Chemical potential measurements constrain models of cholesterol-phosphatidylcholine interactions

ABSTRACT Bilayer membranes composed of cholesterol and phospholipids exhibit diverse forms of non-ideal mixing. In particular, many previous studies document macroscopic liquid-liquid phase separation as well as nanometer-scale heterogeneity in membranes of phosphatidylcholine (PC) lipids and cholesterol. Here, we present experimental measurements of cholesterol chemical potential (μ c ) in binary membranes containing dioleoyl PC (DOPC), 1-palmitoyl-2-oleoyl PC (POPC), or dipalmitoyl PC (DPPC), and in ternary membranes of DOPC and DPPC, adapting a calibrated experimental protocol developed to measure μ c in cells (Ayuyan and Cohen, Biophys. J. 114:904-918). μ c is the thermodynamic quantity that dictates the availability of cholesterol to bind other factors, and notably must be equal between coexisting phases of a phase-separated mixture. It is simply related to concentration under conditions of ideal mixing but is found to be far from ideal for the majority of lipid mixtures investigated. Here we perform experimental measurements of μ c , constraining thermodynamic models of membrane interactions. Our measurements are consistent with models involving cholesterol-phospholipid complexes, but only if complexes are more weakly bound than has been assumed in previous reports. Experimental measurements are also well described by regular solution theory and lattice models with pairwise interactions between components. We find that μ c can vary by ~1.5 k B T at constant cholesterol mole-fraction implying a more than five-fold change in its availability for binding receptors and other reactions. These findings reinforce that μ c depends on membrane composition overall, suggesting avenues for cells to alter the availability of cholesterol without varying cholesterol concentration. SIGNIFICANCE The chemical potential of cholesterol (μ c ) reflects its availability to interact with other molecules. In a complex mixtures this chemical potential can vary dramatically even at fixed cholesterol concentration. In this report, we present measurements of μ c in bilayer membranes composed of mixtures of cholesterol with one or two phospholipids. We find that μ c in these mixtures depends strongly on the phospholipids that are present, with activity varying by a factor of more than five at fixed cholesterol concentration. This suggests that the availability of cholesterol in biological membranes could be tuned without altering cholesterol concentration directly, by adjusting the concentration of other lipid or protein components.