Breakdown of Charged Lipid Asymmetry as a Result of Lipidic Pore Formation

Abstract

Negatively charged lipids are usually located in the inner leaflet of the plasma membrane. Their appearance in the outer leaflet is known to correlate with several physiological and pathological conditions in cells. Understanding how membrane lipids lose their asymmetric transmembrane distribution and achieve their nonrandom distribution in cells is a key challenge in cell biology. Negatively charged lipids do not spontaneously exchange between the two leaflets of a lipid bilayer because the polar headgroups cannot readily cross the hydrophobic membrane interior. We hypothesized that the formation of a transient hydrophilic lipidic pore in the membrane leads to diffusive translocation of negatively charged lipids through the pore to the opposite membrane leaflet. To test this hypothesis, we established a variation of the inner field compensation technique for time resolved measurements of membrane boundary potentials in asymmetric bilayer lipid membranes formed by the Montal-Mueller method. External application of electric fields across the bilayer induced transient conductive states. We observed fast transitions between these different conductance levels, reflecting opening and closing of meta-stable lipidic pores. Comparison of the capacitance minimization potential for different asymmetric membranes before and after pore formation confirmed negatively charged lipids transfer across the bilayer. We also constructed a model governing lipid flow rate based on pore analysis and lipid lateral diffusion rate. Together, our study provides a new tool to monitor loss of membrane asymmetry and our results indicate that lipid transfer can occur through lipidic pores.