Reorganization of the slide helix by cholesterol during KirBac1.1 channel inactivation in membranes

KirBac1.1 is a prokaryotic homolog of mammalian inward-rectifier potassium (Kir) channels, and the functional equilibrium of KirBac1.1 is highly dependent on the type of membrane lipids. Like Kir channels, KirBac1.1 activity is also inhibited by cholesterol, a physiologically relevant lipid in human health and disease. Despite the slide helix is an important functional motif during lipid-dependent gating, the structural dynamics of the slide helix during cholesterol-induced channel inactivation is not well understood and is the focus of this work. Sequence analysis reveals that the slide helix of KirBac1.1 itself is a putative cholesterol-recognition motif. Liposomal K+ flux assays show that transport activity of the wild-type channel in PC/PG membranes is completely abolished at high concentration of cholesterol, whereas this is not observed in some of the single-cysteine mutants of the slide helix, indicating slide helix residues are critical for cholesterol sensitivity. Quenching of intrinsic Trp fluorescence upon increasing cholesterol concentration strongly suggests that the KirBac1.1 functional inhibition by cholesterol is possibly due to its direct interaction with the channel. Membrane penetration depth measurements using NBD-labeled slide helix residues clearly show relatively shallow membrane interfacial localization of the slide helix in cholesterol-containing membranes that is associated with significant structural dynamics changes and altered conformational heterogeneity. Based on distance measurements in varying membrane lipid compositions that stabilize the active and inactive conformations, we hypothesize that slide helix position in the membrane might possibly act as a "conformational switch" in regulating the KirBac1.1 function. These results involving dynamic lipid-protein interactions in lipid-dependent gating might be relevant for other Kir channels.