Macromolecular Crowding Affects Voltage-Dependent Alamethicin Pore Formation in Lipid Bilayer Membranes

Abstract

Macromolecular crowding is known to modulate chemical equilibria, reaction rates, and molecular binding events, both in aqueous solution and at lipid bilayer membranes, natural barriers which enclose the crowded environments of cells and their subcellular compartments. Previous studies on the effects that macromolecular crowding in aqueous compartments have on conduction through membranes have focused on single-channel ionic conduction through previously formed pores at thermodynamic equilibrium. Here, the effects of macromolecular crowding on the mechanism of pore formation itself were studied using the droplet interface bilayer (DIB) technique with the voltage-dependent pore-forming peptide alamethicin (alm). Macromolecular crowding was varied using 8 kDa molecular weight polyethylene glycol (PEG8k) or 500 kDa dextran (DEX500k) in the two aqueous droplets on both sides of the bilayer membrane. In general, voltage thresholds for pore formation in the presence of crowders in the droplets decreased compared to their values in the absence of crowders, due to excluded volume effects, water binding by PEG, and changes in the ordering of water molecules and hydrogen-bonding interactions involving the polar lipid headgroups. In addition, asymmetric crowder loading (e.g., PEG8k/DEX500k on either side of the membrane) resulted in transmembrane osmotic pressure gradients that either enhanced or degraded electric field induced insertion of alm monomers into the membrane and the subsequent formation of conductive pores.