Plasma Membrane Lipid Asymmetry Dynamically Regulates Passive Diffusion

Plasma membrane (PM) phospholipid asymmetry is a fundamental feature of eukaryotic cells that regulates signaling, trafficking, and cell fate. Whether it also controls passive diffusion across the PM remains unknown. Here, we show that phospholipid asymmetry is a critical determinant of PM permeability to hydrophobic molecules. Using genetic and biochemical approaches, we demonstrate that disruption of lipid asymmetry by the Ca^{2 +}-activated scramblase TMEM16F or apoptotic scramblase XKR8 profoundly alters the permeation and intracellular accumulation of diverse hydrophobic compounds, including widely used fluorescent probes, while preserving the barrier to membrane-impermeable molecules. Lipid scrambling is sufficient to remodel membrane permeability to lipophilic molecules, alter membrane biophysical properties, and differentially affect their intracellular accumulation, suggesting selective regulation of passive diffusion rather than nonspecific membrane leakage. Physiological lipid scrambling during T cell activation was also accompanied by altered membrane permeability. Moreover, lipid asymmetry regulates the cellular entry of doxorubicin, 17β-estradiol, and all-trans retinoic acid. Together, our findings identify PM lipid asymmetry as a previously unrecognized regulator of membrane permeability and establish a link between membrane organization and molecular transport.