Membrane potential-driven translocation of a lipid-conjugated rhodamine

The present study demonstrates that the permanently positively charged, lipid-conjugated rhodamine, R18, can be transported from the outer to the inner leaflet of lipid bilayers in response of a transmembrane potential (negative inside). This conclusion was based on the following observations. (i) A fast decrease of the R18 fluorescence, when present at self-quenching concentrations in DOPC large unilamellar vesicles, was revealed upon induction of a valinomycin-induced K(+)-diffusion potential. (ii) Iodide quenching experiments demonstrated that R18 was no longer accessible to externally added aqueous quencher after application of a transmembrane potential. (iii) 2H-NMR measurements, using DOPC, specifically deuterated at the alpha-position of the phosphocholine head group, revealed a massive transbilayer movement of R18 upon induction of a membrane potential. The extent of the fluorescence changes were found to be dependent on the magnitude of the applied transmembrane potential, which opens possibilities for novel applications of R18 as an internal lipid-conjugated membrane potential probe.