Cation-Selectivity of a Self-Assembled Peptide Pore in Planar Phospholipid Bilayers using Electrical Impedance Spectroscopy

Abstract

GALA is a 30-residue, amphipathic peptide that self-assembles into multimeric, transmembrane pores in a pH-dependent fashion. Based on kinetic data and dye release assays, previous studies have reported that in liposomes GALA forms large multimeric pores composed of 8-12 peptides, but no direct structural information is available. In this study, we characterize the pore size and multimeric structure of GALA pores in planar phospholipid bilayers using electrical impedance spectroscopy (EIS) and molecular dynamics (MD) simulations. Specifically, we used a number of alkaline earth cations and the large organic cations, Choline and TEA, to estimate the pore size by measuring concentration-dependent changes in membrane conduction. This data was combined with MD simulations to estimate the multimeric state of GALA pores. We report that in planar tethered lipid bilayers composed of POPC, GALA pores likely consist of six peptide monomers rather than eight to twelve monomers as previously reported in liposomes. We further demonstrate, for the first time, that GALA exhibits cation selectivity, which is based on the free energy of hydration of the ions. We propose that the difference in predicted pore structure between planar bilayers and lipid vesicles exemplifies the importance of phospholipid bilayer structure, which include lipid packing, area per lipid, hydration or charge distribution caused by asymmetry between the inner and outer bilayer leaflets. These bilayer properties could have significant effects on the aggregation of transmembrane helical structures.