Effects of Membrane Potential on the Entry of Cell-Penetrating Peptides Transportan 10 into Single Vesicles

Abstract

Cell-penetrating peptides (CPPs) have an activity to translocate across plasma membranes to enter cytosol of eukaryotic cells without decreasing cell viability. To reveal the mechanism of their translocation, we examined the effect of membrane potential, φm, on the entry of a CPP, transportan 10 (TP10) into the lumen of single giant unilamellar vesicles (GUVs). For this purpose, we used the single GUV method to detect the entry of carboxyfluorescein (CF)-labeled TP10 (CF-TP10) to single GUV lumen. First, we applied various negative membrane potentials on single GUVs containing gramicidin A in their membranes by producing various K+ concentration differences and confirmed these potentials using φ-sensitive fluorescent probe, DiOC6(3). The fluorescence intensity of the GUV membranes (i.e., the rim intensity) due to this dye increased with lφml up to −118 mV, and its dependence on lφml less than −28 mV agreed with a theoretical estimation. Then, we examined the effect of φm on the entry of CF-TP10 into GUVs using single GUVs containing small GUVs or large unilamellar vesicles inside the mother GUV lumen. We found that CF-TP10 entered the GUV without pore formation and the rate of entry of CF-TP10 into the GUV lumen, Ventry, increased with an increase in lφml. The rim intensity due to CF-TP10 also increased with an increase in lφml, indicating that CF-TP10 concentration in the inner leaflet of the GUV increased with lφml. These results indicate that the φm-induced increase in Ventry can be explained by the increase in CF-TP10 concentration in the inner leafletwith lφml. We discussed the mechanism of this effect of membrane potential based on the pre-pore model of the translocation of CF-TP10 across the GUV membrane.