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)P2) 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)P2 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)P2 on the SLB surface as shown by the interaction with the PI(4,5)P2 binding domain of the cortical membrane-cytoskeleton linker protein ezrin. A time-dependent reduction of PI(4,5)P2 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.