Figure 2
Voltage-dependent DPA-induced effects on patch-clamped HEK293 cells expressing plasma membrane-targeted green fluorescent protein (GFPf) carrying a farnesylation tag. (A) Time course of the fluorescent response on voltage pulses (shown on top of the traces) from −120 to +120 mV in the presence of 10 μM DPA. The time at which the monochromator wavelength was changed from 470 nm (corresponding to a zero volt input) to 498 nm and vice versa, is marked as ON and OFF, respectively. Note that no fluorescence is detected at 470 nm due to the cutoff properties of the filter set employed. For details, see Methods. Averaged responses (in arbitrary units) from 30 trials delivered at 1 s intervals are shown. The superimposed line corresponds to a mono-exponential fit to the data. A fluorescence image of the cells (100x oil-immersion objective) demonstrating the prominent plasma membrane distribution of the GFPf signal is shown on the right. (B) Voltage dependence of the DPA-induced effects. A family of superimposed fluorescence traces in response to voltage steps as illustrated at the top including a central pulse from −120 to +120 mV at 10 mV intervals is shown on the upper left panel. Only results from pulses every 20 mV are shown on the graph for simplicity. For every voltage, traces correspond to averaged responses from 10 repetitive trials delivered at 1 s intervals. Separate traces obtained at +120, 0 and −120 mV are shown on the right. A plot of ΔF/Fo versus voltage, obtained from pulse responses such as those at the top, is shown at the bottom. Averaged responses from four individual cells are illustrated in the graph. A Boltzmann curve fitted to the data with V1/2 = −38 mV is also shown.
