Garcia Research Group

Cell Penetrating Peptides

A Mechanism for the Translocation of Cell Penetrating Peptides Across Lipid Bilayers

Figure 1. Binding of the tat-peptides to the lipid phosphate groups.

We have proposed a mechanism for the translocation of the HIV-1 tat-peptide based on molecular dynamics simulations. This mechanism shows how these peptides are able to passively diffuse across the cell membrane (Herce and Garcia, PNAS, 104: 20805-20810 (2007)). The mechanism for translocation can be described as composed of four basic steps (illustrated in Figs. 1-4):

Figure 2. Decrease of the bilayer thickness with the increase in the surface density of the peptide.
Figure 3. Translocation of an arginine side chain across the bilayer
Figure 4. Formation of a transient toroidal pore. The tat-peptide diffuses on the surface of the toroidal pore.
  1. The peptides bind to the surface of the bilayer, attracted by the phosphate groups of the phospholipids. (Fig. 1) This occurs in the 10 ns timescale in MD simulations (at 323 K).
  2. As the surface concentration of peptides increases, the arrangement of lipids is strongly distorted compared to the resting membrane. (Fig. 2) This occurs in a few tens of ns.
  3. At high concentrations of peptides the arginine sidechains do not find enough phosphate groups on the proximal layer within their reach and translocate to the distal layer nucleating the formation of a water pore. (Fig. 3) This occurs in the 100 ns timescale. The fact that the arginine reaches for phosphate groups on the distal layer is energetically possible, since the electric field generated by the charges on the distal layer are poorly screened by the low dielectric medium formed by the lipid carbon chains.
  4. A few lipids translocate (flip) by diffusing on the surface of the pore as the pore closes. (Fig. 4) This occurs in the 500 - 1000 ns timescale. Upon diffusion the peptide carries with it some of the bound lipids, resulting in a mixing of the lipids on the two layers.

Experimental validation of permeabilization of planar phospholipid bilayers and human cells by CPP

Figure 5—Experimental validation of Permeabilization of planar phospholipid bilayers and human cells. Panels a to b show the planar phospholipid bilayer permeabilization induced by addition of CPP. (a) Current versus time. (b) Current as a function of the holding potential before and after adding the peptide to the cis chamber. The phospholipid bilayers are composed of a lipid mixture of DOPC:DOPG (3:1), the ionic concentration is 100mM of KCl and the pH is 7.4. The potential of the cis chamber relative to the trans chamber (the holding potential) is 50mV. The ionic concentration is 100mM of KCl and the pH is 7.4. After the addition of 7µM of Arg-9 to the cis chamber the current increases across the bilayer. The human cells are smooth muscle cells from the human umbilical artery extracted form umbilical cords. Panels c to d show patch-clamp permeabilization measurements on live human cells. (c) Current, averaged over 7 cells, versus time after adding the CPP. (d) Typical current signals before (D(a)), and after (D (b) and D(c) ) adding the peptide.

Molecular dynamics simulations of the translocation of tat-peptides across lipid bilayers suggest that the Arginine rich CPP disturb the lipid bilayer and induce the formation of transient pores on phospholipid membranes. If pores open, ions should be able to move across the membrane and, in presence of a transmembrane potential a net ionic current should be observed. We tested this prediction experimentally on planar phospholipid bilayers using the black lipid membrane method, and on human cells using the patch clamp method. In both cases we see that the peptides destabilize and permeabilize the membranes. The results have been published in: H. D. Herce, A. E. Garcia, J. Litt, R. S. Kane, P. Martin, N. Enrique, A. Rebolledo, and V. Milesi. “Arginine-rich peptides destabilize the plasma membrane, consistent with a pore formation translocation mechanism of cell penetrating peptides.” Biophysical Journal 97, 1917-1925(2009).

Black lipid membrane experiments- The basic idea of the setup for planar phospholipid bilayers is that a bilayer is made on a 100 µm diameter hole that separates two chambers filled with a saline solution. An electrode is introduced into each chamber and creates an electrostatic potential across the membrane. The chamber where the active electrode is introduced is conventionally called the cis chamber and the other chamber is called the trans chamber. In the first column of Figure 5 (a) the permeabilization of a phospholipid bilayer composed of a lipid mixture of DOPC:DOPG (3:1) is shown after the addition of 7µM of Arg-9 to the cis chamber with a potential of 50mV relative to the trans chamber. After the peptide is added to the cis chamber the ionic permeabilization across the membrane increases. The permeabilization increases both continuously and with discrete jumps. In Figure 5 (b) the dependence of current versus voltage before and after adding the peptide is shown.

Patch-clamp experiments on live cells -- Patch clamp experiments have been conducted using smooth muscle cells from the human umbilical artery extracted from umbilical cords. In the second column of Figure 6 (c) the results obtained in the whole cell configuration in which the holding potential is at -50 mV are shown. It can be seen that after the addition of the Arg-9 peptide the current across the cell slowly increases. The average holding current showed a significant increase of the current relative to the control current measured in absence of peptide. The mean increase relative to the control current is 126 ± 45 pA (averaged over 7 cells) for 7 µM of Arg-9. We also observed that in the presence of the peptide, during the recording, the current presents instantaneous jumps like the one shown in Figure 5 (d), which resemble the current jumps observed in planar phospholipid bilayers Figure 5 (a).

Selected Publications:

Retrieved from ""