Formation and Characterization of Supported Lipid Bilayers Containing Phosphatidylinositol-4,5-bisphosphate and Cholesterol as Functional Surfaces

Solid-supported lipid bilayers (SLBs) mimicking a biological membrane are commonly used to investigate lipid-lipid or lipid-protein interactions. Simple binary or ternary lipid systems are well established, whereas more complex model membranes containing biologically important signaling lipids such as phosphatidylinositol-4,5-bisphosphate (PI(4,5)P-2) and cholesterol have not been extensively described yet. Here we report the generation of such bilayers and their relevant biophysical properties and in particular the accessibility of PI(4,5)P-2 for protein binding. Ternary mixtures of POPC with 20% cholesterol and either 3 or 5 mol % dioleoyl-phosphatidylinositol-4,5-bisphosphate were probed by employing the quartz crystal microbalance and atomic force microscopy. We show that these mixtures form homogeneous solid-supported bilayers that exhibit no intrinsic phase separation and are characterized by long-term stability (>8 h). Bilayers were formed in a pH-dependent manner and were characterized by the accessibility of PI(4,5)P-2 on the SLB surface as shown by the interaction with the PI(4,5)P-2 binding domain of the cortical membrane-cytoskeleton linker protein ezrin. A time-dependent reduction of PI(4,5)P-2 levels in the upper leaflet of SLBs was observed, which could be effectively inhibited by the incorporation of a negatively charged lipid such as phosphatidylserine. Furthermore, quartz crystal microbalance measurements revealed that cholesterol affects bilayer adsorption to the solid support.