Ion gradient-induced membrane translocation of model peptides

Abstract

The K+ diffusion potential-induced association of synthetic model peptides carrying a single positive charge originating from the NH2-terminal amino function with large unilamellar vesicles (LUV) consisting of phosphatidylcholine (PC) has been reported previously (de Kroon, A. I. P. M., J. de Gier, and B. de Kruijff. 1989. Biochim. Biophys. Acta. 981:371-373). To determine the vesicle localization of the associated peptides, fluorescence measurements utilizing the peptides' tryptophan residue as intrinsic fluorescent probe were performed. The application in these measurements, of vesicles that exhibit an asymmetric transbilayer distribution of brominated PC which is a quencher of tryptophan fluorescence, unequivocally demonstrated that the peptide H3N(+)-AIMLWA-Ome (AIXme+) is accumulated in the interface of the inner leaflet of the vesicle membrane in response to the valinomycin-induced K+ diffusion potential (negative inside). The relative contributions of the membrane potential (delta psi) and the pH gradient (delta pH, acidic inside) induced by the K+ diffusion potential, to the process have been assessed. An analysis of the pH and delta pH dependencies of the process demonstrated that the K+ diffusion potential-induced peptide accumulation is largely determined by a redistribution of peptide according to the transbilayer pH gradient, in agreement with a translocation across the vesicle membrane of the neutral, deprotonated form of the peptide. The general validity of the mechanism proposed for the vesicle-uptake of AIXme+ has been examined by extending the experiments to peptide analogues with a single negative charge and to peptides with two positive charges, and by investigating the effect of incorporating the acidic phospholipid cardiolipin (CL) into the LUV. The incorporation of CL appeared not to affect the K+ diffusion, potential-induced vesicle uptake of AIXme+. The peptide H3N(+)-RMLWA-Ome (RXme2+) showed a small delta pH independent fluorescence response to the delta psi upon raising the CL content of the vesicles to 25%.

Studies on the cellular uptake of substance P and lysine-rich, KLA-derived model peptides

“…This model is similar to the formation of 'inverted micelles' (Derossi et al, 1996). Several groups have claimed that the presence of a transbilayer potential mediates the translocation of cationic peptides (Binder and Lindblom, 2003;de Kroon et al, 1991;Maduke and Roise, 1993;Terrone et al, 2003). Thoren et al (2000) reported that penetratin translocates lipid bilayers of giant unilamellar vesicles without pore formation, even though the uptake kinetics (fluorescently labelled peptide in the interior of the vesicles was observed after less than 15 min) is seemingly not representative of the very rapid uptake of SP and KLA-derived peptides observed in CLSM studies with live cells, as discussed above.…”

Nuclear Localization of Cell-Penetrating Peptides Is Dependent on Endocytosis Rather Than Cytosolic Delivery in CHO Cells

“…MAP may be innately more resilient against degradation, possibly due to the hydrophobic character. Upon endocytosis MAP may coat or imbed itself into the inner leaflet of the endosomal membrane, making it less accessible to proteases and potentially facilitate its endosomal escape 26 .…”

“…Upon endocytosis MAP may coat or imbed itself into the inner leaflet of the endosomal membrane, making it less accessible to proteases and potentially facilitate its endosomal escape. 29 The internalization and nuclear transport of YG(R) 9 following treatment with lysosomotropic amines showed slightly different results. Ammonium chloride treatment slightly decreases nuclear localization the highly transduced peptide YG(R) 9 , which was expected to remain unaffected since it was previously observed that ammonium chloride had no effect on the nuclear localization of this peptide.…”

Antimicrobial peptides: New candidates in the fight against bacterial infections

“…61 Finally, the large inside negative transmembrane potential of bacterial cells 62 may also facilitate the formation of ion channels. 63,64 Despite all these factors, we should note that there are a number of bacterial species that have already developed resistance to the toxicity of AMPs. One of the major resistance mechanisms is the modification of the bacterial cell envelope components 65,66 (e.g., incorporation of lysine into phosphatidylglycerol).…”