Membrane permeabilization by α-helical peptides: a flow cytometry study

Abstract

The permeabilization by α-helical peptides of nucleated mammalian cells can be monitored by flow cytometry. Ethidium bromide, a non fluorescent and poorly membrane permeant molecule, becomes strongly fluorescent only upon binding to DNA. On this basis, the permeabilization of the plasma membrane of HL60 promyelocytic cells induced by α-helical peptides such as melittin, succinylated melittin and anionic peptides derived from the N-terminus of HA2 subunit of the influenza virus hemagglutinin, was measured. Melittin (GIGAVLKVLTTGLPALISWIKRKRQQ-NH 2) caused a rapid (< 5 min) and dose-dependent (ED 50 = 0.5 μM) permeabilization of HL60 cells at neutral pH, whereas the succinylated derivative induced cell permeabilization only at pH below 4.5 with an ED 50=18 μM. The permeabilization by the anionic E5CA peptide (GLFEAIAEFIEGGWEGLIEGCA) containing 5 glutamic residues occurred (ED 50 = 11 μM) at pH ranging from 6.5 to 6.0; replacing the tryptophan residue in position 14 by a phenylalanine residue decreased by about 1 unit the pH at which membrane permeabilization was effective. The membrane permeabilization activity of the E5CA peptide was reversibly abolished when the peptide was linked to a protein carrier. These results show that α-helical peptide-induced membrane permeabilization can be easily monitored by using flow cytometry in the presence of a non permeant dye. This method allows a rapid screening and an efficient mean of selection of peptides suitable to induce membrane permeabilization.