Tobias Linder

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.

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.

Structure-activity relationships of pentamidine-affected ion channel trafficking and dofetilide mediated rescue.

Drug interference with normal hERG protein trafficking substantially reduces the channel density in the plasma membrane and thereby poses an arrhythmic threat. The chemical substructures important for hERG trafficking inhibition were investigated using pentamidine as a model drug. Furthermore, the relationship between acute ion channel block and correction of trafficking by dofetilide was studied.

Identification of the putative binding pocket of valerenic acid on GABAA receptors using docking studies and site-directed mutagenesis

β2/3‐subunit‐selective modulation of GABAA receptors by valerenic acid (VA) is determined by the presence of transmembrane residue β2/3N265. Currently, it is not known whether β2/3N265 is part of VAs binding pocket or is involved in the transduction pathway of VAs action. The aim of this study was to clarify the localization of VAs binding pocket on GABAA receptors.

Neutralisation of a single voltage sensor affects gating determinants in all four pore-forming S6 segments of Ca V 1.2: a cooperative gating model

Voltage sensors trigger the closed–open transitions in the pore of voltage-gated ion channels. To probe the transmission of voltage sensor signalling to the channel pore of CaV1.2, we investigated how elimination of positive charges in the S4 segments (charged residues were replaced by neutral glutamine) modulates gating perturbations induced by mutations in pore-lining S6 segments. Neutralisation of all positively charged residues in IIS4 produced a functional channel (IIS4N), while replacement of the charged residues in IS4, IIIS4 and IVS4 segments resulted in nonfunctional channels. The IIS4N channel displayed activation kinetics similar to wild type. Mutations in a highly conserved structure motif on S6 segments (“GAGA ring”: G432W in IS6, A780T in IIS6, G1193T in IIIS6 and A1503G in IVS6) induce strong left-shifted activation curves and decelerated channel deactivation kinetics. When IIS4N was combined with these mutations, the activation curves were shifted back towards wild type and current kinetics were accelerated. In contrast, 12 other mutations adjacent to the GAGA ring in IS6–IVS6, which also affect activation gating, were not rescued by IIS4N. Thus, the rescue of gating distortions in segments IS6–IVS6 by IIS4N is highly position-specific. Thermodynamic cycle analysis supports the hypothesis that IIS4 is energetically coupled with the distantly located GAGA residues. We speculate that conformational changes caused by neutralisation of IIS4 are not restricted to domain II (IIS6) but are transmitted to gating structures in domains I, III and IV via the GAGA ring.

Molecular Dynamics Simulations of KirBac1.1 Mutants Reveal Global Gating Changes of Kir Channels

Prokaryotic inwardly rectifying (KirBac) potassium channels are homologous to mammalian Kir channels. Their activity is controlled by dynamical conformational changes that regulate ion flow through a central pore. Understanding the dynamical rearrangements of Kir channels during gating requires high-resolution structure information from channels crystallized in different conformations and insight into the transition steps, which are difficult to access experimentally. In this study, we use MD simulations on wild type KirBac1.1 and an activatory mutant to investigate activation gating of KirBac channels. Full atomistic MD simulations revealed that introducing glutamate in position 143 causes significant widening at the helix bundle crossing gate, enabling water flux into the cavity. Further, global rearrangements including a twisting motion as well as local rearrangements at the subunit interface in the cytoplasmic domain were observed. These structural rearrangements are similar to recently reported KirBac3.1 crystal structures in closed and open conformation, suggesting that our simulations capture major conformational changes during KirBac1.1 opening. In addition, an important role of protein–lipid interactions during gating was observed. Slide-helix and C-linker interactions with lipids were strengthened during activation gating.

Structural insights into trapping and dissociation of small molecules in K⁺ channels.

K(+) channels play a critical role in numerous physiological and pathophysiological processes rendering them an attractive target for therapeutic intervention. However, the hERG K(+) channel poses a special challenge in drug discovery, since block of this channel by a plethora of diverse chemical entities can lead to long QT syndrome and sudden death. Of particular interest is the so-called trapping phenomenon, characterized by capture of a drug behind closed channel gates, which harbors an increased pro-arrhythmic risk. In this study we investigated the influence of trapped blockers on the gating dynamics and probed the state dependence of dissociation in K(+) channels by making use of the quaternary tetrabutylammonium. By applying essential dynamics simulations and two-electrode voltage clamp we obtained detailed insights into the dynamics of trapping in KcsA and hERG. Our simulations suggest that the trapped TBA influences the F656 flexibility during gate closure. Based on these findings, we provide a structural hypothesis for drug trapping. Further our simulations reveal the extent of gate opening necessary for drug dissociation.

Dehydroevodiamine and hortiamine, alkaloids from the traditional Chinese herbal drug Evodia rutaecarpa, are IKr blockers with proarrhythmic effects in vitro and in vivo

Graphical abstract Figure. No caption available. ABSTRACT Evodiae fructus is a widely used herbal drug in traditional Chinese medicine. Evodia extract was found to inhibit hERG channels. The aim of the current study was to identify hERG inhibitors in Evodia extract and to investigate their potential proarrhythmic effects. Dehydroevodiamine (DHE) and hortiamine were identified as IKr (rapid delayed rectifier current) inhibitors in Evodia extract by HPLC‐microfractionation and subsequent patch clamp studies on human embryonic kidney cells. DHE and hortiamine inhibited IKr with IC50s of 253.2 ± 26.3 nM and 144.8 ± 35.1 nM, respectively. In dog ventricular cardiomyocytes, DHE dose‐dependently prolonged the action potential duration (APD). Early afterdepolarizations (EADs) were seen in 14, 67, 100, and 67% of cells after 0.01, 0.1, 1 and 10 &mgr;M DHE, respectively. The proarrhythmic potential of DHE was evaluated in 8 anesthetized rabbits and in 8 chronic atrioventricular block (cAVB) dogs. In rabbits, DHE increased the QT interval significantly by 12 ± 10% (0.05 mg/kg/5 min) and 60 ± 26% (0.5 mg/kg/5 min), and induced Torsade de Pointes arrhythmias (TdP, 0.5 mg/kg/5 min) in 2 rabbits. In cAVB dogs, 0.33 mg/kg/5 min DHE increased QT duration by 48 ± 10% (P < 0.05*) and induced TdP in 2/4 dogs. A higher dose did not induce TdP. In human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs), methanolic extracts of Evodia, DHE and hortiamine dose‐dependently prolonged APD. At 3 &mgr;M DHE and hortiamine induced EADs. hERG inhibition at submicromolar concentrations, APD prolongation and EADs in hiPSC‐CMs and dose‐dependent proarrhythmic effects of DHE at micromolar plasma concentrations in cAVB dogs should increase awareness regarding proarrhythmic effects of widely used Evodia extracts.