Ferutinin can form transient pores in lipid membranes in the presence of magnesium or calcium ions

Abstract

The natural phytoestrogen ferutinin acts as a divalent cation-selective ionophore, promoting the opening of Ca²⁺-activated permeability transition pore (PTP) in mitochondria. Here, we found that Mg²⁺ cations, known to suppress PTP, markedly augmented the ferutinin-induced permeabilization of rat liver mitochondria. To uncover the permeabilization mechanism, we measured calcein flow from large (LUV) and giant (GUV) unilamellar vesicles. In both cases, the ferutinin-induced calcein efflux was significantly enhanced in the presence of Ca²⁺ or Mg²⁺. Calcein influx into GUV stopped within 15-min incubation with ferutinin and Ca²⁺. In electrophysiological experiments, ferutinin caused a smooth increase in the electrical current through planar bilayer lipid membranes (BLM) in the presence of Ca²⁺ or Mg²⁺, followed by burst of fluctuations in the current at its high level. In patch-clamp BLM experiments, we detected transient channel activity upon the addition of ferutinin together with Ca²⁺, diminishing over minutes time scale. We propose permeabilization mechanism which involves adsorption of ferutinin on lipid membranes, its dimerization upon binding Mg²⁺ or Ca²⁺, leading to mechanical stress induction, and ultimately to membrane pore formation. The mechanical stress is then gradually relieved due to translocation of ferutinin dimers and lipid molecules to the opposite lipid monolayer, resulting in pore closure.