataxin 3 is able to dissociate from Munc18b and to form a stable complex with VAMP2, VAMP3 and SNAP23. siRNA experiments indicate that Syntaxin 3, VAMP2, VAMP3 and SNAP23 are required for AQP2 vesicle fusion at the apical membrane. Moreover, Munc18 knockdown induced accumulation of AQP2 at the apical plasma membrane. Conclusion: These findings, taken together, propose a complementary set of SNARE proteins and SNARE-regulators responsible for AQP2 storage vesicle fusion into the apical membrane in the renal collecting duct.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
