The scorpion toxin BeKm-1 blocks hERG cardiac potassium channels using an indispensable arginine residue.

BeKm-1 is a peptide toxin from scorpion venom that blocks the pore of the potassium channel hERG (Kv11.1) in the human heart. Although individual protein structures have been resolved, the structure of the complex between hERG and BeKm-1 is unknown. Here, we used molecular dynamics and ensemble docking, guided by previous double-mutant cycle analysis data, to obtain an in silico model of the hERG-BeKm-1 complex. Adding to the previous mutagenesis study of BeKm-1, our model uncovers the key role of residue Arg20, which forms three interactions (a salt bridge and hydrogen bonds) with the channel vestibule simultaneously. Replacement of this residue even by lysine weakens the interactions significantly. In accordance, the recombinantly produced BeKm-1R20K mutant exhibited dramatically decreased activity on hERG. Our model may be useful for future drug design attempts.

Functional Impact of BeKm-1, a High-Affinity hERG Blocker, on Cardiomyocytes Derived from Human-Induced Pluripotent Stem Cells.

IKr current, a major component of cardiac repolarization, is mediated by human Ether-à-go-go-Related Gene (hERG, Kv11.1) potassium channels. The blockage of these channels by pharmacological compounds is associated to drug-induced long QT syndrome (LQTS), which is a life-threatening disorder characterized by ventricular arrhythmias and defects in cardiac repolarization that can be illustrated using cardiomyocytes derived from human-induced pluripotent stem cells (hiPS-CMs). This study was meant to assess the modification in hiPS-CMs excitability and contractile properties by BeKm-1, a natural scorpion venom peptide that selectively interacts with the extracellular face of hERG, by opposition to reference compounds that act onto the intracellular face. Using an automated patch-clamp system, we compared the affinity of BeKm-1 for hERG channels with some reference compounds. We fully assessed its effects on the electrophysiological, calcium handling, and beating properties of hiPS-CMs. By delaying cardiomyocyte repolarization, the peptide induces early afterdepolarizations and reduces spontaneous action potentials, calcium transients, and contraction frequencies, therefore recapitulating several of the critical phenotype features associated with arrhythmic risk in drug-induced LQTS. BeKm-1 exemplifies an interesting reference compound in the integrated hiPS-CMs cell model for all drugs that may block the hERG channel from the outer face. Being a peptide that is easily modifiable, it will serve as an ideal molecular platform for the design of new hERG modulators displaying additional functionalities.

The macrolide drug erythromycin does not protect the hERG channel from inhibition by thioridazine and terfenadine.

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.

Fluorescent analogues of BeKm-1 with high and specific activity against the hERG channel.

Peptidic toxins that target specifically mammalian channels and receptors can be found in the venom of animals. These toxins are rarely used directly as tools for biochemical experiments, and need to be modified via the attachment of chemical groups (e.g., radioactive or fluorescent moieties). Ideally, such modifications should maintain the toxin specificity and affinity for its target. With the goal of obtaining fluorescent derivatives of BeKm-1, a toxin from the scorpion species Buthus eupeus that selectively inhibits the voltage-gated potassium ion channel hERG, we produced four active analogues using a model of BeKm-1 docking to the outer mouth of the channel. In these BeKm-1 analogues, the natural peptide was linked to the fluorescent cyanine 5 (Cy5) probe via four different linkers at Arg1 or Arg/Lys27. All analogues retained their specificity towards the hERG channel in electrophysiological experiments but displayed a lesser affinity. These results validate our strategy for designing toxin analogues and demonstrate that different chemical groups can be attached to different residues of BeKm-1.