CONICET | Buscador de Institutos y Recursos Humanos

Quantification methods of precise membrane channel number when expressed in Xenopus laevis oocytes is an open question. In the field of aquaporin studies, this determination could be useful to estimate the unitary permeability coefficient of an AQP (pf). In early studies this estimation was performed by using cryofracture techniques in order to quantify the particle density at the membrane. Simultaneously, the macroscopic osmotic permeability coefficient (Pf) is calculated by using videomicroscopy techniques. Then pf is calculated by dividing Pf by the number of channels calculated in the membrane surface of the whole cell.In recent years, fluorescence confocal techniques involving fluctuation analysis were used to evaluate the number of channels at the membrane of mammalian cells , proteoliposomes or GUVs, and these estimations have been used to estimate pf of some AQPs. However, fluorescence correlation spectroscopy (FCS) has been never used to estimate the number of AQPs expressed in Xenopus laevis oocytes.The aim of this work was to use the FCS approach to estimate the number of EYFP-FaPIP2.1 channels expressed in xenopus oocytes. We recorded oocytes without vitelline envelope and registered fluorescence intensity in both point scan and line scan modes. These recordings were used to construct an autocorrelation function (ACF). Fitting ACF to a diffusion model allows calculation of at least two parameters, the diffusion coefficient of the fluorescent probe and another parameter that is proportional to the number of channels (N). The next step will be to improve the measurements to estimate channel density and calculate N and Pf in the same oocyte..