Figure 4
DPA voltage-dependent fluorescence quenching of hERG channels tagged with FPs at different positions. Representative fluorescence traces obtained in response to voltage step sequences such as those illustrated in Figs 1A and 2A, including a central pulse from −120 to +120 mV, are shown at the top. Data from patch-clamped HEK293 cells expressing hERG channels tagged with CFP, cerulean and YFP at the amino and carboxy ends, and with CFP at positions 162 and 1030 of the channel sequence are shown. Excitation wavelengths of 440 (CFP and cerulean) and 510 nm (YFP) were used. Averaged responses from 50 trials delivered at 1 s intervals are illustrated on the graphs. Expanded sections of the fluorescence traces are shown in the insets. In all cases, vertical and horizontal calibration bars correspond to 5% ΔF/Fo and 20 ms, respectively. Superimposed lines corresponding to mono-exponential fits to the data are shown on the upper file. Results from an analogous experiment using cells expressing a channel construct carrying an amino terminal CCPGCC sequence that was subsequently labeled with a FlAsH fluorophore (see text for details), are also shown. A summary of the DPA-dependent effects obtained with all the tested channel constructs is depicted at the bottom, indicating the number of tested cells and with the fluorophore and the point of insertion indicated below the bars of the histogram. A schematic linear diagram of the hERG channel in which the size of each domain is represented proportional to the total length of the protein is shown in the inset. The position of the boundaries between the different channel domains is marked with numbers. The places at which the fluorescent tags tested here were introduced are also indicated (arrows).
