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

Cell-penetrating peptides (CPPs) can translocate across plasma membranes to enter the cytosol of eukaryotic cells without decreasing cell viability. We revealed the mechanism underlying this translocation by examining the effect of membrane potential, phi(m), 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) into the lumen of single GUVs. First, we used various K+ concentration differences to apply different negative membrane potentials on single GUVs containing gramicidin A in their membrane and confirmed these potentials using the phi(m)-sensitive fluorescent probe 3,3'-dihexyloxacarbocyanine iodine. The fluorescence intensity of the GUV membranes (i.e., the rim intensity) due to 3,3'-dihexyloxacarbocyanine iodine increased with vertical bar phi(m)vertical bar up to 118 mV, and its dependence on vertical bar phi(m)vertical bar less than 28 mV agreed with a theoretical estimation (i.e., the dye concentration in the inner leaflet of a GUV is larger than that in the outer leaflet according to the Boltzmann distribution). We then examined the effect of (phi(m) 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 lumen without pore formation and the rate of entry of CF-TP10 into the GUV lumen, V-entry, increased with an increase in 194. The rim intensity due to CF-TP10 increased with an increase in vertical bar phi(m)vertical bar, indicating that the CF-TP10 concentration in the inner leaflet of the GUV increased with vertical bar phi(m)vertical bar. These results indicate that the phi(m)-induced elevation in V-entry can be explained by the increase in CF-TP10 concentration in the inner leaflet with vertical bar phi(m)vertical bar. We discuss the mechanism underlying this effect of membrane potential based on the pre-pore model of the translocation of CF-TP10 across a GUV membrane.