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Background and Purpose: Local anaesthetics block sodium and a variety of potassium channels. Although previous studies identified a residue in the pore signature sequence together with three residues in the S6 segment as a putative binding site, the precise molecular basis of inhibition of Kv channels by local anaesthetics remained unknown. Crystal structures of Kv channels predict that some of these residues point away from the central cavity and face into a drug binding site called side pockets. Thus, the question arises whether the binding site of local anaesthetics is exclusively located in the central cavity or also involves the side pockets.Experimental Approach: A systematic functional alanine mutagenesis approach, scanning 58 mutants, together with in silico docking experiments and molecular dynamics simulations was utilized to elucidate the binding site of bupivacaine and ropivacaine.Key Results: Inhibition of Kv 1.5 channels by local anaesthetics requires binding to the central cavity and the side pockets, and the latter requires interactions with residues of the S5 and the back of the S6 segments. Mutations in the side pockets remove stereoselectivity of inhibition of Kv 1.5 channels by bupivacaine. Although binding to the side pockets is conserved for different local anaesthetics, the binding mode in the central cavity and the side pockets shows considerable variations.Conclusion and Implications: Local anaesthetics bind to the central cavity and the side pockets, which provide a crucial key to the molecular understanding of their Kv channel affinity and stereoselectivity, as well as their spectrum of side effects. [ABSTRACT FROM AUTHOR]

Potassium channels of the tandem of two-pore-domain (K2P) family were among the last potassium channels cloned. However, recent progress in understanding their physiological relevance and molecular pharmacology revealed their therapeutic potential and thus these channels evolved as major drug targets against a large variety of diseases. However, after the initial cloning of the fifteen family members there was a lack of potent and/or selective modulators. By now a large variety of K2P channel modulators (activators and blockers) have been described, especially for TASK-1, TASK-3, TREK-1, TREK2, TRAAK and TRESK channels. Recently obtained crystal structures of K2P channels, alanine scanning approaches to map drug binding sites, in silico experiments with molecular dynamics simulations (MDs) combined with electrophysiological studies to reveal the mechanism of channel inhibition/activation, yielded a good understanding of the molecular pharmacology of these channels. Besides summarizing drugs that were identified to modulate K2P channels, the main focus of this article is on describing the differential binding sites and mechanisms of channel modulation that are utilized by the different K2P channel blockers and activators. [ABSTRACT FROM AUTHOR]

Background/Aims: The two-pore-domain potassium channel TASK-1 regulates atrial action potential duration. Due to the atrium-specific expression of TASK-1 in the human heart and the functional upregulation of TASK-1 currents in atrial fibrillation (AF), TASK-1 represents a promising target for the treatment of AF. Therefore, detailed knowledge of the molecular determinants of TASK-1 inhibition may help to identify new drugs for the future therapy of AF. In the current study, the molecular determinants of TASK-1 inhibition by the potent and antiarrhythmic compound A293 (AVE1231) were studied in detail. Methods: Alanine-scanning mutagenesis together with two-electrode voltage-clamp recordings were combined with in silico docking experiments. Results: Here, we have identified Q126 located in the M2 segment together with L239 and N240 of the M4 segment as amino acids essential for the A293-mediated inhibition of TASK‑1. These data indicate a binding site which is different to that of A1899 for which also residues of the pore signature sequence and the late M4 segments are essential. Using in silico docking experiments, we propose a binding site at the lower end of the cytosolic pore, located at the entry to lateral side fenestrations of TASK-1. Strikingly, TASK-1 inhibition by the low affinity antiarrhythmic TASK‑1 blockers propafenone, amiodarone and carvedilol was also strongly diminished by mutations at this novel binding site. Conclusion: We have identified the A293 binding site in the central cavity of TASK-1 and propose that this site might represent a conserved site of action for many low affinity antiarrhythmic TASK-1 blockers. [ABSTRACT FROM AUTHOR]

Abstract: STIM1 ‘senses’ decreases in endoplasmic reticular (ER) luminal Ca2+ and induces store-operated Ca2+ (SOC) entry through plasma membrane Orai channels. The Ca2+/calmodulin-activated K+ channel KCa3.1 (previously known as SK4) has been implicated as an ‘amplifier’ of the Ca2+-release activated Ca2+ (CRAC) current, especially in T lymphocytes. We have previously shown that human macrophages express KCa3.1, and here we used the whole-cell patch-clamp technique to investigate the activity of these channels during Ca2+ store depletion and store-operated Ca2+ influx. Using RT-PCR, we found that macrophages express the elementary CRAC channel components Orai1 and STIM1, as well as Orai2, Orai3 and STIM2, but not the putatively STIM1-activated channels TRPC1, TRPC3–7 or TRPV6. In whole-cell configuration, a robust Ca2+-induced outwardly rectifying K+ current inhibited by clotrimazole and augmented by DC-EBIO could be detected, consistent with KCa3.1 channel current (also known as intermediate-conductance IK1). Introduction of extracellular Ca2+ following Ca2+ store depletion via P2Y2 receptors induced a robust charybdotoxin (CTX)- and 2-APB-sensitive outward K+ current and hyperpolarization. We also found that SOC entry induced by thapsigargin treatment induced CTX-sensitive K+ current in HEK293 cells transiently expressing KCa3.1. Our data suggest that SOC and KCa3.1 channels are tightly coupled, such that a small Ca2+ influx current induces a much large KCa3.1 channel current and hyperpolarization, providing the necessary electrochemical driving force for prolonged Ca2+ signaling and store repletion. [Copyright &y& Elsevier]

Abstract: Objective : The outward current flowing through the two-pore domain acid-sensitive potassium channel TASK-1 (I TASK) and its inhibition via α1-adrenergic receptors was studied in rat ventricular cardiomyocytes. Methods : Quantitative RT-PCR experiments were carried out with mRNA from rat heart. Patch-clamp recordings were performed in isolated rat cardiomyocytes. TASK-1 and other K+ channels were expressed in Xenopus oocytes to study the pharmacological properties of a new TASK-1 channel blocker, A293. Results : TASK-1 channels were found to be strongly expressed in rat heart. Analysis of the sensitivity of various K+ channels to A293 in Xenopus oocytes showed that at low concentrations A293 was a selective blocker of TASK-1 channels. I TASK in rat cardiomyocytes was dissected by application of A293 and by extracellular acidification to pH 6.0; it had an amplitude of ∼0.30 pA/pF at +30 mV. Application of 200 nM A293 increased action potential duration (APD50) by 31±3% at a stimulation rate of 4 Hz. The plausibility of the effects of A293 on APD50 was checked with a mathematical action potential model. Application of the α1-adrenergic agonist methoxamine inhibited I TASK in Xenopus oocytes co-injected with cRNA for TASK-1 and α1A-receptors. In cardiomyocytes, methoxamine inhibited an outward current with characteristics similar to I TASK. This effect was abolished in the presence of the α1A-antagonist 5-methyl-urapidil. Conclusions : Our results suggest that in rat cardiomyocytes I TASK makes a substantial contribution to the outward current flowing in the plateau range of potentials and that this current component can be inhibited via α1A-adrenergic receptors. [Copyright &y& Elsevier]

TASK channels belong to the two-pore-domain potassium (K2P) channels subfamily. These channels modulate cellular excitability, input resistance, and response to synaptic stimulation. TASK-channel inhibition led to membrane depolarization. TASK-3 is expressed in different cancer cell types and neurons. Thus, the discovery of novel TASK-3 inhibitors makes these bioactive compounds very appealing to explore new cancer and neurological therapies. TASK-3 channel blockers are very limited to date, and only a few heterofused compounds have been reported in the literature. In this article, we combined a pharmacophore hypothesis with molecular docking to address for the first time the rational design, synthesis, and evaluation of 5-(indol-2-yl)pyrazolo[3,4-b]pyridines as a novel family of human TASK-3 channel blockers. Representative compounds of the synthesized library were assessed against TASK-3 using Fluorometric imaging plate reader—Membrane Potential assay (FMP). Inhibitory properties were validated using two-electrode voltage-clamp (TEVC) methods. We identified one active hit compound (MM-3b) with our systematic pipeline, exhibiting an IC50 ≈ 30 μM. Molecular docking models suggest that compound MM-3b binds to TASK-3 at the bottom of the selectivity filter in the central cavity, similar to other described TASK-3 blockers such as A1899 and PK-THPP. Our in silico and experimental studies provide a new tool to predict and design novel TASK-3 channel blockers. [ABSTRACT FROM AUTHOR]

Dysfunction of noradrenergic locus coeruleus (LC) neurons is involved in psychiatric and neurodegenerative diseases and is an early hallmark of Parkinson's disease (PD). The analysis of ion channels underlying the autonomous electrical activity of LC neurons, which is ultimately coupled to cell survival signaling pathways, can lead to a better understanding of the vulnerability of these neurons. In LC neurons somatodendritic Ca 2 + oscillations, mediated by L-type Ca 2 + channels, accompany spontaneous spiking and are linked to mitochondrial oxidant stress. However, the expression and functional implication of low-threshold activated T-type Ca 2 + channels in LC neurons were not yet studied. To this end we performed RT-PCR expression analysis in LC neurons. In addition, we utilized slice patch clamp recordings of in vitro brainstem slices in combination with L-type and T-type Ca 2 + channel blockers. We found the expression of a distinct set of L-type and T-type Ca 2 + channel subtypes mediating a pronounced low-threshold activated Ca 2 + current component. Analyzing spike trains, we revealed that neither L-type Ca 2 + channel nor T-type Ca 2 + channel blockade alone leads to a change in firing properties. In contrast, a combined application of antagonists significantly decreased the afterhyperpolarization amplitude, resulting in an increased firing frequency. Hence, we report the functional expression of T-type Ca 2 + channels in LC neurons and demonstrate their role in increasing the robustness of LC pacemaking by working in concert with Cav1 channels. [ABSTRACT FROM AUTHOR]

Potassium channels of the tandem of two-pore-domain (K 2P ) family were among the last potassium channels cloned. However, recent progress in understanding their physiological relevance and molecular pharmacology revealed their therapeutic potential and thus these channels evolved as major drug targets against a large variety of diseases. However, after the initial cloning of the fifteen family members there was a lack of potent and/or selective modulators. By now a large variety of K 2P channel modulators (activators and blockers) have been described, especially for TASK-1, TASK-3, TREK-1, TREK2, TRAAK and TRESK channels. Recently obtained crystal structures of K 2P channels, alanine scanning approaches to map drug binding sites, in silico experiments with molecular dynamics simulations (MDs) combined with electrophysiological studies to reveal the mechanism of channel inhibition/activation, yielded a good understanding of the molecular pharmacology of these channels. Besides summarizing drugs that were identified to modulate K 2P channels, the main focus of this article is on describing the differential binding sites and mechanisms of channel modulation that are utilized by the different K 2P channel blockers and activators., Competing Interests: The authors have no conflicts of interest to declare., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)