Abstract
The pore forming properties of synthetic cecropin-melittin hybrid peptide (Acetyl-KWKLFKKIGAVLKVL-CONH2; CM15) were investigated by using photoreceptor rod outer segments (OS) isolated from frog retinae obtained by using the whole-cell configuration of the patch-clamp technique. CM15 was applied (and removed) to (from) the OS in ~50 ms with a computer-controlled microperfusion system. Once the main OS endogenous conductance was blocked with light, the OS membrane resistance was ≥1 GΩ, allowing high resolution, low-noise recordings. Different to alamethicines, CM15 produced voltage-independent membrane permeabilisation, repetitive peptide application caused a progressive permeabilisation increase, and no single-channel events were detected at low peptide concentrations. Collectively, these results indicate a toroidal mechanism of pore formation by CM15.
Highlights
The antimicrobial peptides form an essential part of the innate immunity against pathogens; many peptides induce bacterial death by permeabilising their plasma membrane to the point of inducing cell lyses
From the barrel-stave mechanism, the peptides are always associated with the lipid head groups, even when they are perpendicularly inserted in the lipid bilayer
The photoreceptor rod outer segments (OS) mechanically isolated from Rana esculenta have been found suitable to carry on this study, because of their large size (Figure 2, Right panel) and for the commercial availability and low cost of this frog species
Summary
The antimicrobial peptides form an essential part of the innate immunity against pathogens; many peptides induce bacterial death by permeabilising their plasma membrane to the point of inducing cell lyses. The computer controlled microperfusion system, developed in collaboration with an industrial partner (De Angelis s.r.l., Genoa, Italy; Figure 2), allowed us to apply and remove ions, drugs and peptides on the cells in ~50 ms with an accurately controlled timing This allowed to study, besides the ion channel characteristics (such as the presence of gating, its voltage dependence, the unitary conductance, and the selectivity and blockade [8]), the dynamics of pore formation as well, that are of key importance to assess the channel performance and its potential biotherapeutic activity
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