title: Small-molecule screening identifies modulators of aquaporin-2 trafficking.
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.
authors.affiliation: Max Delbrueck Center for Molecular Medicine, Robert-Rössle Strasse, 10 D-13125, Berlin, Germany.
authors.forename: Jana
authors.initials: J
authors.lastname: Bogum
authors.forename: Dörte
authors.initials: D
authors.lastname: Faust
authors.forename: Kerstin
authors.initials: K
authors.lastname: Zühlke
authors.forename: Jenny
authors.initials: J
authors.lastname: Eichhorst
authors.forename: Marie C
authors.initials: MC
authors.lastname: Moutty
authors.forename: Jens
authors.initials: J
authors.lastname: Furkert
authors.forename: Adeeb
authors.initials: A
authors.lastname: Eldahshan
authors.forename: Martin
authors.initials: M
authors.lastname: Neuenschwander
authors.forename: Jens Peter
authors.initials: JP
authors.lastname: von Kries
authors.forename: Burkhard
authors.initials: B
authors.lastname: Wiesner
authors.forename: Christiane
authors.initials: C
authors.lastname: Trimpert
authors.forename: Peter M T
authors.initials: PM
authors.lastname: Deen
authors.forename: Giovanna
authors.initials: G
authors.lastname: Valenti
authors.forename: Walter
authors.initials: W
authors.lastname: Rosenthal
authors.forename: Enno
authors.initials: E
authors.lastname: Klussmann
mesh_terms: D000818:Animals; D051399:Aquaporin 2 / Q000378:metabolism*; D052117:Benzodioxoles / Q000494:pharmacology*; D001572:Benzofurans / Q000494:pharmacology*; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology; D015725:Fluconazole / Q000494:pharmacology; D006056:Golgi Apparatus / Q000378:metabolism; D057166:High-Throughput Screening Assays; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D025262:Vacuolar Proton-Translocating ATPases / Q000037:antagonists & inhibitors*
chemical_list: C582438:4-acetyldiphyllin; C496023:AQP2 protein, human; D051399:Aquaporin 2; D052117:Benzodioxoles; D001572:Benzofurans; D005576:Colforsin; D015725:Fluconazole; D000242:Cyclic AMP; D025262:Vacuolar Proton-Translocating ATPases
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