Interactions between amiodarone and the hERG potassium channel pore determined with mutagenesis and in silico docking

AbstractThe antiarrhythmic drug amiodarone delays cardiac repolarisation through inhibition of hERG-encoded potassium channels responsible for the rapid delayed rectifier potassium current (IKr). This study aimed to elucidate molecular determinants of amiodarone binding to the hERG channel. Whole-cell patch-clamp recordings were made at 37°C of ionic current (IhERG) carried by wild-type (WT) or mutant hERG channels expressed in HEK293 cells. Alanine mutagenesis and ligand docking were used to investigate the roles of pore cavity amino-acid residues in amiodarone binding. Amiodarone inhibited WT outward IhERG tails with a half-maximal inhibitory concentration (IC50) of ∼45nM, whilst inward IhERG tails in a high K+ external solution ([K+]e) of 94mM were blocked with an IC50 of 117.8nM. Amiodarone’s inhibitory action was contingent upon channel gating. Alanine-mutagenesis identified multiple residues directly or indirectly involved in amiodarone binding. The IC50 for the S6 aromatic Y652A mutation was increased to ∼20-fold that of WT IhERG, similar to the pore helical mutant S624A (∼22-fold WT control). The IC50 for F656A mutant IhERG was ∼17-fold its corresponding WT control. Computational docking using a MthK-based hERG model differentiated residues likely to interact directly with drug and those whose Ala mutation may affect drug block allosterically. The requirements for amiodarone block of aromatic residues F656 and Y652 within the hERG pore cavity are smaller than for other high affinity IhERG inhibitors, with relative importance to amiodarone binding of the residues investigated being S624A∼Y652A>F656A>V659A>G648A>T623A

Voltage sensor gating charge transfer in a HERG potassium channel model

AbstractRelaxation of a hERG K+ channel model during molecular-dynamics simulation in a hydrated POPC bilayer was accompanied by transitions of an arginine gating charge across a charge transfer center in two voltage sensor domains. Inspection of the passage of arginine side chains across the charge transfer center suggests that the unique hydration properties of the arginine guanidine cation facilitates charge transfer during voltage sensor responses to changes in membrane potential, and underlies the preference of Arg over Lys as a mobile charge carrier in voltage-sensitive ion channels

Cloning, identification and functional characterization of bovine free fatty acid receptor-1 (FFAR1/GPR40) in neutrophils.

Long chain fatty acids (LCFAs), which are ligands for the G-protein coupled receptor FFAR1 (GPR40), are increased in cow plasma after parturition, a period in which they are highly susceptible to infectious diseases. This study identified and analyzed the functional role of the FFAR1 receptor in bovine neutrophils, the first line of host defense against infectious agents. We cloned the putative FFAR1 receptor from bovine neutrophils and analyzed the sequence to construct a homology model. Our results revealed that the sequence of bovine FFAR1 shares 84% identity with human FFAR1 and 31% with human FFAR3/GPR41. Therefore, we constructed a homology model of bovine FFAR1 using human as the template. Expression of the bovine FFAR1 receptor in Chinese hamster ovary (CHO)-K1 cells increased the levels of intracellular calcium induced by the LCFAs, oleic acid (OA) and linoleic acid (LA); no increase in calcium mobilization was observed in the presence of the short chain fatty acid propionic acid. Additionally, the synthetic agonist GW9508 increased intracellular calcium in CHO-K1/bFFAR1 cells. OA and LA increased intracellular calcium in bovine neutrophils. Furthermore, GW1100 (antagonist of FFAR1) and U73122 (phospholipase C (PLC) inhibitor) reduced FFAR1 ligand-induced intracellular calcium in CHO-K1/bFFAR1 cells and neutrophils. Additionally, inhibition of FFAR1, PLC and PKC reduced the FFAR1 ligand-induced release of matrix metalloproteinase (MMP)-9 granules and reactive oxygen species (ROS) production. Thus, we identified the bovine FFAR1 receptor and demonstrate a functional role for this receptor in neutrophils activated with oleic or linoleic acid

Assessing hERG Pore Models As Templates for Drug Docking Using Published Experimental Constraints: The Inactivated State in the Context of Drug Block

Many structurally and therapeutically diverse drugs interact with the human heart K⁺ channel hERG by binding within the K⁺ permeation pathway of the open channel, leading to drug-induced ‘long QT syndrome’. Drug binding to hERG is often stabilized by inactivation gating. In the absence of a crystal structure, hERG pore homology models have been used to characterize drug interactions. Here we assess potentially inactivated states of the bacterial K⁺ channel, KcsA, as templates for inactivated state hERG pore models in the context of drug binding using computational docking. Although Flexidock and GOLD docking produced low energy score poses in the models tested, each method selected a MthK K⁺ channel-based model over models based on the putative inactivated state KcsA structures for each of the 9 drugs tested. The variety of docking poses found indicates that an optimal arrangement for drug binding of aromatic side chains in the hERG pore can be achieved in several different configurations. This plasticity of the drug “binding site” is likely to be a feature of the hERG inactivated state. The results demonstrate that experimental data on specific drug interactions can be used as structural constraints to assess and refine hERG homology models

Multiscale Workflow for Modeling Ligand Complexes of Zinc Metalloproteins

Zinc metalloproteins are ubiquitous, with protein zinc centers of structural and functional importance, involved in interactions with ligands and substrates and often of pharmacological interest. Biomolecular simulations are increasingly prominent in investigations of protein structure, dynamics, ligand interactions, and catalysis, but zinc poses a particular challenge, in part because of its versatile, flexible coordination. A computational workflow generating reliable models of ligand complexes of biological zinc centers would find broad application. Here, we evaluate the ability of alternative treatments, using (nonbonded) molecular mechanics (MM) and quantum mechanics/molecular mechanics (QM/MM) at semiempirical (DFTB3) and density functional theory (DFT) levels of theory, to describe the zinc centers of ligand complexes of six metalloenzyme systems differing in coordination geometries, zinc stoichiometries (mono- and dinuclear), and the nature of interacting groups (specifically the presence of zinc–sulfur interactions). MM molecular dynamics (MD) simulations can overfavor octahedral geometries, introducing additional water molecules to the zinc coordination shell, but this can be rectified by subsequent semiempirical (DFTB3) QM/MM MD simulations. B3LYP/MM geometry optimization further improved the accuracy of the description of coordination distances, with the overall effectiveness of the approach depending upon factors, including the presence of zinc–sulfur interactions that are less well described by semiempirical methods. We describe a workflow comprising QM/MM MD using DFTB3 followed by QM/MM geometry optimization using DFT (e.g., B3LYP) that well describes our set of zinc metalloenzyme complexes and is likely to be suitable for creating accurate models of zinc protein complexes when structural information is more limited.EPSRCBBSRCSouthWestBiosciences Doctoral Training PartnershipMCIN/AEI/10.13039/501100011033NIHChina Scholarship CouncilRoyal Society of ChemistryDepto. de Química FísicaFac. de Ciencias QuímicasTRUEpu