Effect of a scramblase activator upon lipid and probe scrambling and membrane domain formation in HEK 293T cells

Abstract

Lipid asymmetry, a difference between the lipid composition of the inner and outer leaflets (monolayers) of a membrane, is a characteristic of mammalian plasma membranes. In artificial lipid vesicles, asymmetry can either suppress or induce the formation of coexisting ordered and disordered lipid domains depending on lipid composition. In mammalian plasma membrane preparations, loss of asymmetry induces the formation of ordered domains. In this report, we studied the effect of a scramblase activator, the ionophore BrA23187 (BrA) plus Ca²⁺, upon ordered domain (lipid raft) formation in human embryonic kidney 293T cells. Addition of BrA induced a decrease in FRET between the plasma membrane outer leaflet probe TMADPH, which partly associates with ordered domains, and ODRB, which localizes largely in liquid disordered domains. This is consistent with the formation of coexisting ordered and disordered domains in the plasma membrane. In addition, upon BrA addition, the plasma membrane outer leaflet probe Pro12A exhibited a decrease in the generalized polarization (GP) suggesting a decrease in outer leaflet membrane order, perhaps due to a decrease in outer leaflet cholesterol However, there are other explanations for these observations. To test if BrA induced scrambling of fluorescent membrane probes, which would complicate interpretation of the experiments described above, we measured the effect of BrA upon extractability of outer leaflet probes with MβCD (in most cases, MβCD was more effective for extraction than BSA). These experiments showed that at most a small fraction of probes migrate to the inner leaflet upon addition of BrA. Other experiments raise the possibility that BrA binding to membranes may directly influence ordered domain formation and properties or alter fluorescence by direct interactions with TMADPH, and thus not reflect changes in domain formation.