Insights Into the Molecular Requirements for Cholesterol Binding to Ion Channels

The concept that cholesterol binds to proteins via specific binding motifs, and thereby modulates their function, has emerged two decades ago. When we recently embarked on studies to uncover the putative binding region(s) of cholesterol in the Kir2.1 channel, we carried out an unbiased approach that combines computational and experimental methods. This approach resulted in the identification of novel cholesterol-binding regions distinct from known cholesterol-binding motifs. In recent years, a plethora of structures of proteins complexed with cholesterol have been determined revealing variegated cholesterol-binding regions that can provide invaluable insights into the prerequisites for cholesterol binding. Thus, using this database of structures, the goal of this chapter is to present a comprehensive analysis of representative cholesterol-binding regions, and thereby determine the molecular requirements for cholesterol binding. The analysis demonstrates that the primary requirement for cholesterol binding is a highly hydrophobic environment, and that the interaction with the cholesterol molecule can be stabilized by stacking interactions between its ring structure and hydrophobic aromatic residues, and by hydrogen bonding between its hydroxyl group and a variety of protein residues. This general requirement suggests that the known cholesterol-binding motifs describe a subset of cholesterol-binding regions, and provides a framework for expanding the search for novel cholesterol-binding regions in ion channels.