Abstract
Artificial peptides P4, A1 and A4 are homologous to amphipathic α-helical fragments of the influenza virus M1 protein. P4 and A4 contain the cholesterol recognition sequence CARC, which is absent in A1. As shown previously, P4 and A4 but not A1 have cytotoxic effects on some eukaryotic and bacterial cells. This might be caused by the dysfunction of cholesterol-dependent cellular structures, inhibition of the respiratory chain, or disruption of the membrane. Here, we analyzed the latter hypothesis by studying the uncoupling effect of the peptides on asolectin membranes. The influence of A4 on Δψ pre-formed either by the valinomycin-dependent K(+) diffusion or by the activity of membrane-built cytochrome c oxidase (CcO) was studied on (proteo)liposomes. Also, we investigated the effect of P4, A1 and A4 on liposomes loaded with calcein. It is found that A4 in a submicromolar range causes an immediate and complete dissipation of diffusion Δψ across the liposomal membrane. Uncoupling of the CcO-containing proteoliposomes requires an order of magnitude of higher peptide concentration, which may indicate the sorption of A4 on the enzyme. The presence of cholesterol in the membrane significantly weakens the uncoupling. Submicromolar A4 and P4 cause the release of calcein from liposomes, indicating the formation of membrane pores. The process develops in minutes and is significantly decelerated by cholesterol. Micromolar A1 induces pore formation in a cholesterol-independent manner. We conclude that the peptides P4, A4 and, in higher concentrations, A1 form pores in the asolectin membrane. The CARC-mediated interaction of A4 and P4 with cholesterol impedes the peptide oligomerization necessary for pore formation. The rapid uncoupling effect of A4 is apparently caused by an increase in the proton conductivity of the membrane without pore formation.