Explore the vast range of titles available.

Gating charges in voltage-sensing domains (VSD) of voltage-sensitive ion channels and enzymes are carried on arginine side chains rather than lysine. This arginine preference may result from the unique hydration properties of the side chain guanidinium group which facilitates its movement through a hydrophobic plug that seals the center of the VSD, as suggested by molecular dynamics simulations. To test for side chain interactions implicit in this model we inspected interactions of the side chains of arginine and lysine with each of the 19 non-glycine amino acids in proteins in the protein data bank. The arginine guanidinium interacts with non-polar aromatic and aliphatic side chains above and below the guanidinium plane while hydrogen bonding with polar side chains is restricted to in-plane positions. In contrast, non-polar side chains interact largely with the aliphatic part of the lysine side chain. The hydration properties of arginine and lysine are strongly reflected in their respective interactions with non-polar and polar side chains as observed in protein structures and in molecular dynamics simulations and likely underlie the preference for arginine as a mobile charge carrier in VSD.

The human voltage-sensitive K channel hERG plays a fundamental role in cardiac action potential repolarization, effectively controlling the QT interval of the electrocardiogram. Inherited loss- or gain-of-function mutations in hERG can result in dangerous \"long\" (LQTS) or \"short\" QT syndromes (SQTS), respectively, and the anomalous susceptibility of hERG to block by a diverse range of drugs underlies an acquired LQTS. A recent open channel cryo-EM structure of hERG should greatly advance understanding of the molecular basis of hERG channelopathies and drug-induced LQTS. Here we describe an update of recent research that addresses the nature of the particular gated state of hERG captured in the new structure, and the insight afforded by the structure into the molecular basis for high affinity drug block of hERG, the binding of hERG activators and the molecular basis of hERG's peculiar gating properties. Interpretation of the pharmacology of natural SQTS mutants in the context of the structure is a promising approach to understanding the molecular basis of hERG inactivation, and the structure suggests how voltage-dependent changes in the membrane domain may be transmitted to an extracellular \"turret\" to effect inactivation through aromatic side chain motifs that are conserved throughout the KCNH family of channels.

The lipophilic polycyclic aromatic hydrocarbon (PAH) phenanthrene is relatively abundant in polluted air and water and can access and accumulate in human tissue. Phenanthrene has been reported to interact with cardiac ion channels in several fish species. This study was undertaken to investigate the ability of phenanthrene to interact with hERG (human Ether-à-go-go-Related Gene) encoded Kv11.1 K+ channels, which play a central role in human ventricular repolarization. Pharmacological inhibition of hERG can be proarrhythmic. Whole-cell patch clamp recordings of hERG current (IhERG) were made from HEK293 cells expressing wild-type (WT) and mutant hERG channels. WT IhERG1a was inhibited by phenanthrene with an IC50 of 17.6 ± 1.7 µM, whilst IhERG1a/1b exhibited an IC50 of 1.8 ± 0.3 µM. WT IhERG block showed marked voltage and time dependence, indicative of dependence of inhibition on channel gating. The inhibitory effect of phenanthrene was markedly impaired by the attenuated inactivation N588K mutation. Remarkably, mutations of S6 domain aromatic amino acids (Y652, F656) in the canonical drug binding site did not impair the inhibitory action of phenanthrene; the Y652A mutation augmented IhERG block. In contrast, the F557L (S5) and M651A (S6) mutations impaired the ability of phenanthrene to inhibit IhERG, as did the S624A mutation below the selectivity filter region. Computational docking using a cryo-EM derived hERG structure supported the mutagenesis data. Thus, phenanthrene acts as an inhibitor of the hERG K+ channel by directly interacting with the channel, binding to a distinct site in the channel pore domain.

hERG (human Ether-à-go-go Related Gene)-encoded potassium channels underlie the cardiac rapid delayed rectifier (IKr) potassium current, which is a major target for antiarrhythmic agents and diverse non-cardiac drugs linked to the drug-induced form of long QT syndrome. E-4031 is a high potency hERG channel inhibitor from the methanesulphonanilide drug family. This study utilized a methanesulphonate-lacking E-4031 analogue, “E-4031-17”, to evaluate the role of the methanesulphonamide group in E-4031 inhibition of hERG. Whole-cell patch-clamp measurements of the hERG current (IhERG) were made at physiological temperature from HEK 293 cells expressing wild-type (WT) and mutant hERG constructs. For E-4031, WT IhERG was inhibited by a half-maximal inhibitory concentration (IC50) of 15.8 nM, whilst the comparable value for E-4031-17 was 40.3 nM. Both compounds exhibited voltage- and time-dependent inhibition, but they differed in their response to successive applications of a long (10 s) depolarisation protocol, consistent with greater dissociation of E-4031-17 than the parent compound between applied commands. Voltage-dependent inactivation was left-ward voltage shifted for E-4031 but not for E-4031-17; however, inhibition by both compounds was strongly reduced by attenuated-inactivation mutations. Mutations of S6 and S5 aromatic residues (F656V, Y652A, F557L) greatly attenuated actions of both drugs. The S624A mutation also reduced IhERG inhibition by both molecules. Overall, these results demonstrate that the lack of a methanesulphonate in E-4031-17 is not an impediment to high potency inhibition of IhERG.

Loss of function (LOF) mutations of voltage sensitive K + channel proteins hERG (Kv11.1) and KCNQ1 (Kv7.1) account for the majority of instances of congenital Long QT Syndrome (cLQTS) with the dominant molecular phenotype being a mistrafficking one resulting from protein misfolding. We explored the use of Evolutionary Coupling (EC) analysis, which identifies evolutionarily conserved pairwise amino acid interactions that may contribute to protein structural stability, to identify regions of the channels susceptible to misfolding mutations. Comparison with published experimental trafficking data for hERG and KCNQ1 showed that the method strongly predicts “scaffolding” regions of the channel membrane domains and has useful predictive power for trafficking phenotypes of individual variants. We identified a region in and around the cytoplasmic S2-S3 loop of the hERG Voltage Sensor Domain (VSD) as susceptible to destabilising mutation, and this was confirmed using a quantitative LI-COR ® based trafficking assay that showed severely attenuated trafficking in eight out of 10 natural hERG VSD variants selected using EC analysis. Our analysis highlights an equivalence in the scaffolding structures of the hERG and KCNQ1 membrane domains. Pathogenic variants of ion channels with an underlying mistrafficking phenotype are likely to be located within similar scaffolding structures that are identifiable by EC analysis.

The voltage‐gated hERG (human‐Ether‐à‐go‐go Related Gene) K+ channel plays a fundamental role in cardiac action potential repolarization. Loss‐of‐function mutations or pharmacological inhibition of hERG leads to long QT syndrome, whilst gain‐of‐function mutations lead to short QT syndrome. A recent open channel cryo‐EM structure of hERG represents a significant advance in the ability to interrogate hERG channel structure‐function. In order to suppress protein aggregation, a truncated channel construct of hERG (hERGT) was used to obtain this structure. In hERGT cytoplasmic domain residues 141 to 350 and 871 to 1,005 were removed from the full‐length channel protein. There are limited data on the electrophysiological properties of hERGT channels. Therefore, this study was undertaken to determine how hERGT influences channel function at physiological temperature. Whole‐cell measurements of hERG current (IhERG) were made at 37°C from HEK 293 cells expressing wild‐type (WT) or hERGT channels. With a standard +20 mV activating command protocol, neither end‐pulse nor tail IhERG density significantly differed between WT and hERGT. However, the IhERG deactivation rate was significantly slower for hERGT. Half‐maximal activation voltage (V0.5) was positively shifted for hERGT by ~+8 mV (p < .05 versus WT), without significant change to the activation relation slope factor. Neither the voltage dependence of inactivation, nor time course of development of inactivation significantly differed between WT and hERGT, but recovery of IhERG from inactivation was accelerated for hERGT (p < .05 versus WT). Steady‐state “window” current was positively shifted for hERGT with a modest increase in the window current peak. Under action potential (AP) voltage clamp, hERGT IhERG showed modestly increased current throughout the AP plateau phase with a significant increase in current integral during the AP. The observed consequences for hERGT IhERG of deletion of the two cytoplasmic regions may reflect changes to electrostatic interactions influencing the voltage sensor domain. The hERGTtruncation mutant was made to derive the cryo‐EM structure that is employed to study hERG K+channel structure function. This paper shows that the hERGT mutant exhibits modest differences in hERG current kinetics from the wild‐type channel. Voltage‐dependent activation and “window current” were positively voltage‐shifted, deactivation was slowed and recovery from inactivation was faster for hERGT compared to the wild‐type channel.

Melittin is arguably the most widely studied amphipathic, membrane-lytic α-helical peptide. Although several lines of evidence suggest an interfacial membrane location at low concentrations, melittin’s exact position and depth of penetration into the hydrocarbon core are unknown. Furthermore, the structural basis for its lytic action remains largely a matter of conjecture. Using a novel x-ray absolute-scale refinement method, we have now determined the location, orientation, and likely conformation of monomeric melittin in oriented phosphocholine lipid multilayers. Its helical axis is aligned parallel to the bilayer plane at the depth of the glycerol groups, but its average conformation differs from the crystallographic structure. As observed earlier for another amphipathic α-helical peptide, the lipid perturbations induced by melittin are remarkably modest. Small bilayer perturbations thus appear to be a general feature of amphipathic helices at low concentrations. In contrast, a dimeric form of melittin causes larger structural perturbations under otherwise identical conditions. These results provide direct structural evidence that self-association of amphipathic helices may be the crucial initial step toward membrane lysis.

The macrolide antibiotic erythromycin has been associated with QT interval prolongation and inhibition of the hERG‐encoded channels responsible for the rapid delayed rectifier K+ current I(Kr). It has been suggested that low concentrations of erythromycin may have a protective effect against hERG block and associated drug‐induced arrhythmia by reducing the affinity of the pore‐binding site for high potency hERG inhibitors. This study aimed to explore further the notion of a potentially protective effect of erythromycin. Whole‐cell patch‐clamp experiments were performed in which hERG‐expressing mammalian (Human Embryonic Kidney; HEK) cells were preincubated with low to moderate concentrations of erythromycin (3 or 30 µM) prior to whole‐cell patch clamp recordings of hERG current (IhERG) at 37°C. In contrast to a previous report, exposure to low concentrations of erythromycin did not reduce pharmacological sensitivity of hERG to the antipsychotic thioridazine and antihistamine terfenadine. The IC50 value for IhERG tail inhibition by terfenadine was decreased by ~32‐fold in the presence of 3 µM erythromycin (p < .05 vs. no preincubation). Sensitivity to thioridazine remained unchanged (p > .05 vs. no preincubation). The effects of low concentrations of erythromycin were investigated for a series of pore blocking drugs, and the results obtained were consistent with additive and/or synergistic effects. Experiments with the externally acting blocker BeKm‐1 on WT hERG and a pore mutant (F656V) were used to explore the location of the binding site for erythromycin. Our data are inconsistent with the use of erythromycin for the management of drug‐induced QT prolongation. In contrast to an earlier study that suggested a protective effect of the macrolide antibiotic erythromycin against pharmacological block of recombinant hERG channels overexpressed in Human Embryonic Kidney (HEK) cells, the present report showed that pretreatment with low concentrations of erythromycin did not reduce hERG channel sensitivity to inhibitors. In the presence of erythromycin, hERG displayed increased sensitivity to pharmacological inhibition by three drugs: the antihistamine terfenadine, the antimalarial chloroquine and antiarrythmic dofetilide. hERG block by antipsychotic thioridazine and antifungal ketoconazole was unaffected. This complex pharmacological profile of erythromycin actions may be mediated through two distinct binding sites on the hERG channel.

Potassium channels encoded by human Ether-à-go-go-Related Gene ( hERG ) are inhibited by diverse cardiac and non-cardiac drugs. Disopyramide is a chiral Class Ia antiarrhythmic that inhibits hERG at clinical concentrations. This study evaluated effects of disopyramide enantiomers on hERG current (I hERG ) from hERG expressing HEK 293 cells at 37 °C. S(+) and R(−) disopyramide inhibited wild-type (WT) I hERG with IC 50 values of 3.9 µM and 12.9 µM respectively. The attenuated-inactivation mutant N588K had little effect on the action of S(+) disopyramide but the IC 50 for the R(−) enantiomer was ~ 15-fold that for S(+) disopyramide. The enhanced inactivation mutant N588E only slightly increased the potency of R(−) disopyramide. S6 mutation Y652A reduced S(+) disopyramide potency more than that of R(−) disopyramide (respective IC 50 values ~ 49-fold and 11-fold their WT controls). The F656A mutation also exerted a stronger effect on S(+) than R(−) disopyramide, albeit with less IC 50 elevation. A WT-Y652A tandem dimer exhibited a sensitivity to the enantiomers that was intermediate between that of WT and Y652A, suggesting Y652 groups on adjacent subunits contribute to the binding. Moving the Y (normally at site 652) one residue in the N- terminal (up) direction in N588K hERG markedly increased the blocking potency of R(−) disopyramide. Molecular dynamics simulations using a hERG pore model produced different binding modes for S(+) and R(−) disopyramide consistent with the experimental observations. In conclusion, S(+) disopyramide interacts more strongly with S6 aromatic binding residues on hERG than does R(−) disopyramide, whilst optimal binding of the latter is more reliant on intact inactivation.

The hERG potassium channel influences ventricular action potential duration. Extracellular acidosis occurs in pathological states including cardiac ischaemia. It reduces the amplitude of hERG current and speeds up deactivation, which can alter cardiac excitability. This study aimed to identify the site of action by which extracellular protons regulate the amplitude of macroscopic hERG current. Recordings of macroscopic and single hERG1a and 1b channel activity, mutagenesis, and the recent cryoEM structure for hERG were employed. Single hERG1a and 1b channels displayed open times that decreased with membrane depolarization, suggestive of a blocking mechanism that senses approximately 20% of the membrane electric field. This mechanism was sensitive to pH; extracellular acidosis reduced both hERG1a and1b channel open time and conductance. The effects of acidosis on macroscopic current amplitude and deactivation displayed different sensitivities to protons. Point mutation of a pair of residues (E575/H578) in the pore turret abolished the acidosis-induced decrease of current amplitude, without affecting the change in current deactivation. In single hERG1a channel recordings, the conductance of the double-mutant channel was unaffected by extracellular acidosis. These findings identify residues in the outer turret of the hERG channel that act as a proton sensor to regulate open time and channel conductance.