Experimental and simulated studies on water and solote permeabilities in AQP-1expresing Xenopus oocytes controlling the intracellular and extracellular media

OZU MARCELO; CHARA OSVALDO; DORR RICARDO; PARISI MARIO

Nara, Japón

Congreso; V Internacional Conference of Aquaporins; 2007

The classical method to measure the osmotic water permeability (Pf), (cm.s-1) in Xenopus oocytes does not allow a direct test on the internal side of the membrane and hence in the cytoplasmic side of the water channel. A new technique allows controlling the composition on both sides of the cellular membrane of emptied-out Xenopus oocytes (Ozu et al, J. Biochem. Biophys. Methods (2005) 63:187-200). Specifically developed software was now used to simulate osmotic responses in Xenopus whole-oocytes during the first 50-sec under a hypotonic challenge and to select the curve that better fitted to the experimental results. We tested AQP1-expressing oocytes with or without external HgCl2. The obtained Pf values were then used to predict the osmotic response for the correspondent emptied-out oocytes. Predicted results did not differ significantly from the experimental ones, validating the technique with AQP1-expressing oocytes. Based on our previously published data (cited before) we also adapted our software to predict the time at which each emptied-out oocyte reached its maximal relative volume (1.26 ± 0.07) before explode (i.e. 80 sec). Actually, in experimental emptied-out AQP1-expressing oocytes reaching the expected maximal volume we observed two responses: oocytes exploding around 50 seconds after the osmotic challenge (short response, SR) and other population which did not explode and tended toward a stationary state (long response, LR). The measured Pf did not differ significantly between both populations (0.050 ± 0.008, n=5 vs. 0.032 ± 0.010, n=4, respectively). We observed that the model that only takes into account water movements across the cellular membrane adjusted to the experimental volume vs time curves of the SR-oocytes. However, this model was not sufficient to explain the experimental LR-oocytes curves. When solute movement was also considered as part of the algorithm, the model significantly adjusted to the latter curve (compared by the Akaike´s criterion). Taking advantage of the new methodology we also tested the effect of HgCl2 from the internal side of the membrane on AQP1-expressing oocytes. In this case, the Pf did not differ significantly from that observed without HgCl2. Again, two SR and LR populations were described sharing properties with AQP1-expressing emptied-out oocytes without HgCl2. In summary, we here present experimental results obtained with a new and powerful technique to study the biophysical properties of water transport. Computer simulated results validate the emptied-out methodology and help to explain the behaviour of AQP1-expressing Xenopus oocytes in the presence of osmotic and solute gradients.