Measuring water permeability under controlled conditions on both sides of the membrane in native and aquaporin-1 injected emptied-out Xenopus oocytes

MARCELO OZU; RICARDO DORR; GABRIELA AMODEO; CLAUDIA CAPURRO; MARIO PARISI

Bruselas

Congreso; IV International Conference on Aquaporins; 2005

International Union of Biochemistry and Molecular Biology

Membrane water permeability is usually calculated from volume changes in Xenopus laevis oocytes during external osmotic challenges. Nevertheless, this approach is limited by the uncertainty on the oocyte internal composition. To circumvent this limitation a new experimental approach is introduced where the cell membrane of an emptied-out oocyte was mounted as a diaphragm between two chambers. First, the oocyte was fixed to an acrylic support. In a second step the oocyte was perforated across a hole present on the mentioned support and fully or partially emptied-out. The rest of the cytoplasmic membrane remained intact separating two independent, rigid and hermetic chambers. In its final configuration the oocyte membrane was part of a closed compartment and net water movements induced swelling or shrinking of it. Volume changes were followed by videomicroscopy and digitally recorded. Therefore, water movements could be continuously monitored while controlling chemical composition and hydrostatic pressure on both sides of the membrane. Using this novel experimental set up an increasing hydrostatic pressure gradient was applied to both mature (stage VI) and immature (stage IV) oocytes. The relative maximal volume change tolerated before disruption was similar in both cases (1.26±0.07 and 1.27±0.03 respectively) and similar to those previously reported under maximal osmotic stress. Nevertheless the osmotic permeability coefficient (x 10-3 cm/s) in mature oocytes (1.72±0.58, n=6) was significantly lower than in immature oocytes (5.18±0.59, n=5; p