Large phospholipid-number asymmetry is not required to reproduce plasma membrane physical properties

Plasma membranes are compositionally asymmetric, but whether this lipid-type asymmetry is accompanied by a substantial phospholipid-number imbalance between leaflets remains debated. Here, we use microsecond all-atom molecular dynamics simulations to compare human red blood cell plasma membrane models with either strong phospholipid-number asymmetry and exoplasmic cholesterol enrichment or near-symmetric phospholipid and cholesterol numbers but preserved lipid-type asymmetry. Both asymmetric models reproduce a densely packed, ordered exoplasmic leaflet and a more fluid cytoplasmic leaflet. Strong phospholipid-number asymmetry, however, drives extensive cholesterol enrichment in the exoplasmic leaflet and amplifies membrane asymmetry, leading to large cholesterol-rich clusters, enhanced shallow hydrophobic exposure, reduced exoplasmic lipid mobility, lower ethanol permeability, and increased area compressibility. Comparison with available diffusion and alcohol-permeability measurements indicates that the strongly asymmetric model overestimates the immobilization and barrier properties of the exoplasmic leaflet, whereas the near-symmetric-number model better captures these dynamic observables. Our results suggest that lipid-type asymmetry is sufficient to reproduce many physical hallmarks of plasma membranes without requiring a large phospholipid-number imbalance.