Human Aquaporin-1: Furosemide Docking and Protein-Protein Interactions

DORR, RICARDO A.; ROSI, PABLO; OZU, MARCELO; COSTA ALMAR, FLORENCIA; TORIANO, ROXANA

Sierra de la Ventana

Congreso; SAB 2014: XLIII Reunión Anual de la Sociedad Argentina de Biofísica; 2014

Sociedad Argentina de Biofísica

In our previous work we showed that the water permeability ofhuman aquaporin-1 (hAQP1) is specifically inhibited by thepresence of intracellular furosemide and modulated by the tensionof the plasma membrane. These data were obtained in theXenopus laevis oocyte membrane expressing water channels. Nowwe use different docking protocols to locate sites of inhibition andmodifiers of membrane tension. The first strategy was to perform ablind docking (with AutoDock) against the monomeric structure ofhAQP1 obtained from electron-crystallographic data (1FQY PDBcode). The results indicate that furosemide binds to both cytosolicand outside areas of the channel, but never at sites in thetransmembrane region. Coincidentally with the in vitro results, thecytosolic region was more favored than the extracellular bindingregion. To evaluate the influence of protein conformationalchanges, we did an unrestricted  -carbon molecular dynamicsimulation (50ns in explicit aqueous solvent, repeating dockingprotocol for 10 frames separated by 5ns each). A significantchange in the angle of the  -helices and a pore occlusion(quantified with PoreWalker server) could be observed. Underthese conditions, the furosemide binding sites are lost quickly. Tosimulate hAQP1 in an environment that resembles protein includedin a lipid bilayer,  -carbon fixation was used. In such condition, thedistinction between furosemide binding sites remains. Once againthe intracellular binding site was favored. Based on published datain which the presence of Na+ channel ENaC modifies themembrane tension, we also studied the co-expression of hAQP1and ENaC in Xenopus oocytes. The in vitro data plus the possibilityof formation of a protein-protein interaction (that was found usingthe ClusPro server) lead us to hypothesize a physiological andmorphological interaction between the two proteins.