Identification of Novel Cholesterol Binding Regions in the Transmembrane Domain of Kir2.1

Abstract

Kir channels are important in setting the resting membrane potential and modulating membrane excitability. A common feature of Kir channels that has emerged in recent years is that they are regulated by cholesterol. Furthermore, accumulating evidence indicates that cholesterol may also regulate ion channel function via direct binding. Our studies demonstrated that specific sterol-protein interactions are responsible for the suppression of Kir channels and that cholesterol binds to purified KirBac1.1 channels. We thus sought to identify the binding site of cholesterol in Kir2 channels. Our earlier studies identified a series of cytosolic residues that are crucial for the sensitivity of Kir2 channels to cholesterol. However, based on computational analysis none of these residues form a cholesterol-binding site.In this study, we used a combined computational-experimental approach independent of known cholesterol binding motifs to identify putative cholesterol binding regions in Kir2.1 channels. We show that cholesterol may bind to two nonanular hydrophobic regions in the transmembrane domain of Kir2.1 located in between adjacent subunits.Cholesterol-binding region 1 is located at the center of the transmembrane domain and region 2 is located at the interface of the transmembrane and cytosolic domains. Analysis of the binding enthalpy and free energy suggest that cholesterol may bind stronger to region 1. With the critical residues that affect cholesterol sensitivity being primarily non polar aliphatic, these cholesterol binding regions differ from previously identified cholesterol binding motifs. Thus, our results identify novel nonannular cholesterol-binding regions in Kir2.1 that have no correspondence to any of the established cholesterol-binding motifs. Furthermore, the location of the binding regions suggests that cholesterol modulates channel function by affecting the hinging motion at the center of the pore-lining transmembrane helix that underlies channel gating.