title: Identification of a cholesterol-binding pocket in 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. Lipid-sterol interactions are not enantioselective, and the enantiomer of cholesterol (ent-cholesterol) does not inhibit Kir channel activity, suggesting that inhibition results from direct enantiospecific binding to the channel, and not indirect effects of changes to the bilayer. Furthermore, conservation of the effect of cholesterol among prokaryotic and eukaryotic Kir channels suggests an evolutionary conserved cholesterol-binding pocket, which we aimed to identify. Computational experiments were performed by docking cholesterol to the atomic structures of Kir2.2 (PDB: 3SPI) and KirBac1.1 (PDB: 2WLL) using Autodock 4.2. Poses were assessed to ensure biologically relevant orientation and then clustered according to location and orientation. The stability of cholesterol in each of these poses was then confirmed by molecular dynamics simulations. Finally, mutation of key residues (S95H and I171L) in this putative binding pocket found within the transmembrane domain of Kir2.1 channels were shown to lead to a loss of inhibition by cholesterol. Together, these data provide support for this location as a biologically relevant pocket.
authors.affiliation: Département de Physiologie Moléculaire et Intégrative and Groupe d'Étude des Protéines Membranaires (GÉPROM), Université de Montréal, Montreal, Quebec, Canada.
authors.forename: Oliver
authors.initials: O
authors.lastname: Fürst
authors.affiliation: Department of Cell Biology and Physiology and Center for Investigation of Membrane Excitabiltiy Diseases, Washington University School of Medicine, St. Louis, Missouri.
authors.forename: Colin G
authors.initials: CG
authors.lastname: Nichols
authors.affiliation: Department of Chemistry and Biochemistry, Centre for Research in Molecular Modeling (CERMM), Groupe d'Étude des Protéines Membranaires (GÉPROM), Concordia University, Montreal, Quebec, Canada.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Lamoureux
authors.affiliation: Département de Physiologie Moléculaire et Intégrative and Groupe d'Étude des Protéines Membranaires (GÉPROM), Université de Montréal, Montreal, Quebec, Canada. Electronic address: nazzareno.d.avanzo@umontreal.ca.
authors.forename: Nazzareno
authors.initials: N
authors.lastname: D'Avanzo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding
chemical_list: D024661:Potassium Channels, Inwardly Rectifying; D002784:Cholesterol