Anna Stary-Weinzinger

Grayanotoxin poisoning: 'mad honey disease' and beyond.

Many plants of the Ericaceae family, Rhododendron, Pieris, Agarista and Kalmia, contain diterpene grayanotoxins. Consumption of grayanotoxin containing leaves, flowers or secondary products as honey may result in intoxication specifically characterized by dizziness, hypotension and atrial-ventricular block. Symptoms are caused by an inability to inactivate neural sodium ion channels resulting in continuous increased vagal tone. Grayanotoxin containing products are currently sold online, which may pose an increasing risk. In humans, intoxication is rarely lethal, in contrast to cattle and pet poisoning cases. Scientific evidence for the medicinal properties of grayanotoxin containing preparations, such as honey or herbal preparation in use in folk medicine, is scarce, and such use may even be harmful.

Computer simulations of structure-activity relationships for hERG channel blockers.

The hERG potassium channel is of major pharmaceutical importance, and its blockade by various compounds, potentially causing serious cardiac side effects, is a major problem in drug development. Despite the large amounts of existing biochemical data on blockade of hERG by drugs and druglike compounds, relatively little is known regarding the structural basis of binding of blockers to the channel. Here, we have used a recently developed homology model of hERG to conduct molecular docking experiments with a series of channel blockers, followed by molecular dynamics simulations of the complexes and evaluation of binding free energies with the linear interaction energy method. The calculations yield a remarkably good agreement with experimental binding affinities and allow for a rationalization of three-dimensional structure-activity relationships in terms of a number of key interactions. Two main interaction regions of the channel are thus identified with implications for further mutagenesis experiments and design of new compounds.

Distinct interactions of Na+ and Ca2+ ions with the selectivity filter of the bacterial sodium channel NaVAb

Rapid and selective ion transport is essential for the generation and regulation of electrical signaling pathways in living organisms. In this study, we use molecular dynamics simulations and free energy calculations to investigate how the bacterial sodium channel Na(V)Ab (Arcobacter butzleri) differentiates between Na(+) and Ca(2+) ions. Multiple nanosecond molecular dynamics simulations revealed distinct binding patterns for these two cations in the selectivity filter and suggested a high affinity calcium binding site formed by backbone atoms of residues Leu-176 and Thr-175 (S(CEN)) in the sodium channel selectivity filter.

Molecular Determinants for Activation of Human Ether-à-go-go-related Gene 1 Potassium Channels by 3-Nitro-N-(4-phenoxyphenyl) Benzamide

Human ether-à-go-go-related gene 1 (hERG1) channels mediate repolarization of cardiac action potentials. Inherited long QT syndrome (LQTS) caused by loss-of-function mutations, or unintended blockade of hERG1 channels by many drugs, can lead to severe arrhythmia and sudden death. Drugs that activate hERG1 are a novel pharmacological approach to treat LQTS. 3-Nitro-n-(4-phenoxyphenyl) benzamide [ICA-105574 (ICA)] has been discovered to activate hERG1 by strong attenuation of pore-type inactivation. Here, we used scanning mutagenesis of hERG1 to identify the molecular determinants of ICA action. Three mutations abolished the activator effects of 30 μM ICA, including L622C in the pore helix, F557L in the S5 segment, and Y652A in the S6 segment. One mutation in S6 (A653M) switched the activity of ICA from an activator to an inhibitor, revealing its partial agonist activity. This was confirmed by showing that the noninactivating mutant hERG1 channel (G628C/S631C) was inhibited by ICA and that the addition of the F557L mutation rendered the channel drug-insensitive. Simulated molecular docking of ICA to homology models of hERG1 corroborated the scanning mutagenesis findings. Together, our findings indicate that ICA is a mixed agonist of hERG1 channels. Activation or inhibition of currents is mediated by the same or overlapping binding site located in the pore module between two adjacent subunits of the homotetrameric channel.

Timothy mutation disrupts the link between activation and inactivation in Ca(V)1.2 protein.

The Timothy syndrome mutations G402S and G406R abolish inactivation of CaV1.2 and cause multiorgan dysfunction and lethal arrhythmias. To gain insights into the consequences of the G402S mutation on structure and function of the channel, we systematically mutated the corresponding Gly-432 of the rabbit channel and applied homology modeling. All mutations of Gly-432 (G432A/M/N/V/W) diminished channel inactivation. Homology modeling revealed that Gly-432 forms part of a highly conserved structure motif (G/A/G/A) of small residues in homologous positions of all four domains (Gly-432 (IS6), Ala-780 (IIS6), Gly-1193 (IIIS6), Ala-1503 (IVS6)). Corresponding mutations in domains II, III, and IV induced, in contrast, parallel shifts of activation and inactivation curves indicating a preserved coupling between both processes. Disruption between coupling of activation and inactivation was specific for mutations of Gly-432 in domain I. Mutations of Gly-432 removed inactivation irrespective of the changes in activation. In all four domains residues G/A/G/A are in close contact with larger bulky amino acids from neighboring S6 helices. These interactions apparently provide adhesion points, thereby tightly sealing the activation gate of CaV1.2 in the closed state. Such a structural hypothesis is supported by changes in activation gating induced by mutations of the G/A/G/A residues. The structural implications for CaV1.2 activation and inactivation gating are discussed.

Efficient and specific cardiac IK1 inhibition by a new pentamidine analogue

AIMS In excitable cells, KIR2.x ion-channel-carried inward rectifier current (IK₁) is thought to set the negative and stable resting membrane potential, and contributes to action potential repolarization. Loss- or gain-of-function mutations correlate with cardiac arrhythmias and pathological remodelling affects normal KIR2.x protein levels. No specific IK1 inhibitor is currently available for in vivo use, which severely hampers studies on the precise role of IK1 in normal cardiac physiology and pathophysiology. The diamine antiprotozoal drug pentamidine (P) acutely inhibits IK₁ by plugging the cytoplasmic pore region of the channel. We aim to develop more efficient and specific IK₁ inhibitors based on the P structure. METHODS AND RESULTS We analysed seven pentamidine analogues (PA-1 to PA-7) for IK₁ blocking potency at 200 nM using inside-out patches from KIR2.1 expressing HEK-293 cells. PA-6 showed the highest potency and was tested further. PA-6 blocked KIR2.x currents of human and mouse with low IC₅₀ values (12-15 nM). Modelling indicated that PA-6 had less electrostatic but more lipophilic interactions with the cytoplasmic channel pore than P, resulting in a higher channel affinity for PA-6 (ΔG -44.1 kJ/Mol) than for P (ΔG -31.7 kJ/Mol). The involvement of acidic amino acid residues E224 and E299 in drug-channel interaction was confirmed experimentally. PA-6 did not affect INav1.5, ICa-L, IKv4.3, IKv11.1, and IKv7.1/minK currents at 200 nM. PA-6 inhibited the inward (50 nM 40%; 100 nM 59%; 200 nM 77%) and outward (50 nM 40%; 100 nM 76%; 200 nM 100%) components of IK₁ in isolated canine adult-ventricular cardiomyocytes (CMs). PA-6 prolonged action potential duration of CMs by 8 (n = 9), 26 (n = 5), and 34% (n = 11) at 50, 100, and 200 nM, respectively. Unlike P, PA-6 had no effect on KIR2.1 channel expression at concentrations from 0.1 to 3 μM. However, PA-6 at 10 μM increased KIR2.1 expression levels. Also, PA-6 did not affect the maturation of hERG, except when applied at 10 μM. CONCLUSION PA-6 has higher efficiency and specificity to KIR2.x-mediated current than P, lengthens action potential duration, and does not affect channel trafficking at concentrations relevant for complete IK₁ block.

New potential binding determinant for hERG channel inhibitors.

Human ether-à-go-go related gene (hERG) 1 channels conduct the rapid delayed rectifier K+ current (IKr) and are essential for the repolarization of the cardiac action potential. hERG1 inhibition by structurally diverse drugs may lead to life threatening arrhythmia. Putative binding determinants of hERG1 channel blockers include T623, S624 and V625 on the pore helix, and residues G648, Y652 and F656, located on segment S6. We and others have previously hypothesized that additional binding determinants may be located on helix S5, which is in close contact with the S6 segments. In order to test this hypothesis, we performed a detailed investigation combining ionic current measurements with two-microelectrode voltage clamp and molecular modeling techniques. We identified a novel aromatic high affinity binding determinant for blockers located in helix S5, F557, which is equally potent as Y652. Modeling supports a direct interaction with the outer pore helix.

Timothy mutation disrupts link between activation and inactivation in CaV1.2

The Timothy syndrome mutations G402S and G406R abolish inactivation of CaV1.2 and cause multiorgan dysfunction and lethal arrhythmias. To gain insights into the consequences of the G402S mutation on structure and function of the channel, we systematically mutated the corresponding Gly-432 of the rabbit channel and applied homology modeling. All mutations of Gly-432 (G432A/M/N/V/W) diminished channel inactivation. Homology modeling revealed that Gly-432 forms part of a highly conserved structure motif (G/A/G/A) of small residues in homologous positions of all four domains (Gly-432 (IS6), Ala-780 (IIS6), Gly-1193 (IIIS6), Ala-1503 (IVS6)). Corresponding mutations in domains II, III, and IV induced, in contrast, parallel shifts of activation and inactivation curves indicating a preserved coupling between both processes. Disruption between coupling of activation and inactivation was specific for mutations of Gly-432 in domain I. Mutations of Gly-432 removed inactivation irrespective of the changes in activation. In all four domains residues G/A/G/A are in close contact with larger bulky amino acids from neighboring S6 helices. These interactions apparently provide adhesion points, thereby tightly sealing the activation gate of CaV1.2 in the closed state. Such a structural hypothesis is supported by changes in activation gating induced by mutations of the G/A/G/A residues. The structural implications for CaV1.2 activation and inactivation gating are discussed.

Probing the energy landscape of activation gating of the bacterial potassium channel KcsA.

The bacterial potassium channel KcsA, which has been crystallized in several conformations, offers an ideal model to investigate activation gating of ion channels. In this study, essential dynamics simulations are applied to obtain insights into the transition pathways and the energy profile of KcsA pore gating. In agreement with previous hypotheses, our simulations reveal a two phasic activation gating process. In the first phase, local structural rearrangements in TM2 are observed leading to an intermediate channel conformation, followed by large structural rearrangements leading to full opening of KcsA. Conformational changes of a highly conserved phenylalanine, F114, at the bundle crossing region are crucial for the transition from a closed to an intermediate state. 3.9 µs umbrella sampling calculations reveal that there are two well-defined energy barriers dividing closed, intermediate, and open channel states. In agreement with mutational studies, the closed state was found to be energetically more favorable compared to the open state. Further, the simulations provide new insights into the dynamical coupling effects of F103 between the activation gate and the selectivity filter. Investigations on individual subunits support cooperativity of subunits during activation gating.

In silico analysis of conformational changes induced by mutation of aromatic binding residues: consequences for drug binding in the hERG K+ channel.

Pharmacological inhibition of cardiac hERG K+ channels is associated with increased risk of lethal arrhythmias. Many drugs reduce hERG current by directly binding to the channel, thereby blocking ion conduction. Mutation of two aromatic residues (F656 and Y652) substantially decreases the potency of numerous structurally diverse compounds. Nevertheless, some drugs are only weakly affected by mutation Y652A. In this study we utilize molecular dynamics simulations and docking studies to analyze the different effects of mutation Y652A on a selected number of hERG blockers. MD simulations reveal conformational changes in the binding site induced by mutation Y652A. Loss of π-π-stacking between the two aromatic residues induces a conformational change of the F656 side chain from a cavity facing to cavity lining orientation. Docking studies and MD simulations qualitatively reproduce the diverse experimentally observed modulatory effects of mutation Y652A and provide a new structural interpretation for the sensitivity differences.