Water _flux through .a human aquaporin: inhiúition by intracellular furosemide and gating by membrane tension. An invitro and in silico approach

MARCELO OZU; RICARDO A. DORR; FACUNDO GUTIERREZ; M. TERESA POLITI; ROXANA TORIANO

San Miguel de Tucumán

Congreso; XLI Reunión Anual de la Sociedad Argentina de Biofísica; 2012

Sociedad Argentina de Biofísica

This work presents studies on the water permeability properties of human aquaporin 1 (hAQP1) expressed in Xenopus laevis oocyte membranes, using a technique where the cellular content is replaced with a known medium, and with the possibility of measuring internal pressure (Pi). Since specific inhibitors for AQPs are nowadays been searching for, we decided to test the effect of furosemide, a non-toxic drug that is clinically used in humans to induce diuresis. A previous work has demonstrated that intracellular-injected furosemide inhibits AQP4. Thus, we tested the effect of furosemide on hAQP1 but without interference of cytoplasmic content. Our results demonstrate that furosemide is also an intracellular inhibitor of hAQP1. In other experimental series, consequences on water transport produced by well-known anisotonic gradients were also tested. Experiments imposing anisotonic mannitol gradients with constant ionic strength showed that the relationship between water flux and the applied mannitol gradient deflects from a perfect osmometer response. An explanation for this behavior is considering hAQP1 as a membrane tension-gated channel. To test this hypothesis, studies of the elastic properties of the membrane were done, measuring Pi under full-controlled conditions. As result, a function between internal volume (Vi) and Pi was obtained. This function was used to design a model in which the osmotic permeability coefficient (Pf) could be studied as a Pi-dependent variable. The unique parameter of the model is the initial Pf, which was obtained by model-dependent fitting. Results demonstrate that this model can reproduce and predict Vi changes that occur with high hypo-osmotic gradients combined with high membrane water permeability. The Pf values obtained by simulation are similar to those experimentally obtained. The predicted Pi, Vi, and Pf changes indicate that the hAQP1 water channels transit from a high water permeability state to a closed state, with a cooperative effect among monomers. We conclude that hAQP1 is a constitutively open channel, which is inhibited by intracellular furosemide, and closes mediated by membrane tension increments.