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

Abstract

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.

Selective Regulation of Maize Plasma Membrane Aquaporin Trafficking and Activity by the SNARE SYP121

“…Our observation that SYP121-Sp2 fragments from Arabidopsis and maize decreased the plasma membrane trafficking of mYFP:ZmPIP2;5 in tobacco epidermal cells and maize mesophyll protoplasts demonstrates that the mechanism of SYP121-mediated plasma membrane anchoring of PIP2 aquaporins is conserved between monocots and dicots. In addition, the localization of the human AQP2 at the apical membrane of epithelial cells of the collecting duct is also specifically regulated by the syntaxin-3, SNARE-associated protein Snapin and SNAP33 complex (Procino et al, 2008;Mistry et al, 2009), suggesting that the relative abundance of plasma membrane aquaporins is regulated by SNARE complexes in all eukaryotes.…”

Aquaporin 2 Promotes Cell Migration and Epithelial Morphogenesis

“…4,5 Critical protein/protein interactions that orchestrate the regulated and constitutive trafficking of AQP2 have been extensively investigated and include actin and microtubules, SNAREs, Rab proteins, heat shock protein 70, clathrin, and others. [6][7][8][9] However, emerging data from AQP2 knockout and transgenic animals suggested an unusual aspect of AQP2 biology. As expected, induction of AQP2 deficiency in mice results in a urinary concentrating defect known as diabetes insipidus (DI).…”

Fluvastatin Increases AQP2 Urine Excretion in a Dyslipidemic Patient with Nephrogenic Diabetes Insipidus: An In Vivo and In Vitro Study

“…A mouse collecting duct cell line stably transfected with human AQP2, MDC4 cells, was used to further investigate the effect of fluvastatin on AQP2 exosome excretion. We previously showed that in this cell line incubation with fluvastatin nicely accumulates AQP2 at the apical plasma membrane [27,31]. Cells were either left untreated (Ctr) or treated with forskolin 100 µM (FK) or fluvastatin 5 µM (Flu) for 16 hours in the culture medium, then subjected to immunofluorescence protocol to visualize AQP2 cellular distribution.…”