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Potassium channel mutations play an important role in neurological diseases, such as spinocerebellar ataxia (SCA). SCA is a heterogeneous autosomal-dominant neurodegenerative disorder with multiple sub-entities, such as SCA13, which is characterized by mutations in the voltage-gated potassium channel Kv3.3 ( KCNC3 ). In this study, we present a rare and atypical case of SCA13 with a predominant episodic central rotational vertigo, while the patient suffered only from mild progressive cerebellar symptoms, such as dysarthria, ataxia of gait and stand, and recently a cognitive impairment. In this patient, we identified a heterozygous variant in KCNC3 (c.2023G > A, p.Glu675Lys) by next-generation sequencing. This Kv3.3 E675K variant was studied using voltage-clamp recordings in Xenopus oocytes. While typical SCA13 variants are dominant-negative, show shifts in the voltage-dependence of activation or an altered TBK1 regulation, the Kv3.3 E675K variant caused only a reduction in current amplitude and a more pronounced cumulative inactivation. Thus, the differences to phenotypes observed in patients with classical SCA13 mutations may be related to the mechanism of the observed Kv3.3 loss-of-function. Treatment of our patient with riluzole, a drug that is known to also activate potassium channels, turned out to be partly beneficial. Strikingly, we found that the Kv3.3 and Kv3.3 E675K inactivation and the frequency-dependent cumulative inactivation was antagonized by increased extracellular potassium levels. Thus, and most importantly, carefully elevated plasma potassium levels in the physiological range, or novel drugs attenuating Kv3.3 inactivation might provide novel therapeutic approaches to rescue potassium currents of SCA13 variants per se . In addition, our findings broaden the phenotypic spectrum of Kv3.3 variants, expanding it to atypical phenotypes of Kv3.3-associated neurological disorders., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Bernhard, Schütte, Heidenblut, Oehme, Rinné and Decher.)

Two-pore domain K + (K 2P ) channel activity was previously thought to be controlled primarily via a selectivity filter (SF) gate. However, recent crystal structures of TASK-1 and TASK-2 revealed a lower gate at the cytoplasmic pore entrance. Here, we report functional evidence of such a lower gate in the K 2P channel K2P17.1 (TALK-2, TASK-4). We identified compounds (drugs and lipids) and mutations that opened the lower gate allowing the fast modification of pore cysteine residues. Surprisingly, stimuli that directly target the SF gate (i.e., pH e ., Rb + permeation, membrane depolarization) also opened the cytoplasmic gate. Reciprocally, opening of the lower gate reduced the electric work to open the SF via voltage driven ion binding. Therefore, it appears that the SF is so rigidly locked into the TALK-2 protein structure that changes in ion occupancy can pry open a distant lower gate and, vice versa, opening of the lower gate concurrently promote SF gate opening. This concept might extent to other K + channels that contain two gates (e.g., voltage-gated K + channels) for which such a positive gate coupling has been suggested, but so far not directly demonstrated., (© 2024. The Author(s).)

TASK-5 (KCNK15) belongs to the acid-sensitive subfamily of two-pore domain potassium (K 2P ) channels, which includes TASK-1 and TASK-3. TASK-5 stands out as K 2P channel for which there is no functional data available, since it was reported in 2001 as non-functional and thus "silent". Here we show that TASK-5 channels are indeed non-functional as homodimers, but are involved in the formation of functional channel complexes with TASK-1 and TASK-3. TASK-5 negatively modulates the surface expression of TASK channels, while the heteromeric TASK-5-containing channel complexes located at the plasma membrane are characterized by changes in single-channel conductance, Gq-coupled receptor-mediated channel inhibition, and sensitivity to TASK modulators. The unique pharmacology of TASK-1/TASK-5 heterodimers, affected by a common polymorphism in KCNK15, needs to be carefully considered in the future development of drugs targeting TASK channels. Our observations provide an access to study TASK-5 at the functional level, particularly in malignant cancers associated with KCNK15., (© 2024. The Author(s).)

Low temperatures and cooling agents like menthol induce cold sensation by activating the peripheral cold receptors TRPM8 and TRPA1, cation channels belonging to the TRP channel family, while the reduction of potassium currents provides an additional and/or synergistic mechanism of cold sensation. Despite extensive studies over the past decades to identify the molecular receptors that mediate thermosensation, cold sensation is still not fully understood and many cold-sensitive peripheral neurons do not express the well-established cold sensor TRPM8. We found that the voltage-gated potassium channel KCNQ1 (Kv7.1), which is defective in cardiac LQT1 syndrome, is, in addition to its known function in the heart, a highly relevant and sex-specific sensor of moderately cold temperatures. We found that KCNQ1 is expressed in skin and dorsal root ganglion neurons, is sensitive to menthol and cooling agents, and is highly sensitive to moderately cold temperatures, in a temperature range at which TRPM8 is not thermosensitive. C-fiber recordings from KCNQ1 -/- mice displayed altered action potential firing properties. Strikingly, only male KCNQ1 -/- mice showed substantial deficits in cold avoidance at moderately cold temperatures, with a strength of the phenotype similar to that observed in TRPM8 -/- animals. While sex-dependent differences in thermal sensitivity have been well documented in humans and mice, KCNQ1 is the first gene reported to play a role in sex-specific temperature sensation. Moreover, we propose that KCNQ1, together with TRPM8, is a key instrumentalist that orchestrates the range and intensity of cold sensation., Competing Interests: Competing interests statement:The authors declare no competing interest.

Popeye domain containing (POPDC) proteins are predominantly expressed in the heart and skeletal muscle, modulating the K 2P potassium channel TREK-1 in a cAMP-dependent manner. POPDC1 and POPDC2 variants cause cardiac conduction disorders with or without muscular dystrophy. Searching for POPDC2-modulated ion channels using a functional co-expression screen in Xenopus oocytes, we found POPDC proteins to modulate the cardiac sodium channel Nav1.5. POPDC proteins downregulate Nav1.5 currents in a cAMP-dependent manner by reducing the surface expression of the channel. POPDC2 and Nav1.5 are both expressed in different regions of the murine heart and consistently POPDC2 co-immunoprecipitates with Nav1.5 from native cardiac tissue. Strikingly, the knock-down of popdc2 in embryonic zebrafish caused an increased upstroke velocity and overshoot of cardiac action potentials. The POPDC modulation of Nav1.5 provides a new mechanism to regulate cardiac sodium channel densities under sympathetic stimulation, which is likely to have a functional impact on cardiac physiology and inherited arrhythmias., Competing Interests: L.F. has received institutional research grants and non-financial support from European Union, DFG, British Heart Foundation, Medical Research Council (UK), NIHR, and several biomedical companies. L.F. is listed as inventor on two patents held by the academic employer (Atrial Fibrillation Therapy WO 2015140571, Markers for Atrial Fibrillation WO 2016012783)., (© 2024 The Author(s).)

Small conductance calcium-activated potassium (SK) channel activity has been proposed to play a role in the pathology of several neurological diseases. Besides regulating plasma membrane excitability, SK channel activation provides neuroprotection against ferroptotic cell death by reducing mitochondrial Ca 2+ uptake and reactive oxygen species (ROS). In this study, we employed a multifaceted approach, integrating structure-based and computational techniques, to strategically design and synthesize an innovative class of potent small-molecule SK2 channel modifiers through highly efficient multicomponent reactions (MCRs). The compounds' neuroprotective activity was compared with the well-studied SK positive modulator, CyPPA. Pharmacological SK channel activation by selected compounds confers neuroprotection against ferroptosis at low nanomolar ranges compared to CyPPA, that mediates protection at micromolar concentrations, as shown by an MTT assay, real-time cell impedance measurements and propidium iodide staining (PI). These novel compounds suppress increased mitochondrial ROS and Ca 2+ level induced by ferroptosis inducer RSL3. Moreover, axonal degeneration was rescued by these novel SK channel activators in primary mouse neurons and they attenuated glutamate-induced neuronal excitability, as shown via microelectrode array. Meanwhile, functional afterhyperpolarization of the novel SK2 channel modulators was validated by electrophysiological measurements showing more current change induced by the novel modulators than the reference compound, CyPPA. These data support the notion that SK2 channel activation can represent a therapeutic target for brain diseases in which ferroptosis and excitotoxicity contribute to the pathology., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The TWIK-related spinal cord K + channel (TRESK, K 2P 18.1) is a K 2P channel contributing to the maintenance of membrane potentials in various cells. Recently, physiological TRESK function was identified as a key player in T-cell differentiation rendering the channel a new pharmacological target for treatment of autoimmune diseases. The channel activator cloxyquin represents a promising lead compound for the development of a new class of immunomodulators. Identification of cloxyquin binding site and characterization of the molecular activation mechanism can foster the future drug development. Here, we identify the cloxyquin binding site at the M2/M4 interface by mutational scan and analyze the molecular mechanism of action by protein modeling as well as in silico and in vitro electrophysiology using different permeating ion species (K + / Rb + ). In combination with kinetic analyses of channel inactivation, our results suggest that cloxyquin allosterically stabilizes the inner selectivity filter facilitating the conduction process subsequently activating hTRESK., (© 2023. The Author(s).)

CACNA1C encodes the pore-forming α1C subunit of the L-type Ca 2+ channel, Cav1.2. Mutations and polymorphisms of the gene are associated with neuropsychiatric and cardiac disease. Haploinsufficient Cacna1c +/- rats represent a recently developed model with a behavioral phenotype, but its cardiac phenotype is unknown. Here, we unraveled the cardiac phenotype of Cacna1c +/- rats with a main focus on cellular Ca 2+ handling mechanisms. Under basal conditions, isolated ventricular Cacna1c +/- myocytes exhibited unaltered L-type Ca 2+ current, Ca 2+ transients (CaTs), sarcoplasmic reticulum (SR) Ca 2+ load, fractional release, and sarcomere shortenings. However, immunoblotting of left ventricular (LV) tissue revealed reduced expression of Cav1.2, increased expression of SERCA2a and NCX, and augmented phosphorylation of RyR2 (at S2808) in Cacna1c +/- rats. The β-adrenergic agonist isoprenaline increased amplitude and accelerated decay of CaTs and sarcomere shortenings in both Cacna1c +/- and WT myocytes. However, the isoprenaline effect on CaT amplitude and fractional shortening (but not CaT decay) was impaired in Cacna1c +/- myocytes exhibiting both reduced potency and efficacy. Moreover, sarcolemmal Ca 2+ influx and fractional SR Ca 2+ release after treatment with isoprenaline were smaller in Cacna1c +/- than in WT myocytes. In Langendorff-perfused hearts, the isoprenaline-induced increase in RyR2 phosphorylation at S2808 and S2814 was attenuated in Cacna1c +/- compared to WT hearts. Despite unaltered CaTs and sarcomere shortenings, Cacna1c +/- myocytes display remodeling of Ca 2+ handling proteins under basal conditions. Mimicking sympathetic stress with isoprenaline unmasks an impaired ability to stimulate Ca 2+ influx, SR Ca 2+ release, and CaTs caused, in part, by reduced phosphorylation reserve of RyR2 in Cacna1c +/- cardiomyocytes.

Tonic current through hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels is influencing neuronal firing properties and channel function is strongly influenced by the brain-specific auxiliary subunit tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b). Since Kv1.2 channels and TRIP8b were also suggested to interact, we assessed brain Kv1.2 mRNA and protein expression as well as the reduction of K + outward currents by Kv1.2-blocking compounds (Psora-4; tityustoxin-Kα, TsTX-Kα) in different brain areas of TRIP8b -deficient ( TRIP8b -/- ) compared to wildtype (WT) mice. We found that transcription levels of Kv1.2 channels were not different between genotypes. Furthermore, Kv1.2 current amplitude was not affected upon co-expression with TRIP8b in oocytes. However, Kv1.2 immunofluorescence was stronger in dendritic areas of cortical and hippocampal neurons. Furthermore, the peak net outward current was increased and the inactivation of the Psora-4-sensitive current component was less pronounced in cortical neurons in TRIP8b -/- mice. In current clamp recordings, application of TsTX increased the excitability of thalamocortical (TC) neurons with increased number of elicited action potentials upon step depolarization. We conclude that TRIP8b may not preferentially influence the amplitude of current through Kv1.2 channels but seems to affect current inactivation and channel localization. In TRIP8b -/- a compensatory upregulation of other Kv channels was observed., (© 2023 the author(s), published by De Gruyter, Berlin/Boston.)

K V 1.5 channels are key players in the regulation of vascular tone and atrial excitability and their impairment is associated with cardiovascular diseases including pulmonary arterial hypertension (PAH) and atrial fibrillation (AF). Unfortunately, pharmacological strategies to improve K V 1.5 channel function are missing. Herein, we aimed to study whether the chaperone sigma-1 receptor (S1R) is able to regulate these channels and represent a new strategy to enhance their function. By using different electrophysiological and molecular techniques in X. laevis oocytes and HEK293 cells, we demonstrate that S1R physically interacts with K V 1.5 channels and regulate their expression and function. S1R induced a bimodal regulation of K V 1.5 channel expression/activity, increasing it at low concentrations and decreasing it at high concentrations. Of note, S1R agonists (PRE084 and SKF10047) increased, whereas the S1R antagonist BD1047 decreased, K V 1.5 expression and activity. Moreover, PRE084 markedly increased K V 1.5 currents in pulmonary artery smooth muscle cells and attenuated vasoconstriction and proliferation in pulmonary arteries. We also show that both K V 1.5 channels and S1R, at mRNA and protein levels, are clearly downregulated in samples from PAH and AF patients. Moreover, the expression of both genes showed a positive correlation. Finally, the ability of PRE084 to increase K V 1.5 function was preserved under sustained hypoxic conditions, as an in vitro PAH model. Our study provides insight into the key role of S1R in modulating the expression and activity of K V 1.5 channels and highlights the potential role of this chaperone as a novel pharmacological target for pathological conditions associated with K V 1.5 channel dysfunction., Competing Interests: Conflict of interest None., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The KCNQ1 gene encodes the α-subunit of the cardiac voltage-gated potassium (Kv) channel KCNQ1, also denoted as Kv7.1 or KvLQT1. The channel assembles with the ß-subunit KCNE1, also known as minK, to generate the slowly activating cardiac delayed rectifier current I Ks , a key regulator of the heart rate dependent adaptation of the cardiac action potential duration (APD). Loss-of-function variants in KCNQ1 cause the congenital Long QT1 (LQT1) syndrome, characterized by delayed cardiac repolarization and a QT interval prolongation in the surface electrocardiogram (ECG). Autosomal dominant loss-of-function variants in KCNQ1 result in the LQT syndrome called Romano-Ward syndrome (RWS), while autosomal recessive variants affecting function, lead to Jervell and Lange-Nielsen syndrome (JLNS), associated with deafness. The aim of this study was the characterization of novel KCNQ1 variants identified in patients with RWS to widen the spectrum of known LQT1 variants, and improve the interpretation of the clinical relevance of variants in the KCNQ1 gene. We functionally characterized nine human KCNQ1 variants using the voltage-clamp technique in Xenopus laevis oocytes, from which we report seven novel variants. The functional data was taken as input to model surface ECGs, to subsequently compare the functional changes with the clinically observed QTc times, allowing a further interpretation of the severity of the different LQTS variants. We found that the electrophysiological properties of the variants correlate with the severity of the clinically diagnosed phenotype in most cases, however, not in all. Electrophysiological studies combined with in silico modelling approaches are valuable components for the interpretation of the pathogenicity of KCNQ1 variants, but assessing the clinical severity demands the consideration of other factors that are included, for example in the Schwartz score.

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., (© 2023. The Author(s).)

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., (©2022 The Authors. Published under the terms of the CC BY 4.0 license.)

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 -N -(2-ethyl-phenyl)-benzamide or F3 , blocking TASK-1 with an IC 50 of 148 nM, showing a reduced inhibition of TASK-3 channels and not a significant effect on different K + channels. We identified putative F3-binding sites in the TASK-1 channel by molecular modeling studies. Mutation of seven residues to A (I118A, L122A, F125A, Q126A, L232A, I235A, and L239A) markedly decreased the F3-induced inhibition of TASK-1 channels, consistent with the molecular modeling predictions. F3 blocks cell proliferation and viability in the MCF-7 cancer cell line but not in TASK-1 knockdown MCF-7 cells, indicating that it is acting in TASK-1 channels. These results indicated that TASK-1 is necessary to drive proliferation in the MCF-7 cancer cell line.

Cav1.3 voltage-gated L-type calcium channels (LTCCs) are involved in cardiac pacemaking, hearing and hormone secretion, but are also expressed postsynaptically in neurons. So far, homozygous loss of function mutations in CACNA1D encoding the Cav1.3 α 1 -subunit are described in congenital sinus node dysfunction and deafness. In addition, germline mutations in CACNA1D have been linked to neurodevelopmental syndromes including epileptic seizures, autism, intellectual disability and primary hyperaldosteronism. Here, a three-generation family with a syndromal phenotype of sinus node dysfunction, idiopathic epilepsy and attention deficit hyperactivity disorder (ADHD) is investigated. Whole genome sequencing and functional heterologous expression studies were used to identify the disease-causing mechanisms in this novel syndromal disorder. We identified a heterozygous non-synonymous variant (p.Arg930His) in the CACNA1D gene that cosegregated with the combined clinical phenotype in an autosomal dominant manner. Functional heterologous expression studies showed that the CACNA1D variant induces isoform-specific alterations of Cav1.3 channel gating: a gain of ion channel function was observed in the brain-specific short CACNA1D isoform (Cav1.3 S ), whereas a loss of ion channel function was seen in the long (Cav1.3 L ) isoform. The combined gain-of-function (GOF) and loss-of-function (LOF) induced by the R930H variant are likely to be associated with the rare combined clinical and syndromal phenotypes in the family. The GOF in the Cav1.3 S variant with high neuronal expression is likely to result in epilepsy, whereas the LOF in the long Cav1.3 L variant results in sinus node dysfunction.

The voltage-dependent L-type calcium channel isoform Ca V 1.2 is critically involved in many physiological processes, e.g., in cardiac action potential formation, electromechanical coupling and regulation of insulin secretion by beta cells. Gain-of-function mutations in the calcium voltage-gated channel subunit alpha 1 C ( CACNA1C ) gene, encoding the Ca V 1.2 α 1 -subunit, cause Timothy syndrome (TS), a multisystemic disorder that includes autism spectrum disorders and long QT (LQT) syndrome. Strikingly, TS patients frequently suffer from hypoglycemia of yet unproven origin. Using next-generation sequencing, we identified a novel heterozygous CACNA1C mutation in a patient with congenital hyperinsulinism (CHI) and associated hypoglycemic episodes. We characterized the electrophysiological phenotype of the mutated channel using voltage-clamp recordings and in silico action potential modeling experiments. The identified Ca V 1.2 L566P mutation causes a mixed electrophysiological phenotype of gain- and loss-of-function effects. In silico action potential modeling supports that this mixed electrophysiological phenotype leads to a tissue-specific impact on beta cells compared to cardiomyocytes. Thus, CACNA1C variants may be associated with non-syndromic hyperinsulinemic hypoglycemia without long-QT syndrome, explained by very specific electrophysiological properties of the mutated channel. We discuss different biochemical characteristics and clinical impacts of hypoglycemia in the context of CACNA1C variants and show that these may be associated with significant morbidity for Timothy Syndrome patients. Our findings underline that the potential of hypoglycemia warrants careful attention in patients with CACNA1C variants, and such variants should be included in the differential diagnosis of non-syndromic congenital hyperinsulinism.

Atrial fibrillation (AF) is the most common cardiac arrhythmia. Its treatment includes antiarrhythmic drugs (AADs) to modulate the function of cardiac ion channels. However, AADs have been limited by proarrhythmic effects, non-cardiovascular toxicities as well as often modest antiarrhythmic efficacy. Theoretical models showed that a combined blockade of Na v 1.5 (and its current, I Na ) and K v 1.5 (and its current, I Kur ) ion channels yield a synergistic anti-arrhythmic effect without alterations in ventricles. We focused on K v 1.5 and Na v 1.5 to search for structural similarities in their binding site (BS) for flecainide (a common blocker and widely prescribed AAD) as a first step for prospective rational multi-target directed ligand (MTDL) design strategies. We present a computational workflow for a flecainide BS comparison in a flecainide-K v 1.5 docking model and a solved structure of the flecainide-Na v 1.5 complex. The workflow includes docking, molecular dynamics, BS characterization and pattern matching. We identified a common structural pattern in flecainide BS for these channels. The latter belongs to the central cavity and consists of a hydrophobic patch and a polar region, involving residues from the S6 helix and P-loop. Since the rational MTDL design for AF is still incipient, our findings could advance multi-target atrial-selective strategies for AF treatment.

Aims: TASK-1 (K2P3.1) two-pore-domain potassium channels are atrial-specific and significantly up-regulated 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 this 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 days 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 days 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., Conclusion: Repurposing doxapram might yield a promising new antiarrhythmic drug to treat AF in patients., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: journals.permissions@oup.com.)

Loss-of-function mutations in K v 7.1 often lead to long QT syndrome (LQTS), a cardiac repolarization disorder associated with arrhythmia and subsequent sudden cardiac death. The discovery of agonistic I Ks modulators may offer a new potential strategy in pharmacological treatment of this disorder. The benzodiazepine derivative (R)-L3 potently activates K v 7.1 channels and shortens action potential duration, thus may represent a starting point for drug development. However, the molecular mechanisms underlying modulation by (R)-L3 are still unknown. By combining alanine scanning mutagenesis, non-canonical amino acid incorporation, voltage-clamp electrophysiology and fluorometry, and in silico protein modelling, we show that (R)-L3 not only stimulates currents by allosteric modulation of the pore domain but also alters the kinetics independently from the pore domain effects. We identify novel (R)-L3-interacting key residues in the lower S4-segment of K v 7.1 and observed an uncoupling of the outer S4 segment with the inner S5, S6 and selectivity filter segments., (© 2022. The Author(s).)

The identification of similar three-dimensional (3D) amino acid patterns among different proteins might be helpful to explain the polypharmacological profile of many currently used drugs. Also, it would be a reasonable first step for the design of novel multitarget compounds. Most of the current computational tools employed for this aim are limited to the comparisons among known binding sites, and do not consider several additional important 3D patterns such as allosteric sites or other conserved motifs. In the present work, we introduce Geomfinder2.0, which is a new and improved version of our previously described algorithm for the deep exploration and discovery of similar and druggable 3D patterns. As compared with the original version, substantial improvements that have been incorporated to our software allow: (i) to compare quaternary structures, (ii) to deal with a list of pairs of structures, (iii) to know how druggable is the zone where similar 3D patterns are detected and (iv) to significantly reduce the execution time. Thus, the new algorithm achieves up to 353x speedup as compared to the previous sequential version, allowing the exploration of a significant number of quaternary structures in a reasonable time. In order to illustrate the potential of the updated Geomfinder version, we show a case of use in which similar 3D patterns were detected in the cardiac ions channels NaV1.5 and TASK-1. These channels are quite different in terms of structure, sequence and function and both have been regarded as important targets for drugs aimed at treating atrial fibrillation. Finally, we describe the in vitro effects of tafluprost (a drug currently used to treat glaucoma, which was identified as a novel putative ligand of NaV1.5 and TASK-1) upon both ion channels' activity and discuss its possible repositioning as a novel antiarrhythmic drug., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Valdés-Jiménez, Jiménez-González, Kiper, Rinné, Decher, González, Reyes-Parada and Núñez-Vivanco.)

Parkinson's disease (PD) is clinically defined by the presence of the cardinal motor symptoms, which are associated with a loss of dopaminergic nigrostriatal neurons in the substantia nigra pars compacta (SNpc). While SNpc neurons serve as the prototypical cell-type to study cellular vulnerability in PD, there is an unmet need to extent our efforts to other neurons at risk. The noradrenergic locus coeruleus (LC) represents one of the first brain structures affected in Parkinson's disease (PD) and plays not only a crucial role for the evolving non-motor symptomatology, but it is also believed to contribute to disease progression by efferent noradrenergic deficiency. Therefore, we sought to characterize the electrophysiological properties of LC neurons in two distinct PD models: (1) in an in vivo mouse model of focal α-synuclein overexpression; and (2) in an in vitro rotenone-induced PD model. Despite the fundamental differences of these two PD models, α-synuclein overexpression as well as rotenone exposure led to an accelerated autonomous pacemaker frequency of LC neurons, accompanied by severe alterations of the afterhyperpolarization amplitude. On the mechanistic side, we suggest that Ca 2+ -activated K + (SK) channels are mediators of the increased LC neuronal excitability, as pharmacological activation of these channels is sufficient to prevent increased LC pacemaking and subsequent neuronal loss in the LC following in vitro rotenone exposure. These findings suggest a role of SK channels in PD by linking α-synuclein- and rotenone-induced changes in LC firing rate to SK channel dysfunction., (© 2022. The Author(s).)

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 K v channels by local anaesthetics remained unknown. Crystal structures of K v 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 K v 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 K v 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 K v channel affinity and stereoselectivity, as well as their spectrum of side effects., (© 2021 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

TASK channels belong to the two-pore-domain potassium (K 2P ) 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 IC 50 ≈ 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.

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.)

KCNQ1 encodes the voltage-gated potassium (Kv) channel KCNQ1, also known as KvLQT1 or Kv7.1. Together with its ß-subunit KCNE1, also denoted as minK, this channel generates the slowly activating cardiac delayed rectifier current I Ks , which is a key regulator of the heart rate dependent adaptation of the cardiac action potential duration (APD). Loss-of-function mutations in KCNQ1 cause congenital long QT1 (LQT1) syndrome, characterized by a delayed cardiac repolarization and a prolonged QT interval in the surface electrocardiogram. Autosomal dominant loss-of-function mutations in KCNQ1 result in long QT syndrome, called Romano-Ward Syndrome (RWS), while autosomal recessive mutations lead to Jervell and Lange-Nielsen syndrome (JLNS), associated with deafness. Here, we identified a homozygous KCNQ1 mutation, c.1892_1893insC (p.P631fs*20), in a patient with an isolated LQT syndrome (LQTS) without hearing loss. Nevertheless, the inheritance trait is autosomal recessive, with heterozygous family members being asymptomatic. The results of the electrophysiological characterization of the mutant, using voltage-clamp recordings in Xenopus laevis oocytes, are in agreement with an autosomal recessive disorder, since the I Ks reduction was only observed in homomeric mutants, but not in heteromeric I Ks channel complexes containing wild-type channel subunits. We found that KCNE1 rescues the KCNQ1 loss-of-function in mutant I Ks channel complexes when they contain wild-type KCNQ1 subunits, as found in the heterozygous state. Action potential modellings confirmed that the recessive c.1892_1893insC LQT1 mutation only affects the APD of homozygous mutation carriers. Thus, our study provides the molecular mechanism for an atypical autosomal recessive LQT trait that lacks hearing impairment.

TWIK-related K + channel (TREK-1) two-pore-domain potassium (K 2P ) 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., Competing Interests: Declaration of competing interest The authors declare that they have no competing financial interests or personal relationships that could be perceived to have influenced the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The hyperpolarization-activated cation current I f 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.

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 (POPDC2 W188⁎ ), 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 (K 2P ) channel TREK-1 is preferentially expressed in the atrioventricular node. Co-expression studies in Xenopus oocytes revealed that POPDC2 W188⁎ causes a loss-of-function with impaired TREK-1 modulation. Consistent with the high expression level of POPDC2 in the murine sinoatrial node, POPDC2 W188⁎ 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 POPDC2 W188⁎ 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., Competing Interests: Declaration of Competing Interest L.Fa has received institutional research grants from European Union, British Heart Foundation, Medical Research Council (UK), DFG and Gilead. L.Fa is listed as inventor on two patents held by University of Birmingham (Atrial Fibrillation Therapy WO 2015140571, Markers for Atrial Fibrillation WO 2016012783)., (Copyright © 2020 Elsevier Ltd. All rights reserved.)