Regulation of phospholipid scramblases by cholesterol

The activity of membrane proteins is often regulated by their lipid environment, either via physical properties of the membrane or by direct interaction with specific lipids. Testing these effects with purified proteins reconstituted into lipid vesicles is challenging because of the compositional heterogeneity of the vesicle sample. Cholesterol influences membrane properties and binds to a variety of membrane proteins, thereby regulating their activity. To study the effect of cholesterol on the phospholipid scramblase activity of G protein-coupled receptors (GPCRs), we developed a protocol to introduce cholesterol into vesicles after reconstituting the protein. This approach sidesteps the problem of having to account for the possible effect of cholesterol on the reconstitution process itself, enabling direct evaluation of the effect of cholesterol on activity. Here we describe the cholesterol loading protocol and how to quantify the amount loaded by colorimetric assays and membrane fluidity measurements. We provide sample data on the effect of cholesterol on GPCRs. Our protocol is broadly applicable and can be used in any study of the effect of cholesterol on a reconstituted membrane protein.