Biomedical Engineering Reference
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Nevertheless, they illustrate the possible complexity of the translocation mecha-
nism: the simulations showed an accumulation of peptides at the boundary of the
polar region that lead to bilayer destabilization and pore nucleation which was con-
sistent with the carpet model. But they also suggested that the entry of the peptides
was lead by the phosphate-basic residue interactions and that the first step of the
pore nucleation is the entry of the CPP in the bilayer hydrophobic core to create an
interaction with distant phosphates: this is more in tune with the concepts developed
in the hydrophobic counterion model mentioned above. This emphasizes the fact
that the classification presented here distinguishes models for clarity purposes but
their borders may be permeable. Another illustration of this complexity is the lipid
segregation model. It starts similarly to the carpet model with accumulation of pep-
tides on the membrane surface. But if the peptide binds preferentially to certain
lipids (such as anionic lipids), this binding is likely to entail domain formation or
more generally a modification of the lateral organization of the lipids in the mem-
brane. These domains may show packing defects at their boundaries and these sites
are likely to be more favorable to peptide entry in the membrane or even act as
nucleation sites for pore formation. This model again, suggested for AMPs (Epand
et al.
2006
), may be relevant for CPPs. Recent DSC measurements have shown that
penetratin, by segregating cardiolipin in a DDPC/cardiolipin mixtures, was able to
induce the formation of domains in an otherwise homogenous membrane (Joanne
et al.
2009
). Finally, another model related to the carpet model is the electroporation
mechanism proposed by Lindblom and collaborators (Binder and Lindblom
2003
):
they propose that the destabilization of the membrane by the CPP carpet is due (in
the case of penetratin) to the asymmetrical distribution of the charged CPPs between
the outer and inner surfaces of the bilayer causing a transmembrane electrical field,
which alters the lateral and the curvature stresses acting within the membrane.
Certain mechanisms include the formation of an aqueous pore following CPP
addition to a membrane. This step is somewhat easier to check experimentally
because it can manifest itself trough leakage of hydrophilic markers or ionic current
flow. Lactate dehydrogenase release assays conducted on HeLa cells incubated 3 h
with 100 mM with Tat, HIV1-rev-(34-50) or arginine octamers showed no signifi-
cant leak. Absence of leakage of CHO cells treated with penetratin or RL16
(RRLRRLLRRLLRRLRR) was confirmed with a similar assay (1 h, 10 mM)
(Joanne et al.
2009
). However electrophysiological experiments conducted on
DOPC: DOPG 3:1 planar bilayers and HUA smooth muscle cells in presence of
arginine nonamers showed significant ionic current revealing membrane perme-
ation (Herce et al.
2009
). These results seem controversial but must be compared
with the sensitivity of the technique in mind (electrophysiological measurements
being more sensitive). It seemed to date that major leaking induced by CPPs can be
ruled out while the possibility of transient, rapid, small aqueous pores cannot.
Finally, another question often mentioned in the literature regarding the mecha-
nism of CPP entry is that of the driving force for the uptake of the peptides. This role
is generally attributed to the transmembrane potential across cell membrane, a natural
candidate for these polycationic peptides. Experiments on vesicules showing mem-
brane potential dependant translocation of penetratin are consistent with this role of
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