XB-IMG-148469
Xenbase Image ID: 148469
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Fig. 2. KCNE1 inhibits KCNQ1 currents and depolarizes embryonic cells. (A) Tail currents were analyzed at −120 mV and normalized to the value followed after the 60-mV depolarizing pulse to estimate the voltage dependence of channel activation (n = 10 or 11). At voltages of 0 mV or less, addition of KCNE1 reduced the currents compared with KCNQ1 alone. (B) KCNQ1 (5 ng of mRNA) was expressed alone or together with 0.2 ng of KCNE1 mRNA. Each lane was loaded with equal amounts of protein (as estimated by Bradford test and Ponceau staining). Four consecutive biotinylation Western blots detected no clear alterations of KCNQ1 protein in the plasma membrane (densitometric analysis of all four gels normalized to the respective KCNQ1 band gave 1.0061 ± 0.1025 for KCNQ1 + KCNE1 injections). Analysis of membrane potential in vivo using DiSBAC voltage reporter dye reveals that KCNE1 mRNA expression depolarizes cells in the neurulating embryo. (C) Histograms showing normalized frequencies corresponding to normalized fluorescence intensities were created for each embryo (n = 37). One example each of the histograms from controls (injected with mRNA encoding Xenopus Ductin, a subunit of the V-ATPase ion pump) and KCNE1-injected embryos are shown. The peak value is the value of relative intensity corresponding to a frequency of 1.0. Also indicated is the half-maximum (frequency = 0.5) position at which width was measured for statistical comparisons. Area is computed as the peak times the width. These two histograms were chosen because their peak and width values are closest to the mean values; however, there was large variation in shapes of the histograms. (D) Bar chart showing the difference between three descriptors of histograms from xDuctin- and KCNE1-injected embryos. Control xDuctin-injected histograms had a mean peak at 0.255 ± 0.093 (mean ± 99% confidence intervals), a mean width of 0.150 ± 0.042, and a mean area of 0.044 ± 0.030, whereas the mean peak for KCNE1-injected embryos was at 0.786 ± 0.134, the mean width was 0.627 ± 0.192, and mean area was 0.552 ± 0.308. Two-tailed t tests revealed highly significant differences for each comparison: peaks, P = 8 × 10−10; widths, P = 2 × 10−6; areas, P = 4 × 10−4. Control n = 15; KCNE1 n = 22. Image published in: Morokuma J et al. (2008) Copyright © 2008. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View |