A Combined Effect of Proteins SP-B and SP-C and Membrane Curvature on Cholesterol Partition in Lung Surfactant Membranes: Answers from Fluorescence

Abstract

Cholesterol is the major neutral lipid present in lung surfactant, a lipid-protein complex responsible for the stabilization of the air-liquid interface in mammalian lungs, which is essential for the breathing function. It has been proposed that cholesterol content is tightly regulated in surfactant and that protein SP-C could have a role in this respect. To further analyse the affinity of surfactant membranes for cholesterol in the absence or presence of proteins, we have used cyclodextrin as a cholesterol solubilising agent and a fluorescent analog of cholesterol, cholestatrienol (CTL), whose partition can be followed in membrane vesicles with different lipid/protein compositions and sizes. Our results suggest that there is a direct effect of vesicle size/curvature on the affinity of membranes for CTL. This is translated into lower partition coefficients (Kx) for membranes with higher curvatures, possibly caused by an increased exposure of CTL in more curved bilayers. Surfactant hydrophobic proteins SP-B and SP-C were found to have a curvature-independent effect on CTL mobility, as well as in the mobility of other probes such as transparinaric acid-sphingomyelin (tPA-SM) or diphenylhexatriene-phosphatidylcholine (DPH-PC) in membranes of the same composition. Furthermore, in the absence of SP-B, SP-C-containing liposomes were found to spontaneously split into 30 nm vesicles increasing membrane curvature regardless lipid composition, which correlated with a reduction of Kx compared to pure lipid systems. Taken all together, our results indicate that curvature alters cholesterol accessibility in surfactant lipid bilayers and that lung surfactant proteins disturb membranes in cholesterol containing domains, which may have important physiological consequences for cholesterol mobilization in lung surfactant membranes and films.