Electrophysiological measurement of translocation of LF through a single pore. A horizontal lipid bilayer system was used, which separates compartments with different pH values; pH 5.6 for the upper (cis) chamber and pH 6.6 for the lower (trans) chamber as described previously (Groulx et al., 2010; see Materials and methods). (A) Translocation of LF through PA63. To test function of the proteins and verify translocation of LF through PA63, PA63 was added to the cis chamber, and once a macroscopic current at 80 mV was observed, the voltage was switched to 20 mV and LF or LFN was added (arrow). At 20 mV, LF will block the pore without being translocated. Blockage of the pore was evident by reduced current. Switching the membrane potential back to 80 mV enabled translocation of LF/LFN through the pore, and once complete, the current increased again. (B) Translocation times for analysis were determined from single-channel recordings; example traces of single translocation events are shown for LFN (top) and LF (middle and bottom). The dashed lines mark the translocation event, and the arrows indicate short (<1 s) spontaneous closings that were also observed in the absence of LF/LFN and were thus ignored in the analysis. The dotted lines indicate the blocked PA. (C) The duration of single pore closings (translocations) at various concentrations of full-length LF is shown in a cloud plot. Each point represents a single measurement such as shown in B. Pore closure times at high concentrations were statistically different to the lower concentrations of LF (P < 0.05). (D) Pore closure times for LFN showed no concentration dependence, and the data were pooled. Closure times for LFN were statistically different from those of full-length LF (P < 0.005). Statistical significance was tested with one-way ANOVA.
