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Aims TASK-1 (K2P3.1) two-pore domain potassium channels are atrial-specific and significantly upregulated in atrial fibrillation (AF) patients, contributing to AF-related electrical remodelling. Inhibition of TASK-1 in cardiomyocytes of AF patients was shown to counteract AF-related action potential duration shortening. Doxapram was identified as a potent inhibitor of the TASK-1 channel. In the present study, we investigated the antiarrhythmic efficacy of doxapram in a porcine model of AF. Methods and results Doxapram successfully cardioverted pigs with artificially induced episodes of AF. We established a porcine model of persistent AF in domestic pigs via intermittent atrial burst stimulation using implanted pacemakers. All pigs underwent catheter-based electrophysiological investigations prior to and after 14 d of doxapram treatment. Pigs in the treatment group received intravenous administration of doxapram once per day. In doxapram-treated AF pigs, the AF burden was significantly reduced. After 14 d of treatment with doxapram, TASK-1 currents were still similar to values of sinus rhythm animals. Doxapram significantly suppressed AF episodes and normalized cellular electrophysiology by inhibition of the TASK-1 channel. Patch-clamp experiments on human atrial cardiomyocytes, isolated from patients with and without AF could reproduce the TASK-1 inhibitory effect of doxapram. Conclusions Repurposing doxapram might yield a promising new antiarrhythmic drug to treat AF in patients. Translational perspective Pharmacological suppression of atrial TASK 1 potassium currents prolongs atrial refractoriness with no effects on ventricular repolarization, resulting in atrial-specific class III antiarrhythmic effects. In our preclinical pilot study the respiratory stimulant doxapram was successfully administered for cardioversion of acute AF as well as rhythm control of persistent AF in a clinically relevant porcine animal model.

The Popeye domain containing (POPDC) genes encode sarcolemma-localized cAMP effector proteins. Mutations in blood vessel epicardial substance (BVES) also known as POPDC1 and POPDC2 have been associated with limb-girdle muscular dystrophy and cardiac arrhythmia. Muscle biopsies of affected patients display impaired membrane trafficking of both POPDC isoforms. Biopsy material of patients carrying mutations in BVES were immunostained with POPDC antibodies. The interaction of POPDC proteins was investigated by co-precipitation, proximity ligation, bioluminescence resonance energy transfer and bimolecular fluorescence complementation. Site-directed mutagenesis was utilised to map the domains involved in protein–protein interaction. Patients carrying a novel homozygous variant, BVES (c.547G > T, p.V183F) displayed only a skeletal muscle pathology and a mild impairment of membrane trafficking of both POPDC isoforms. In contrast, variants such as BVES p.Q153X or POPDC2 p.W188X were associated with a greater impairment of membrane trafficking. Co-transfection analysis in HEK293 cells revealed that POPDC proteins interact with each other through a helix-helix interface located at the C-terminus of the Popeye domain. Site-directed mutagenesis of an array of ultra-conserved hydrophobic residues demonstrated that some of them are required for membrane trafficking of the POPDC1–POPDC2 complex. Mutations in POPDC proteins that cause an impairment in membrane localization affect POPDC complex formation while mutations which leave protein–protein interaction intact likely affect some other essential function of POPDC proteins.

Chemical structures of selective blockers of TASK channels contain aromatic groups and amide bonds. Using this rationale, we designed and synthesized a series of compounds based on 3-benzamidobenzoic acid. These compounds block TASK-1 channels by binding to the central cavity. The most active compound is 3-benzoylamino

BackgroundThe Popeye domain containing (POPDC) genes encode sarcolemma-localised cAMP effector proteins. Mutations inBVES (POPDC1)andPOPDC2have been associated with limb-girdle muscular dystrophy and cardiac arrhythmia. Muscle biopsies of affected patients display impaired membrane trafficking of both POPDC isoforms.MethodsBiopsy material of patients carrying mutations inBVESwere immunostained with POPDC antibodies. The interaction of POPDC proteins was investigated by co-precipitation, proximity ligation, bioluminescence resonance energy transfer and bimolecular fluorescence complementation. Site-directed mutagenesis was utilised to map the domains involved in protein interaction.FindingsPatients carrying a novel homozygous variant,BVES(c.547G>T, p.V183F) displayed only a skeletal muscle pathology and a mild impairment of membrane trafficking of both POPDC isoforms. This is in contrast to variants such asBVESp.Q153X orPOPDC2p.W188X, which were associated with a greater impairment of membrane trafficking. Co-transfection analysis in HEK293 cells revealed that POPDC proteins interact with each other through a helix-helix interface located at the C-terminus of the Popeye domain. Site-directed mutagenesis of an array of ultra-conserved hydrophobic residues demonstrated that some of them are required for membrane trafficking of the POPDC1-POPDC2 complex.InterpretationMutations in POPDC proteins that cause an impairment in membrane localisation affect POPDC complex formation while mutations which leave the protein interaction intact likely affect some other essential function of POPDC proteins.FundingThis study was funded by an EPSRC/British Heart Foundation co-funded Imperial Institute of Chemical Biology (ICB) Centre for Doctoral Training (CDT) PhD studentship (EP/S023518/1), a project grant of the British Heart Foundation (PG19/13/34247) and the Deutsche Forschungsgemeinschaft (DE1482/9-1).Research in ContextEvidence before this studySeveral biallelic missense and nonsense variants inBVES (POPDC1)have been described and are associated with heart and skeletal muscle disease. Skeletal muscle biopsies of homozygous carriers of these variants display a loss of sarcolemmal localisation of POPDC1 and POPDC2.Added value of this studyWe demonstrate that POPDC1 and POPDC2 form a heteromeric complex and that complex formation is required for plasma membrane trafficking of POPDC proteins. Transfection of different disease variants in HEK293 cells replicates their defective membrane targeting observed in biopsy material. Structural modelling and site-directed mutagenesis identifies an interface of strongly conserved hydrophobic residues in POPDC proteins, which likely mediate the interaction of POPDC proteins.Implications of all the available evidenceThese data provide novel insight into the membrane targeting requirements of POPDC proteins. We recommend testing the membrane targeting properties of any novel variant in POPDC isoforms using a newly developed co-transfection assay in HEK293 cells to characterise its pathogenicity. Our novel insight into the requirement of heterodimerization for proper membrane targeting may also offer novel opportunities to treat patients carrying mutations in POPDC proteins.

The establishment of macromolecular complexes by scaffolding proteins is key to the local production of cAMP by anchored adenylyl cyclase (AC) and the subsequent cAMP signaling necessary for cardiac functions. We identify a novel AC scaffold, the Popeye domain-containing (POPDC) protein. The POPDC family of proteins is important for cardiac pacemaking and conduction, due in part to their cAMP-dependent binding and regulation of TREK-1 potassium channels. We show that TREK-1 binds the AC9:POPDC1 complex and copurifies in a POPDC1-dependent manner with AC9 activity in heart. Although the AC9:POPDC1 interaction is cAMP-independent, TREK-1 association with AC9 and POPDC1 is reduced upon stimulation of the β-adrenergic receptor (βAR). AC9 activity is required for βAR reduction of TREK-1 complex formation with AC9:POPDC1 and in reversing POPDC1 enhancement of TREK-1 currents. Finally, deletion of the gene-encoding AC9 (Adcy9) gives rise to bradycardia at rest and stress-induced heart rate variability, a milder phenotype than the loss of Popdc1 but similar to the loss of Kcnk2 (TREK-1). Thus, POPDC1 represents a novel adaptor for AC9 interactions with TREK-1 to regulate heart rate control.

© 2021 The Authors., [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 Kv1.5 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 Kv1.5 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., The study was supported by the Ministerio de Ciencia e Innovación (MICINN, Spain) Grants SAF2016-75021-R and PID2019-104366RB-C21 (to C.V. and T.G.); the European Regional Development Fund (Fondo Europeo de Desarrollo Regional [FEDER]) and the Instituto de Salud Carlos III CIBERCV programme CB/11/00222 (to C.V. and T.G.); the Consejo Superior de Investigaciones Científicas (CSIC) Grants PIE201820E104 and 2019AEP148 (to C.V.); the Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT) 1191133 and the Fondo de Equipamiento Científico y Tecnológico (FONDEQUIP) 160063 grants from ANID (to W.G.); and the Deutsche Forschungsgemeinschaft (DFG) Grant DE1482-4/1 to N.D.

TWIK-related K+ channel (TREK-1) two-pore-domain potassium (K2P) channels mediate background potassium currents and regulate cellular excitability in many different types of cells. Their functional activity is controlled by a broad variety of different physiological stimuli, such as temperature, extracellular or intracellular pH, lipids and mechanical stress. By linking cellular excitability to mechanical stress, TREK-1 currents might be important to mediate parts of the mechanoelectrical feedback described in the heart. Furthermore, TREK-1 currents might contribute to the dysregulation of excitability in the heart in pathophysiological situations, such as those caused by abnormal stretch or ischaemia-associated cell swelling, thereby contributing to arrhythmogenesis. In this review, we focus on the functional role of TREK-1 in the heart and its putative contribution to cardiac mechanoelectrical coupling. Its cardiac expression among different species is discussed, alongside with functional evidence for TREK-1 currents in cardiomyocytes. In addition, evidence for the involvement of TREK-1 currents in different cardiac arrhythmias, such as atrial fibrillation or ventricular tachycardia, is summarized. Furthermore, the role of TREK-1 and its interaction partners in the regulation of the cardiac heart rate is reviewed. Finally, we focus on the significance of TREK-1 in the development of cardiac hypertrophy, cardiac fibrosis and heart failure.

The hyperpolarization-activated cation current If is a key determinant for cardiac pacemaker activity. It is conducted by subunits of the hyperpolarization-activated cyclic nucleotide–gated (HCN) channel family, of which HCN4 is predominant in mammalian heart. Both loss-of-function and gain-of-function mutations of the HCN4 gene are associated with sinus node dysfunction in humans; however, their functional impact is not fully understood yet. Here, we sought to characterize a HCN4 V759I variant detected in a patient with a family history of sick sinus syndrome. The genomic analysis yielded a mono-allelic HCN4 V759I variant in a 49-year-old woman presenting with a family history of sick sinus syndrome. This HCN4 variant was previously classified as putatively pathogenic because genetically linked to sudden infant death syndrome and malignant epilepsy. However, detailed electrophysiological and cell biological characterization of HCN4 V759I in Xenopus laevis oocytes and embryonic rat cardiomyocytes, respectively, did not reveal any obvious abnormality. Voltage dependence and kinetics of mutant channel activation, modulation of cAMP-gating by the neuronal HCN channel auxiliary subunit PEX5R, and cell surface expression were indistinguishable from wild-type HCN4. In good agreement, the clinically likewise affected mother of the patient does not exhibit the reported HCN4 variance. HCN4 V759I resembles an innocuous genetic HCN channel variant, which is not sufficient to disturb cardiac pacemaking. Once more, our work emphasizes the importance of careful functional interpretation of genetic findings not only in the context of hereditary cardiac arrhythmias.

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.

OBJECTIVE: The Popeye domain containing 3 (POPDC3) gene encodes a membrane protein involved in cyclic adenosine monophosphate (cAMP) signaling. Besides gastric cancer, no disease association has been described. We describe a new muscular dystrophy associated with this gene.METHODS: We screened 1,500 patients with unclassified limb girdle weakness or hyperCKemia for pathogenic POPDC3 variants. Five patients carrying POPDC3 variants were examined by muscle magnetic resonance imaging (MRI), muscle biopsy, and cardiac examination. We performed functional analyses in a zebrafish popdc3 knockdown model and heterologous expression of the mutant proteins in Xenopus laevis oocytes to measure TREK-1 current.RESULTS: We identified homozygous POPDC3 missense variants (p.Leu155His, p.Leu217Phe, and p.Arg261Gln) in 5 patients from 3 ethnically distinct families. Variants affected highly conserved residues in the Popeye (p.Leu155 and p.Leu217) and carboxy-terminal (p.Arg261) domains. The variants were almost absent from control populations. Probands' muscle biopsies were dystrophic, and serum creatine kinase levels were 1,050 to 9,200U/l. Muscle weakness was proximal with adulthood onset in most patients and affected lower earlier than upper limbs. Muscle MRI revealed fat replacement of paraspinal and proximal leg muscles; cardiac investigations were unremarkable. Knockdown of popdc3 in zebrafish, using 2 different splice-site blocking morpholinos, resulted in larvae with tail curling and dystrophic muscle features. All 3 mutants cloned in Xenopus oocytes caused an aberrant modulation of the mechano-gated potassium channel, TREK-1.INTERPRETATION: Our findings point to an important role of POPDC3 for skeletal muscle function and suggest that pathogenic variants in POPDC3 are responsible for a novel type of autosomal recessive limb girdle muscular dystrophy. ANN NEUROL 2019;86:832-843.

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 KA 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.Inhibition of KLocal anaesthetics bind to the central cavity and the side pockets, which provide a crucial key to the molecular understanding of their K

TWIK-related acid-sensitive potassium (TASK) channels—members of the two pore domain potassium (K2P) channel family—are found in neurons1, cardiomyocytes2–4 and vascular smooth muscle cells5, where they are involved in the regulation of heart rate6, pulmonary artery tone5,7, sleep/wake cycles8 and responses to volatile anaesthetics8–11. K2P channels regulate the resting membrane potential, providing background K+ currents controlled by numerous physiological stimuli12–15. Unlike other K2P channels, TASK channels are able to bind inhibitors with high affinity, exceptional selectivity and very slow compound washout rates. As such, these channels are attractive drug targets, and TASK-1 inhibitors are currently in clinical trials for obstructive sleep apnoea and atrial fibrillation16. In general, potassium channels have an intramembrane vestibule with a selectivity filter situated above and a gate with four parallel helices located below; however, the K2P channels studied so far all lack a lower gate. Here we present the X-ray crystal structure of TASK-1, and show that it contains a lower gate—which we designate as an ‘X-gate’—created by interaction of the two crossed C-terminal M4 transmembrane helices at the vestibule entrance. This structure is formed by six residues (243VLRFMT248) that are essential for responses to volatile anaesthetics10, neurotransmitters13 and G-protein-coupled receptors13. Mutations within the X-gate and the surrounding regions markedly affect both the channel-open probability and the activation of the channel by anaesthetics. Structures of TASK-1 bound to two high-affinity inhibitors show that both compounds bind below the selectivity filter and are trapped in the vestibule by the X-gate, which explains their exceptionally low washout rates. The presence of the X-gate in TASK channels explains many aspects of their physiological and pharmacological behaviour, which will be beneficial for the future development and optimization of TASK modulators for the treatment of heart, lung and sleep disorders. The X-ray crystal structure of the potassium channel TASK-1 reveals the presence of an X-gate, which traps small-molecule inhibitors in the intramembrane vestibule and explains their low washout rates from the channel.

TWIK-related K

Despite recent progress in the understanding of cardiac ion channel function and its role in inherited forms of ventricular arrhythmias, the molecular basis of cardiac conduction disorders often remains unresolved. We aimed to elucidate the genetic background of familial atrioventricular block (AVB) using a whole exome sequencing (WES) approach. In monozygotic twins with a third-degree AVB and in another, unrelated family with first-degree AVB, we identified a heterozygous nonsense mutation in the POPDC2 gene causing a premature stop at position 188 (POPDC2W188*), deleting parts of its cAMP binding-domain. Popeye-domain containing (POPDC) proteins are predominantly expressed in the skeletal muscle and the heart, with particularly high expression of POPDC2 in the sinoatrial node of the mouse. We now show by quantitative PCR experiments that in the human heart the POPDC-modulated two-pore domain potassium (K2P) channel TREK 1 is preferentially expressed in the atrioventricular node. Co-expression studies in Xenopus oocytes revealed that POPDC2W188* causes a loss-of-function with impaired TREK-1 modulation. Consistent with the high expression level of POPDC2 in the murine sinoatrial node, POPDC2W188* knock-in mice displayed stress-induced sinus bradycardia and pauses, a phenotype that was previously also reported for POPDC2 and TREK-1 knock-out mice. We propose that the POPDC2W188* loss-of-function mutation contributes to AVB pathogenesis by an aberrant modulation of TREK 1, highlighting that POPDC2 represents a novel arrhythmia gene for cardiac conduction disorders.

The genetic underpinnings that orchestrate the vertebrate heart rate are not fully understood yet, but of high clinical importance, since diseases of cardiac impulse formation and propagation are common and severe human arrhythmias. To identify novel regulators of the vertebrate heart rate, we deciphered the pathogenesis of the bradycardia in the homozygous zebrafish mutant hiphop (hip) and identified a missense-mutation (N851K) in Na+/K+-ATPase α1-subunit (atp1a1a.1). N851K affects zebrafish Na+/K+-ATPase ion transport capacity, as revealed by in vitro pump current measurements. Inhibition of the Na+/K+-ATPase in vivo indicates that hip rather acts as a hypomorph than being a null allele. Consequently, reduced Na+/K+-ATPase function leads to prolonged QT interval and refractoriness in the hip mutant heart, as shown by electrocardiogram and in vivo electrical stimulation experiments. We here demonstrate for the first time that Na+/K+-ATPase plays an essential role in heart rate regulation by prolonging myocardial repolarization.

The dorsal part of the lateral geniculate nucleus (dLGN) is the main thalamic site for state-dependent transmission of visual information. Non-retinal inputs from the ascending arousal system and inhibition provided by γ-aminobutyric acid (GABA)ergic local circuit interneurons (INs) control neuronal activity within the dLGN. In particular, acetylcholine (ACh) depolarizes thalamocortical relay (TC) neurons by inhibiting two-pore domain potassium (K2P) channels. Conversely, ACh also hyperpolarizes INs via an as-yet-unknown mechanism. By using whole cell patch-clamp recordings in brain slices and appropriate pharmacological tools we here report that stimulation of type 2 muscarinic ACh receptors (M2AChRs) induces IN hyperpolarization by recruiting the G beta-gamma complex (Gβγ), class-1A phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), and cellular and sarcoma (c-Src) tyrosine kinase (TK), leading to activation of two-pore domain weakly inwardly rectifying K+ channel (TWIK)-related acid-sensitive K+ (TASK)-1 channels. The latter was confirmed by the use of TASK-1 deficient mice. Furthermore inhibition of phospholipase Cβ (PLCβ) as well as an increase in the intracellular level of phosphatidylinositol-3,4,5-trisphosphate (PIP3) facilitated the muscarinic effect. Our results have uncovered a previously unknown role of c-Src TK in regulating IN function in the brain and identified a novel mechanism by which TASK-1 channels are activated in neurons. This article is protected by copyright. All rights reserved

In a patient with right ventricular outflow tract (RVOT) tachycardia, we identified a heterozygous point mutation in the selectivity filter of the stretch‐activated K2P potassium channel TREK‐1 ( KCNK2 or K2P2.1). This mutation introduces abnormal sodium permeability to TREK‐1. In addition, mutant channels exhibit a hypersensitivity to stretch‐activation, suggesting that the selectivity filter is directly involved in stretch‐induced activation and desensitization. Increased sodium permeability and stretch‐sensitivity of mutant TREK‐1 channels may trigger arrhythmias in areas of the heart with high physical strain such as the RVOT. We present a pharmacological strategy to rescue the selectivity defect of the TREK‐1 pore. Our findings provide important insights for future studies of K2P channel stretch‐activation and the role of TREK‐1 in mechano‐electrical feedback in the heart. ![][1] A point mutation in the selectivity filter of the stretch‐activated K2P potassium channel TREK‐1 was identified in a patient with right ventricular outflow tract tachycardia. The mutation most likely causes arrhythmias through abnormal sodium permeability and hypersensitivity to stretch‐activation. [1]: /embed/graphic-1.gif

TASK-3 is a two-pore domain potassium (K2P) channel highly expressed in the hippocampus, cerebellum, and cortex. TASK-3 has been identified as an oncogenic potassium channel and it is overexpressed in different cancer types. For this reason, the development of new TASK-3 blockers could influence the pharmacological treatment of cancer and several neurological conditions. In the present work, we searched for novel TASK-3 blockers by using a virtual screening protocol that includes pharmacophore modeling, molecular docking, and free energy calculations. With this protocol, 19 potential TASK-3 blockers were identified. These molecules were tested in TASK-3 using patch clamp, and one blocker (DR16) was identified with an IC50 = 56.8 &plusmn, 3.9 &mu, M. Using DR16 as a scaffold, we designed DR16.1, a novel TASK-3 inhibitor, with an IC50 = 14.2 &plusmn, 3.4 &mu, M. Our finding takes on greater relevance considering that not many inhibitory TASK-3 modulators have been reported in the scientific literature until today. These two novel TASK-3 channel inhibitors (DR16 and DR16.1) are the first compounds found using a pharmacophore-based virtual screening and rational drug design protocol.

TASK channels are unusual members of the two-pore domain potassium (K2P) channel family, with unique and unexplained physiological and pharmacological characteristics. TASKs are found in neurons1,2, cardiomyocytes3–5 and vascular smooth muscle cells6 where they are involved in regulation of heart rate7, pulmonary artery tone6,8, sleep/wake cycles9 and responses to volatile anaesthetics9–12. K2P channels regulate the resting membrane potential, providing background K+ currents controlled by numerous physiological stimuli13,14. Unlike other K2P channels, TASK channels have the capacity to bind inhibitors with high affinity, exceptional selectivity and very slow compound washout rates. These characteristics make the TASK channels some of the the most easily druggable potassium channels, and indeed TASK-1 inhibitors are currently in clinical trials for obstructive sleep apnea (OSA) and atrial fibrillation (Afib)15 (The DOCTOS and SANDMAN Trials). Generally, potassium channels have an intramembrane vestibule with a selectivity filter above and a gate with four parallel helices below. However, K2P channels studied to date all lack a lower gate. Here we present the structure of TASK-1, revealing a unique lower gate created by interaction of the two crossed C-terminal M4 transmembrane helices at the vestibule entrance, which we designate as an ‟X-gate”. This structure is formed by six residues (V243LRFMT248) that are essential for responses to volatile anaesthetics11, neuro-transmitters16 and G-protein coupled receptors16. Interestingly, mutations within the X-gate and surrounding regions drastically affect both open probability and activation by anaesthetics. Structures of TASK-1 with two novel, high-affinity blockers, shows both inhibitors bound below the selectivity filter, trapped in the vestibule by the X-gate, thus explaining their exceptionally low wash-out rates. Thus, the presence of the X-gate in TASK channels explains many aspects of their unusual physiological and pharmacological behaviour, which is invaluable for future development and optimization of TASK modulators for treatment of heart, lung and sleep disorders.

TWIK-related acid-sensitive potassium (TASK) channels-members of the two pore domain potassium (K

Rational drug design targeting ion channels is an exciting and always evolving research field. New medicinal chemistry strategies are being implemented to explore the wild chemical space and unravel the molecular basis of the ion channels modulators binding mechanisms. TASK channels belong to the two-pore domain potassium channel family and are modulated by extracellular acidosis. They are extensively distributed along the cardiovascular and central nervous systems, and their expression is up- and downregulated in different cancer types, which makes them an attractive therapeutic target. However, TASK channels remain unexplored, and drugs designed to target these channels are poorly selective. Here, we review TASK channels properties and their known blockers and activators, considering the new challenges in ion channels drug design and focusing on the implementation of computational methodologies in the drug discovery process.

TASK-3 potassium (K+) channels are highly expressed in the central nervous system, regulating the membrane potential of excitable cells. TASK-3 is involved in neurotransmitter action and has been identified as an oncogenic K+ channel. For this reason, the understanding of the action mechanism of pharmacological modulators of these channels is essential to obtain new therapeutic strategies. In this study we describe the binding mode of the potent antagonist PK-THPP into the TASK-3 channel. PK-THPP blocks TASK-1, the closest relative channel of TASK-3, with almost nine-times less potency. Our results confirm that the binding is influenced by the fenestrations state of TASK-3 channels and occurs when they are open. The binding is mainly governed by hydrophobic contacts between the blocker and the residues of the binding site. These interactions occur not only for PK-THPP, but also for the antagonist series based on 5,6,7,8 tetrahydropyrido[4,3-d]pyrimidine scaffold (THPP series). However, the marked difference in the potency of THPP series compounds such as 20b, 21, 22 and 23 (PK-THPP) respect to compounds such as 17b, inhibiting TASK-3 channels in the micromolar range is due to the presence of a hydrogen bond acceptor group that can establish interactions with the threonines of the selectivity filter.

Two-pore-domain potassium (K2P) channels are key regulators of many physiological and pathophysiological processes and thus emerged as promising drug targets. As for other potassium channels, there is a lack of selective blockers, since drugs preferentially bind to a conserved binding site located in the central cavity. Thus, there is a high medical need to identify novel drug-binding sites outside the conserved lipophilic central cavity and to identify new allosteric mechanisms of channel inhibition. Here, we identified a novel binding site and allosteric inhibition mechanism, disrupting the recently proposed K+-flux gating mechanism of K2Pchannels, which results in an unusual voltage-dependent block of leak channels belonging to the TASK subfamily. The new binding site and allosteric mechanism of inhibition provide structural and mechanistic insights into the gating of TASK channels and the basis for the drug design of a new class of potent blockers targeting specific types of K2Pchannels.

Inherited forms of sinus node dysfunction (SND) clinically include bradycardia, sinus arrest, and chronotropic incompetence and may serve as disease models to understand sinus node physiology and impulse generation. Recently, a gain-of-function mutation in the G-protein gene GNB2 led to enhanced activation of the GIRK (G-protein activated inwardly rectifying KWe performed a combined approach of targeted sequencing of KCNJ3 and KCNJ5 in 52 patients with idiopathic SND and subsequent whole exome sequencing of additional family members in a genetically affected patient. A putative novel disease-associated gene variant was functionally analyzed by voltage-clamp experiments using various heterologous cell expression systems (Xenopus oocytes, CHO cells, and rat atrial cardiomyocytes).In a 3-generation family with SND we identified a novel variant in KCNJ5 which leads to an amino acid substitution (p.Trp101Cys) in the first transmembrane domain of the Kir3.4 subunit of the cardiac GIRK channel. The identified variant cosegregated with the disease in the family and was absent in the Exome Variant Server and Exome Aggregation Consortium databases. Expression of mutant Kir3.4 (±native Kir3.1) in different heterologous cell expression systems resulted in increased GIRK currents ( IFor the first time, an inherited gain-of-function mutation in the human GIRK3.4 causes familial human SND. The increased activity of GIRK channels is likely to lead to a sustained hyperpolarization of pacemaker cells and thereby reduces heart rate. Modulation of human GIRK channels may pave a way for further treatment of cardiac pacemaking.