Assess the nature of cholesterol-lipid interactions through the chemical potential of cholesterol in phosphatidylcholine bilayers

Abstract

Cholesterol plays a vital role in determining the physiochemical properties of cell membranes. However, the detailed nature of cholesterol-lipid interactions is a subject of ongoing debate. Existing conceptual models, including the Condensed Complex Model, the Superlattice Model, and the Umbrella Model, identify different molecular mechanisms as the key to cholesterol-lipid interactions. In this work, the compositional dependence of the chemical potential of cholesterol in cholesterol/phosphatidylcholine mixtures was systematically measured at high resolution at 37 degrees C by using an improved cholesterol oxidase (COD) activity assay. The chemical potential of cholesterol was found to be much higher in di18:1-PC bilayers than in di16:0-PC bilayers, indicating a more favorable interaction between cholesterol and saturated chains. More significantly, in 16:0,18:1-PC and di18:1-PC bilayers, the COD initial-reaction rate displays a series of distinct jumps near the cholesterol mole fractions (chi(C)) of 0.15, 0.25, 0.40, 0.50, and 0.57 and a peak at the cholesterol maximum solubility limit of 0.67. These jumps have been identified as the thermodynamic signatures of stable cholesterol regular distributions. In contrast, no such jumps were evident in di16:0-PC bilayers below chi(C) of 0.57. The observed chemical potential profile is in excellent agreement with previous Monte Carlo simulations based on the Umbrella Model but not with the predictions from the other models. The data further indicate that the cholesterol regular distribution domains (superlattices) are not the hypothesized condensed complexes. Those complexes were mainly implicated from studies on lipid monolayer that may not be relevant to the lipid bilayer in cell membranes.

Fig. 1.

Schematic of the μ_C as a function of χ_C predicted by various models. (a) The Condensed Complex Model predicted a jump in μ_C at a stoichiometric composition (7). (b) The Superlattice Model predicted dips in free energy at superlattice compositions (13), which also implied sharp spikes in μ_C. (c) The Umbrella Model predicted a cascade of jump in μ_C, and each jump corresponds to a stable cholesterol regular distribution (14).

Fig. 2.

Six regular distributions of cholesterol that have been successfully simulated previously by using MC simulation based on the Umbrella Model. Filled circles, cholesterol; open circles, acyl chains of PC. (a) χ_C = 0.154. (b) χ_C = 0.25. (c) χ_C = 0.40. (d) Monomer pattern at χ_C = 0.50. (e) Dimer pattern at χ_C = 0.571. (f) Maze pattern at χ_C = 0.667, which is the maximum solubility limit of cholesterol in PC.

Fig. 3.

COD initial-reaction rate as a function of cholesterol mole fraction in the high-cholesterol region. The shaded bars indicate the locations of expected jumps at 0.50 and 0.571 and the cholesterol maximum solubility limit at 0.667 predicted by the Umbrella Model. The width of the bars reflects the experimental uncertainty in χ_C in our samples (±0.015). (a) Average curves, each obtained from three independent sample sets. (Inset) COD reaction progress curves of DOPC/cholesterol mixtures. The reaction rate is higher at χ_C = 0.60 than that at 0.50. (b) Individual curves. The jumps at 0.5 and 0.57 appear sharper.

Fig. 4.

COD initial-reaction rate as a function of χ_C in the low-cholesterol region. The shaded bars indicate the locations of expected jumps at 0.154, 0.25, and 0.40 predicted by the Umbrella Model. The width of the bars reflects the experimental uncertainty in χ_C.