Studies of a Channel-Forming Peptide Inserted into Liposomes formed by POPC:POPS and POPC:POPE

Abstract

Peptide-based channel replacement therapy attempts to use channel-forming peptides to establish new ion conductive pathways through the cell membrane. In the present study, a 22 residue amino acid peptide derived from the second transmembrane segment of the alpha-1-subunit of the glycine receptor (M2GlyR) was investigated because it has previously shown high conductance as a channel-forming peptide in MDCK cells monolayers in Ussing chamber experiments.As membrane models, liposomes formed by different phospholipids common to mammalian plasma membranes were made by extrusion at different ratios and then the ion channel peptide was added into the artificial bilayer in order to study the peptide-lipid interactions. These mixtures were analyzed using Trp fluorescence blue shifts, which is an indication of peptide insertion, Transmission Electron Microscopy (TEM) as well as Circular Dichroism (CD).The Trp fluorescence results showed the highest blue shift for POPC:POPS and POPC:POPE at 7:3 and 6:4 ratios respectively. These results indicated that the peptide was inserted into the artificial bilayer in a phospholipids’ ratio and composition dependent manner. TEM analysis showed the formation of Small Unilamellar Vesicles (SUVs) and Large Unilamellar Vesicles (LUVs) in a size range of 80-120 nm with both systems. The circular dichroism spectra showed an alpha helical content for the peptide without liposomes under 40% TFE and SDS. However, the CD for the peptide/liposomes mixtures showed predominantly random coiled structures.Taken together, these results suggest that an artificial membrane model containing different phospholipids can be a useful tool of studying this channel-forming peptide insertion and peptide/lipid interaction.