Phase Boundaries in Phosphatidylcholine Membranes Saturated and Oversaturated with Cholesterol

Abstract

Conventional and saturation-recovery EPR along with differential scanning calorimetry (DSC) were used to determine the cholesterol/phosphatidylcholine (Chol/PC) mixing ratio at which cholesterol bilayer domains (CBDs) and cholesterol crystals (CR) start to form in dimyristoyl-PC (DMPC) and 1-palmitoyl-2-oleoy-PC (POPC) membranes. The Chol/PC mixing ratio was changed from 0 to 3. Lipid multilamellar dispersions investigated in this work were prepared using the rapid solvent exchange method to preserve compositional homogeneity throughout the suspension. EPR spin-labeling methods were used with spin-labeled cholesterol analogues (cholestane spin label [CSL] and androstane spin label [ASL]) as well as with a spin-labeled phospholipid (1-palmitoyl-2-(5-doxylstearoyl)phosphatidylcholine (5-PC)) to locate the phase boundary for CBD formation. DSC was used to locate the phase boundary for the formation of CRs. Results showed that in both DMPC and POPC membranes the CBD is already formed at a Chol/PC mixing ratio of 1, while CRs are formed only when the Chol/PC mixing ratio exceeds the value of 2. Previous results and data presented here indicate that when the Chol/PC mixing ratio increases, the PC bilayer becomes saturated with cholesterol first. This phospholipid bilayer, saturated with cholesterol, possesses unique physical properties. Increase in cholesterol content beyond the Chol/PC mixing ratio of 1 causes formation of the CBD, and further increase, beyond the Chol/PC mixing ratio of 2, causes formation of CRs (presumably outside of the membrane). Thus, the phase boundary at a Chol/PC mixing ratio of 1 separates the region with a single liquid-ordered phase and the region with a coexisting liquid-ordered phase and CBD. The cholesterol solubility threshold at a Chol/PC mixing ratio of 2 separates the region with a coexisting liquid-ordered phase and CBD from the region in which CRs are formed.