Counting EGFP-Marked Ion-Channels of Voltage-Clamped Oocytes to Deduce the Single Channel Gating Charge

Abstract

Recently we indirectly determined the gating charge of single Na-channels (qNa) to be about 6 e (Gamal El-Din et al., 2008). We compared the ratios of total fluorescence intensity to total gating charge (Ft/Qt) of EGFP marked K- and Na-channels expressed in Xenopus laevis oocytes. This allowed to deduce qNa from the better known K-channels (qK = 13-14 e) without the need to know the fluorescence intensity of a single EGFP (Fs). Since we used EGFP to mark and count both channel types, Fs cancelled out in the comparison.We now attempt to determine and apply Fs to obtain the total number of channels (Nt = Ft/Fs) and from that q = Qt/Nt directly. As outlined in the above cited study, Ft was extrapolated to the whole oocyte's surface from a defined region along the circumference in order to minimize contributions from submembraneous channels and cytoplasmic autofluorescence. Micrographs of EGFP solutions of known concentration in a hemocytometer chamber provided a figure for Fs. However, this figure cannot simply be transferred to the situation of the oocyte images because it holds true only for EGFP molecules in frontal areas, accessible for the entire opening of the microscope objective. At lateral areas of the oocyte the fluorescence intensity is attenuated. The attenuation was determined by measuring the fluorescence intensity of control oocytes whose surface was strongly and homogeneously stained with a fluorescent dye such that autofluorescence from the cytoplasm could be neglected. Comparison of the extrapolation from either the lateral or the frontal area to the total fluorescence of the oocyte (Ft) gave an attenuation factor of 3 to 6 depending on the optics. This factor was used to obtain the number of EGFP marked channels.