Rapid transbilayer movement of spin-labeled steroids in human erythrocytes and in liposomes

The transbilayer movement and distribution of spin-labeled analogs of the steroids androstane (SLA) and cholestane (SLC) were investigated in the human erythrocyte and in liposomes. Membranes were labeled with SLA or SLC, and the analogs in the outer leaflet were selectively reduced at 4C using 6-O-phenylascorbic acid. As shown previously, 6-O-phenylascorbic acid reduces rapidly nitroxides exposed on the outer leaflet, but its permeation of membranes is comparatively slow and thus does not interfere with the assay. From the reduction kinetics, we infer that transbilayer movement of SLA in erythrocytes is rapid at 4C with a half-time of approximately 4.3 min and that the probe distributes almost symmetrically between both halves of the plasma membrane. We have no indication that a protein-mediated transport is involved in the rapid transbilayer movement of SLA because 1) pretreatment of erythrocytes with N-ethyl maleimide affected neither flip-flop nor transbilayer distribution of SLA and 2) flip-flop of SLA was also rapid in pure lipid membranes. The transbilayer dynamics of SLC in erythrocyte membranes could not be resolved by our assay. Thus, the rate of SLC flip-flop must be on the order of, or even faster than, that of probe reduction rate on the exoplasmic leaflet (half-time approximately 0.5 min). The results are discussed with regard to the transbilayer dynamics of cholesterol.