Interleaflet Translocation of Second-Harmonic-Generation-Active Dye Molecules in Phospholipid Bilayers with Transmembrane Pores

Second harmonic generation (SHG) measurements using SHG-active dye molecules have recently attracted attention as a method to detect the formation of pores in phospholipid bilayers. The bilayers, in which the dye molecules are embedded in the outer leaflet, exhibit a noncentrosymmetric structure, generating SHG signals. However, when pores form, these dye molecules translocate through the pores into the inner leaflet, leading to a more centrosymmetric structure and the subsequent loss of the SHG signals. A decrease in the SHG signals has been experimentally observed in membranes subjected to electrical stimuli. However, the characteristics of the interleaflet translocation of SHG-active dye molecules through pores remain unclear, hindering quantitative estimation of the membrane conditions, such as the pore size and density, based on the SHG signal reduction. In this study, we investigated the interleaflet translocation characteristics of Ap3, an SHG-active dye molecule, using molecular dynamics (MD) simulations and two-dimensional random-walk (RW) simulations. The MD simulations revealed that Ap3 molecules only translocate between the leaflets along the pore sidewalls. We determined the lateral diffusion coefficient of Ap3 within the membrane plane and its propensity for interleaflet movement at the pore wall. Based on these movement characteristics, the RW model successfully reproduced the characteristic time scale of the interleaflet translocation observed in the MD simulations. By varying the pore size and density in the RW simulations, we estimated that the characteristic time scale of interleaflet translocation depends on the −0.31 power of the pore radius and the −1.13 power of the pore density. Using these findings, we estimated the number of pores that probably formed in membranes during previous electroporation experiments. These results indicate the potential of optical measurement of the dye molecule movement for the indirect quantitative estimation of the pore size and number, which are challenging to measure optically.