title: Novel Kv7.1-phosphatidylinositol 4,5-bisphosphate interaction sites uncovered by charge neutralization scanning.
abstract: Kv7.1 to Kv7.5 α-subunits belong to the family of voltage-gated potassium channels (Kv). Assembled with the β-subunit KCNE1, Kv7.1 conducts the slowly activating potassium current IKs, which is one of the major currents underlying repolarization of the cardiac action potential. A known regulator of Kv7 channels is the lipid phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 increases the macroscopic current amplitude by stabilizing the open conformation of 7.1/KCNE1 channels. However, knowledge about the exact nature of the interaction is incomplete. The aim of this study was the identification of the amino acids responsible for the interaction between Kv7.1 and PIP2. We generated 13 charge neutralizing point mutations at the intracellular membrane border and characterized them electrophysiologically in complex with KCNE1 under the influence of diC8-PIP2. Electrophysiological analysis of corresponding long QT syndrome mutants suggested impaired PIP2 regulation as the cause for channel dysfunction. To clarify the underlying structural mechanism of PIP2 binding, molecular dynamics simulations of Kv7.1/KCNE1 complexes containing two PIP2 molecules in each subunit at specific sites were performed. Here, we identified a subset of nine residues participating in the interaction of PIP2 and Kv7.1/KCNE1. These residues may form at least two binding pockets per subunit, leading to the stabilization of channel conformations upon PIP2 binding.
authors.affiliation: Department of Biochemistry I-Cation Channel Group, Ruhr University Bochum, 44801 Bochum, Germany; International Graduate School of Neuroscience, Ruhr University Bochum, 44801 Bochum, Germany; Ruhr University Bochum Research School, and Ruhr University Bochum, 44801 Bochum, Germany.
authors.forename: Karina
authors.initials: K
authors.lastname: Eckey
authors.affiliation: IfGH-Myocellular Electrophysiology, Department of Cardiovascular Medicine, University Hospital of Münster, 48149 Münster, Germany, and.
authors.forename: Eva
authors.initials: E
authors.lastname: Wrobel
authors.affiliation: IfGH-Myocellular Electrophysiology, Department of Cardiovascular Medicine, University Hospital of Münster, 48149 Münster, Germany, and.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Strutz-Seebohm
authors.affiliation: Institute of Physiology, Ruhr University Bochum, 44801 Bochum, Germany.
authors.forename: Lutz
authors.initials: L
authors.lastname: Pott
authors.affiliation: Danish National Research Foundation Centre for Cardiac Arrhythmia, University of Copenhagen, 1165 Copenhagen, Denmark.
authors.forename: Nicole
authors.initials: N
authors.lastname: Schmitt
authors.affiliation: International Graduate School of Neuroscience, Ruhr University Bochum, 44801 Bochum, Germany; Ruhr University Bochum Research School, and Ruhr University Bochum, 44801 Bochum, Germany; IfGH-Myocellular Electrophysiology, Department of Cardiovascular Medicine, University Hospital of Münster, 48149 Münster, Germany, and. Electronic address: guiscard.seebohm@ukmuenster.de.
authors.forename: Guiscard
authors.initials: G
authors.lastname: Seebohm
mesh_terms: D000200:Action Potentials / Q000502:physiology; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D006801:Humans; D051657:KCNQ1 Potassium Channel / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D014982:Xenopus laevis
chemical_list: D051657:KCNQ1 Potassium Channel; C496909:KCNQ1 protein, human; D019269:Phosphatidylinositol 4,5-Diphosphate
keywords: Electrophysiology; Heart; Molecular Modeling; Phosphatidylinositol; Phosphoinositide; Phospholipid; Potassium Channel; Structural Biology; Xenopus