New approach using Fluorescence Correlation Spectroscopy (FCS) for channel number quantification of aquaporins (AQPs) expressed in Xenopus laevis oocytes

LUCIANO GALIZIA; FLORENCIA GUASTAFERRI; OLIVER GIBSON; AGUSTÍN CAVIGLIA; GABRIELA AMODEO; MARCELO OZU

Buenos Aires

Congreso; Primeras Jornadas Virtuales de la Sociedad Argentina de Biofísica; 2020

Sociedad Argentina de Biofísica

Quantification methods of precise membrane channel number when expressed inXenopus laevis oocytes is an open question. In the field of aquaporin studies, thisdetermination could be useful to estimate the unitary permeability coefficient of an AQP(pf). In early studies this estimation was performed by using cryofracture techniques inorder to quantify the particle density at the membrane. Simultaneously, the macroscopicosmotic permeability coefficient (Pf) is calculated by using videomicroscopy techniques.Then pf is calculated by dividing P f by the number of channels calculated in themembrane surface of the whole cell.In recent years, fluorescence confocal techniques involving fluctuation analysis wereused 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 someAQPs. However, fluorescence correlation spectroscopy (FCS) has been never used toestimate 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 EYFPFaPIP2.1channels expressed in xenopus oocytes. We recorded oocytes without vitellineenvelope and registered fluorescence intensity in both point scan and line scan modes.These recordings were used to construct an autocorrelation function (ACF). Fitting ACF toa diffusion model allows calculation of at least two parameters, the diffusion coefficientof the fluorescent probe and another parameter that is proportional to the number ofchannels (N). The next step will be to improve the measurements to estimate channeldensity and calculate N and Pf in the same oocyte.