Replacement of the cytoplasm in an animal cell to study aquaporin-mediated water permeability

MARCELO OZU; RICARDO A. DORR; MARIO PARISI; ROXANA TORIANO

Buzios, Brasil

Congreso; VII Iberoamerican Congress of Biophysics; 2009

SBBF, SOBLA, SAB, LAFEBS, SPB, SEB

Xenopus laevis oocyte is the typical system to study osmotic water permeability (Pf) of membranes expressing water channels (aquaporins). Pf is calculated from changes of the whole oocyte volume during the initial swelling after an extracellular hypotonic challenge. Although this method has been convenient to identify aquaporins and to study some of their biophysical properties, this technique is limited because of the inability to have direct access to and full control over the cytoplasm of the oocyte. In this way, classic water permeability measurements are based on several assumptions valid in aqueous solutions or considering that the cytoplasm is a simple, homogeneous, well-defined solution and an ideal-mixing model. We report an improved method that allows replacing the oocyte cytoplasm and controlling the intracellular pressure in oocytes that express aquaporin-1 (AQP1). We tested the consequences of the cytoplasm disintegration, the imposition of really-known anisotonic gradients and the intracellular effects of aquaporin inhibitors. To analyze water movements triggered by the presence of different mannitol concentrations we propose an analogy with the classical current-potential curves used to describe the properties of ionic channels. The results reflect the bidirectional properties of AQP1, with a symmetrical response when inward and outward water fluxes are compared (Pf = 0.019±0.001 cm.s-1). The oocyte membrane that expresses AQP1 behaves as a perfect osmometer until 100mM manitol gradients, but higher gradients provoke a significant Pf decrement (Pf = 0.011±0.0001 cm.s-1). Since our technique implies the absence of the cytoplasm, metabolic control on AQP1 can be excluded; therefore a direct effect of osmotic gradients on the channel can be proposed.