Identification of a Cholesterol Binding Pocket in Bacterial and Eukaryotic Inward Rectifier K+ (Kir) Channels

Abstract

Cholesterol is the major sterol component of all mammalian plasma membranes. Recent studies have shown that cholesterol inhibits both bacterial (KirBac1.1 and KirBac3.1) and eukaryotic (Kir2.1) inward rectifier K+ (Kir) channels but the molecular basis of this interaction has yet to be determined. Biophysical experiments show that lipid-sterol interactions are not enantioselective, but only cholesterol and not the enantiomer of cholesterol (ent-cholesterol) inhibits Kir and KirBac channel activity, implying that the effects of cholesterol in both prokaryotic and eukaryotic channels must be due to direct enantiospecific binding to the channel, and not indirect effects of changes to the bilayer properties. Furthermore, conservation of the effect of cholesterol among prokaryotic and eukaryotic Kir channels suggests an evolutionarily conserved “cholesterol binding pocket”. To identify potential sites, computational experiments were performed in which cholesterol was docked to atomic structures of Kir2.2 (PDB: 3YJC) and KirBac1.1 (PDB: 3WLL) using Autodock 4.2. Poses were assessed to ensure biologically relevant orientation and then clustered according to location and orientation. Binding pockets that interacted with cholesterol and ent-cholesterol were eliminated. Residues identified as to solely interacting with cholesterol are being targeted for mutagenesis studies using purified channels. These experiments will identify a molecular basis for cholesterol-Kir channel interactions, and may provide a “model binding site” that can be used to identify putative cholesterol pockets in other channels.