GALA is a 30-residue, amphipathic peptide that self-assembles into multimeric, transmembrane pores in a pH-dependent fashion. In this study, we characterize the size, multimeric structure and cation selectivity of GALA pores in planar phospholipid bilayers using electrical impedance spectroscopy and molecular dynamics simulations. We demonstrate that in planar bilayers GALA pores are likely formed by six peptide monomers rather than eight to twelve monomers as previously reported in lipid vesicles. Besides, we show that in planar bilayers GALA pores exhibit previously unreported cation selectivity. We propose that the difference in predicted pore structure between planar bilayers and lipid vesicles exemplifies the importance of phospholipid bilayer structural properties on the aggregation of transmembrane helical structures.

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Swept frequency electrical impedance spectroscopy (EIS) can be used in conjunction with tethered bilayer lipid membranes to monitor the membrane permeability of ions in real-time (Deplazes et al. J Phys Chem Lett 11:6353-6358, 2020). Conductance readings, as determined by EIS, are a measure of the ability of ions to be transported across membranes. Recording the change in conductance as a function of cation concentration and a comparison between a range of cations permits conclusions to be made about the specificity of cation transport through pores. An estimate for upper pore size and cation selectivity of ion channels can be established using this method.