Most small-molecule inhibitors of voltage-gated ion channels display poor subtype specificity because they bind to highly conserved residues located in the channel's central cavity. Using a combined approach of scanning mutagenesis, electrophysiology, chemical ligand modification, chemical cross-linking, MS/MS-analyses and molecular modelling, we provide evidence for the binding site for adamantane derivatives and their putative access pathway in Kv7.1/KCNE1 channels. The adamantane compounds, exemplified by JNJ303, are highly potent gating modifiers that bind to fenestrations that become available when KCNE1 accessory subunits are bound to Kv7.1 channels. This mode of regulation by auxiliary subunits may facilitate the future development of potent and highly subtype-specific Kv channel inhibitors.
Adamantane/analogs & derivatives , Adamantane/pharmacology , Ion Channel Gating/drug effects , KCNQ1 Potassium Channel/antagonists & inhibitors , Potassium Channel Blockers/pharmacology , Potassium Channels, Voltage-Gated/antagonists & inhibitors , Adamantane/chemistry , Allosteric Regulation/drug effects , Animals , Binding Sites , Cross-Linking Reagents/chemistry , Humans , KCNQ1 Potassium Channel/genetics , KCNQ1 Potassium Channel/metabolism , Models, Molecular , Mutagenesis , Mutation , Oocytes , Potassium Channel Blockers/chemistry , Potassium Channels, Voltage-Gated/genetics , Potassium Channels, Voltage-Gated/metabolism , Tandem Mass Spectrometry , Xenopus laevis
Kv7.1 to Kv7.5 α-subunits belong to the family of voltage-gated potassium channels (Kv). Assembled with the ß-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.
KCNQ1 Potassium Channel/metabolism , Phosphatidylinositol 4,5-Diphosphate/metabolism , Action Potentials/physiology , Amino Acid Substitution , Animals , Binding Sites , Humans , KCNQ1 Potassium Channel/chemistry , KCNQ1 Potassium Channel/genetics , Phosphatidylinositol 4,5-Diphosphate/chemistry , Phosphatidylinositol 4,5-Diphosphate/genetics , Point Mutation , Protein Binding , Xenopus laevis
RATIONALE: The plateau phase of the ventricular action potential is the result of balanced Ca(2+) influx and K(+) efflux. The action potential is terminated by repolarizing K(+) currents. Under ß-adrenergic stimulation, both the Ca(2+)-influx and the delayed rectifier K(+) currents I(K) are stimulated to adjust the cardiac action potential duration to the enhanced heart rate and to ascertain adequate increase in net Ca(2+) influx. Intracellularly, a Calsequestrin2 (CASQ2)-ryanodine receptor complex serves as the most effective Ca(2+) reservoir/release system to aid the control of intracellular Ca(2+) levels. Currently, it is unclear if disease-associated CASQ2 gene variants alter intracellular free Ca(2+) concentrations and if cardiac ion channels are affected by it. OBJECTIVE: The goal of this study is to test if CASQ2 determines intracellular free Ca(2+) concentrations and to identify cardiac ion channels that are affected by it. Further, we aim to study disease-associated CASQ2 gene variants in this context. METHODS AND RESULTS: Here, we study the effects of the CASQ2 mutations R33Q, F189L, and D307H, located in highly conserved regions, on the functions of cardiac potassium channels in Xenopus oocytes using two electrode voltage clamp. As a result, CASQ2 wild type and CASQ2-mutants modulated hERG functions differently. Free Ca(2+) measurements and molecular dynamics simulations imply alterations in Ca(2+) buffer capacity paralled by changes in the dynamic behavior of the CASQ2-mutants compared to CASQ2 wild type. CONCLUSIONS: These in vitro and in silico data suggest a regulatory role of CASQ2 on cytosolic Ca(2+) and hERG channels which may contribute to the etiology of CPVT.
Calsequestrin/physiology , Ether-A-Go-Go Potassium Channels/metabolism , Action Potentials/physiology , Amino Acid Substitution , Animals , Calcium/metabolism , Calsequestrin/genetics , Calsequestrin/metabolism , Humans , Molecular Dynamics Simulation , Mutation , Oocytes/metabolism , Potassium/metabolism , Protein Structure, Tertiary , Ryanodine Receptor Calcium Release Channel/metabolism , Tachycardia, Ventricular/etiology , Xenopus/growth & development
