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Small-molecule screening identifies modulators of aquaporin-2 trafficking

Item Type:Article
Title:Small-molecule screening identifies modulators of aquaporin-2 trafficking
Creators Name:Bogum, J. and Faust, D. and Zühlke, K. and Eichhorst, J. and Moutty, M.C. and Furkert, J. and Eldahshan, A. and Neuenschwander, M. and von Kries, J.P. and Wiesner, B. and Trimpert, C. and Deen, P.M. and Valenti, G. and Rosenthal, W. and Klussmann, E.
Abstract:In the principal cells of the renal collecting duct, arginine vasopressin (AVP) stimulates the synthesis of cAMP, leading to signaling events that culminate in the phosphorylation of aquaporin-2 water channels and their redistribution from intracellular domains to the plasma membrane via vesicular trafficking. The molecular mechanisms that control aquaporin-2 trafficking and the consequent water reabsorption, however, are not completely understood. Here, we used a cell-based assay and automated immunofluorescence microscopy to screen 17,700 small molecules for inhibitors of the cAMP-dependent redistribution of aquaporin-2. This approach identified 17 inhibitors, including 4-acetyldiphyllin, a selective blocker of vacuolar H(+)-ATPase that increases the pH of intracellular vesicles and causes accumulation of aquaporin-2 in the Golgi compartment. Although 4-acetyldiphyllin did not inhibit forskolin-induced increases in cAMP formation and downstream activation of protein kinase A (PKA), it did prevent cAMP/PKA-dependent phosphorylation at serine 256 of aquaporin-2, which triggers the redistribution to the plasma membrane. It did not, however, prevent cAMP-induced changes to the phosphorylation status at serines 261 or 269. Last, we identified the fungicide fluconazole as an inhibitor of cAMP-mediated redistribution of aquaporin-2, but its target in this pathway remains unknown. In conclusion, our screening approach provides a method to begin dissecting molecular mechanisms underlying AVP-mediated water reabsorption, evidenced by our identification of 4-acetyldiphyllin as a modulator of aquaporin-2 trafficking.
Keywords:Aquaporin 2, Benzodioxoles, Benzofurans, Cultured Cells, Cyclic AMP, Fluconazole, Fluorescence Microscopy, Forskolin, Golgi Apparatus, High-Throughput Screening Assays, Phosphorylation, Protein Transport, Vacuolar Proton-Translocating ATPases, Animals, Mice, Rats
Source:Journal of the American Society of Nephrology
Publisher:American Society of Nephrology
Page Range:744-758
Date:April 2013
Official Publication:https://doi.org/10.1681/ASN.2012030295
PubMed:View item in PubMed

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