Publications by authors named "Guiscard Seebohm"

138 Publications

[2.2]Paracyclophane-based TCN-201 analogs as GluN2A-selective NMDA receptor antagonists.

ChemMedChem 2021 Jul 15. Epub 2021 Jul 15.

University Hospital Munster: Universitatsklinikum Munster, Medicine, Münster, GERMANY.

Recent studies have shown the involvement of GluN2A subunit-containing NMDA receptors in various neurological and pathological disorders. In the X-ray crystal structure, TCN-201 ( 1 ) and analogous pyrazine derivatives 2 and 3 adopt a U-shape (hairpin) conformation within the binding site formed by the ligand binding domains of the GluN1 and GluN2A subunits. In order to mimic the resulting π/π-interactions of two aromatic rings in the binding site, a [2.2]paracyclophane system was designed to lock these aromatic rings in a parallel orientation. Acylation of [2.2]paracyclophane ( 5 ) with oxalyl chloride and chloroacetyl chloride and subsequent transformations led to the oxalamide 7 , triazole 10 and benzamides 12 . The GluN2A inhibitory activities of the paracyclophane derivatives were tested with two-electrode voltage clamp electrophysiology using Xenopus laevis oocytes expressing selectively functional NMDA receptors with GluN2A subunit. The o -iodobenzamide 12b with the highest similarity to TCN-201 showed the highest GuN2A inhibitory activity of this series of compounds. At a concentration of 10 µM, 12b reached 36% of the inhibitory activity of TCN-201 ( 1 ). This result indicates that the [2.2]paracyclophane system is well accepted by the TCN-201 binding site.
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http://dx.doi.org/10.1002/cmdc.202100400DOI Listing
July 2021

Cardiac K Channels and Channelopathies.

Handb Exp Pharmacol 2021 Jul 11. Epub 2021 Jul 11.

Department of Cardiovascular Medicine, Institute for Genetics of Heart Diseases (IfGH), University Hospital Münster, Münster, Germany.

The physiological heart function is controlled by a well-orchestrated interplay of different ion channels conducting Na, Ca and K. Cardiac K channels are key players of cardiac repolarization counteracting depolarizating Na and Ca currents. In contrast to Na and Ca, K is conducted by many different channels that differ in activation/deactivation kinetics as well as in their contribution to different phases of the action potential. Together with modulatory subunits these K channel α-subunits provide a wide range of repolarizing currents with specific characteristics. Moreover, due to expression differences, K channels strongly influence the time course of the action potentials in different heart regions. On the other hand, the variety of different K channels increase the number of possible disease-causing mutations. Up to now, a plethora of gain- as well as loss-of-function mutations in K channel forming or modulating proteins are known that cause severe congenital cardiac diseases like the long-QT-syndrome, the short-QT-syndrome, the Brugada syndrome and/or different types of atrial tachyarrhythmias. In this chapter we provide a comprehensive overview of different K channels in cardiac physiology and pathophysiology.
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http://dx.doi.org/10.1007/164_2021_513DOI Listing
July 2021

Can Unlikely Neanderthal Chloride Channel CLC-2 Gene Variants Provide Insights in Modern Human Infertility?

Cell Physiol Biochem 2021 Jun;55(3):301-310

Cellular Electrophysiology and Molecular Biology, Institute for Genetics of Heart Diseases (IfGH), University Hospital Münster, Münster, Germany,

Background/aims: Neanderthals, although well adapted to local environments, were rapidly replaced by anatomically modern humans (AMH) for unknown reasons. Genetic information on Neanderthals is limited restricting applicability of standard population genetics.

Methods: Here, we apply a novel combination of restricted genetic analyses on preselected physiological key players (ion channels), electrophysiological analyses of gene variants of unclear significance expressed in Xenopus laevis oocytes using two electrode voltage clamp and transfer of results to AMH genetics. Using genetic screening in infertile men identified a loss of CLC-2 associated with sperm deficiency.

Results: Increased genetic variation caused functionally impaired Neanderthals CLC-2 channels.

Conclusion: Increased genetic variation could reflect an adaptation to different local salt supplies at the cost of reduced sperm density. Interestingly and consistent with this hypothesis, lack of CLC-2 protein in a patient associates with high blood K concentration and azoospermia.
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http://dx.doi.org/10.33594/000000376DOI Listing
June 2021

Beyond Hot and Spicy: TRPV Channels and their Pharmacological Modulation.

Cell Physiol Biochem 2021 May;55(S3):108-130

Institute for Genetics of Heart Diseases (IfGH), Department of Cardiovascular Medicine, University Hospital Münster, Münster, Germany.

Transient receptor potential vanilloid (TRPV) channels are part of the TRP channel superfamily and named after the first identified member TRPV1, that is sensitive to the vanillylamide capsaicin. Their overall structure is similar to the structure of voltage gated potassium channels (K) built up as homotetramers from subunits with six transmembrane helices (S1-S6). Six TRPV channel subtypes (TRPV1-6) are known, that can be subdivided into the thermoTRPV (TRPV1-4) and the Ca-selective TRPV channels (TRPV5, TRPV6). Contrary to K channels, TRPV channels are not primary voltage gated. All six channels have distinct properties and react to several endogenous ligands as well as different gating stimuli such as heat, pH, mechanical stress, or osmotic changes. Their physiological functions are highly diverse and subtype as well as tissue specific. In many tissues they serve as sensors for different pain stimuli (heat, pressure, pH) and contribute to the homeostasis of electrolytes, the maintenance of barrier functions and the development of macrophages. Due to their fundamental role in manifold physiological and pathophysiological processes, TRPV channels are promising targets for drug development. However, drugs targeting specific TRPV channels, that are suitable for drug therapy, are rare. Moreover, selective and potent compounds for further research at TRPV channels are often lacking. In this review different aspects of the structure, the different gating stimuli, the expression pattern, the physiological and pathophysiological roles as well as the modulating mechanisms of synthetic, natural and endogenous ligands are summarized.
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http://dx.doi.org/10.33594/000000358DOI Listing
May 2021

Deconstruction - Reconstruction: Analysis of the Crucial Structural Elements of GluN2B-Selective, Negative Allosteric NMDA Receptor Modulators with 3-Benzazepine Scaffold.

Cell Physiol Biochem 2021 Mar;55(S3):1-13

GRK 2515, Chemical biology of ion channels (Chembion), Westfälische Wilhelms-Universität Münster, Münster, Germany,

Background/aims: The NMDA receptor plays a key role in the pathogenesis of neurodegenerative disorders including Alzheimer's and Huntington's disease, as well as depression and drug or alcohol dependence. Due to its participation in these pathologies, the development of selective modulators for this ion channel is a promising strategy for rational drug therapy. The prototypical negative allosteric modulator ifenprodil inhibits selectively GluN2B subunit containing NMDA receptors. It was conformationally restricted as 2-methyl-3-(4-phenylbutyl)-2,3,4,5-tetrahydro-1H-3-benzazepine-1,7-diol, which showed high GluN2B affinity and inhibitory activity. For a better understanding of the relevance of the functional groups and structural elements, the substituents of this 3-benzazepine were removed successively (deconstruction). Then, additional structural elements were introduced (reconstruction) with the aim to analyze, which additional modifications were tolerated by the GluN2B receptor.

Methods: The GluN2B affinity was recorded in radioligand receptor binding studies with the radioligand [H]ifenprodil. The activity of the ligands was determined in two-electrode voltage clamp experiments using Xenopus laevis oocytes transfected with cRNA encoding the GluN1-1a and GluN2B subunits of the NMDA receptor. Docking studies showed the crucial interactions with the NMDA receptor protein.

Results: The deconstruction approach showed that removal of the methyl moiety and the phenolic OH moiety in 7-positon resulted in almost the same GluN2B affinity as the parent 3-benzazepine. A considerably reduced GluN2B affinity was found for the 3-benzazepine without further substituents. However, removal of one or both OH moieties led to considerably reduced NMDA receptor inhibition. Introduction of a NO moiety or bioisosteric replacement of the phenol by a benzoxazolone resulted in comparable GluN2B affinity, but almost complete loss of inhibitory activity. An O-atom, a carbonyl moiety or a F-atom in the tetramethylene spacer led to 6-7-fold reduced ion channel inhibition.

Conclusion: The results reveal an uncoupling of affinity and activity for the tested 3-benzazepines. Strong inhibition of [H]ifenprodil binding by a test compound does not necessarily translate into strong inhibition of the ion flux through the NMDA receptor associated ion channel. 3-(4-Phenylbutyl)-2,3,4,5-tetrahydro-1H-3-benzazepine- 1,7-diol (WMS-1410) shows high GluN2B affinity and strong inhibition of the ion channel. Deconstruction by removal of one or both OH moieties reduced the inhibitory activity proving the importance of the OH groups for ion channel blockade. Reconstruction by introduction of various structural elements into the left benzene ring or into the tetramethylene spacer reduced the NMDA receptor inhibition. It can be concluded that these modifications are not able to translate binding into inhibition.
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http://dx.doi.org/10.33594/000000335DOI Listing
March 2021

Ifenprodil Stereoisomers: Synthesis, Absolute Configuration, and Correlation with Biological Activity.

J Med Chem 2021 01 10;64(2):1170-1179. Epub 2021 Jan 10.

GRK 2515, Chemical Biology of Ion Channels (Chembion), Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany.

Ifenprodil () is a potent GluN2B-selective -methyl-d-aspartate (NMDA) receptor antagonist that is used as a cerebral vasodilator and has been examined in clinical trials for the treatment of drug addiction, idiopathic pulmonary fibrosis, and COVID-19. To correlate biological data with configuration, all four ifenprodil stereoisomers were prepared by diastereoselective reduction and subsequent separation of enantiomers by chiral HPLC. The absolute configuration of ifenprodil stereoisomers was determined by X-ray crystal structure analysis of (1,2)- and (1,2)-. GluN2B affinity, ion channel inhibitory activity, and selectivity over α, σ, and 5-HT receptors were evaluated. (1,2)-Ifenprodil ((1,2)-) showed the highest affinity toward GluN2B-NMDA receptors ( = 5.8 nM) and high inhibition of ion flux in two-electrode voltage clamp experiments (IC = 223 nM). Whereas the configuration did not influence considerably the GluN2B-NMDA receptor binding, (1)-configuration is crucial for elevated inhibitory activity. (1,2)-Configured ifenprodil (1,2)- exhibited high selectivity for GluN2B-NMDA receptors over adrenergic, serotonergic, and σ receptors.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01912DOI Listing
January 2021

Functional characterization of novel alpha-helical rod domain desmin (DES) pathogenic variants associated with dilated cardiomyopathy, atrioventricular block and a risk for sudden cardiac death.

Int J Cardiol 2021 04 26;329:167-174. Epub 2020 Dec 26.

Institute for Genetics of Heart Disease (IfGH), Department of Cardiovascular Medicine, University Hospital Muenster, Muenster, Germany.

Background: Desmin is the major intermediate filament (IF) protein in human heart and skeletal muscle. So-called 'desminopathies' are disorders due to pathogenic variants in the DES gene and are associated with skeletal myopathies and/or various types of cardiomyopathies. So far, only a limited number of DES pathogenic variants have been identified and functionally characterized.

Methods And Results: Using a Sanger- and next generation sequencing (NGS) approach in patients with various types of cardiomyopathies, we identified two novel, non-synonymous missense DES variants: p.(Ile402Thr) and p.(Glu410Lys). Mutation carriers developed dilated (DCM) or arrhythmogenic cardiomyopathy (ACM), and cardiac conduction disease, leading to spare out the exercise-induced polymorphic ventricular tachycardia; we moved this variant to data in brief. To investigate the functional impact of these four DES variants, transfection experiments using SW-13 and H9c2 cells with native and mutant desmin were performed and filament assembly was analyzed by confocal microscopy. The DES_p.(Ile402Thr) and DES_p.(Glu410Lys) cells showed filament assembly defects forming cytoplasmic desmin aggregates. Furthermore, immunohistochemical and ultrastructural analysis of myocardial tissue from mutation carriers with the DES_p.(Glu410Lys) pathogenic variant supported the in vitro results.

Conclusions: Our in vitro results supported the classification of DES_p.(Ile402Thr) and DES_p.(Glu410Lys) as novel pathogenic variants and demonstrated that the cardiac phenotypes associated with DES variants are diverse and cell culture experiments improve in silico analysis and genetic counseling because the pathogenicity of a variant can be clarified.
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http://dx.doi.org/10.1016/j.ijcard.2020.12.050DOI Listing
April 2021

Synthesis of GluN2A-selective NMDA receptor antagonists with an electron-rich aromatic B-ring.

Eur J Med Chem 2021 Jan 15;209:112939. Epub 2020 Oct 15.

Institut für Pharmazeutische und Medizinische Chemie der Westfälischen Wilhelms-Universität Münster, Corrensstraße 48, D-48149, Münster, Germany; Cells-in-Motion Cluster of Excellence (EXC 1003 - CiM), Westfälische Wilhelms-Universität Münster, D-48149, Münster, Germany. Electronic address:

Glutamatergic N-Methyl-d-aspartate (NMDA) receptors are heterotetrameric ion channels that can be comprised of different subunits. GluN2A subunit-containing NMDA receptors are associated with diseases like anxiety, depression, and schizophrenia. However, the exact contribution of these NMDA receptor subtypes is still unclear. To understand better the role of the GluN2A-containing receptors, novel ligands were designed. In co-crystallization with the isolated binding site, TCN-201 (1) and analogs adopt a U-shape conformation with parallel orientation of rings A and B. In order to increase the π/π-interactions between these rings, ring B of TCN-201 was replaced bioisosterically by different electron-rich thiazole, oxazole, and isoxazole heterocycles. The inhibitory activity was measured by two-electrode voltage clamp experiments with Xenopus laevis oocytes expressing GluN2A-containing NMDA receptors. It was found that 21c, 31a, 37a, and 37b were able to inhibit the ion channel. The isoxazole derivative 37b was the most potent negative allosteric modulator displaying 40% of the TCN-201 activity at a concentration of 10 μM.
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http://dx.doi.org/10.1016/j.ejmech.2020.112939DOI Listing
January 2021

A fully automated high-throughput workflow for 3D-based chemical screening in human midbrain organoids.

Elife 2020 11 3;9. Epub 2020 Nov 3.

Department for Cell and Developmental Biology, Max Planck Institute for molecular Biomedicine, Münster, Germany.

Three-dimensional (3D) culture systems have fueled hopes to bring about the next generation of more physiologically relevant high-throughput screens (HTS). However, current protocols yield either complex but highly heterogeneous aggregates ('organoids') or 3D structures with less physiological relevance ('spheroids'). Here, we present a scalable, HTS-compatible workflow for the automated generation, maintenance, and optical analysis of human midbrain organoids in standard 96-well-plates. The resulting organoids possess a highly homogeneous morphology, size, global gene expression, cellular composition, and structure. They present significant features of the human midbrain and display spontaneous aggregate-wide synchronized neural activity. By automating the entire workflow from generation to analysis, we enhance the intra- and inter-batch reproducibility as demonstrated via RNA sequencing and quantitative whole mount high-content imaging. This allows assessing drug effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow.
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http://dx.doi.org/10.7554/eLife.52904DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7609049PMC
November 2020

The first versatile human iPSC-based model of ectopic virus induction allows new insights in RNA-virus disease.

Sci Rep 2020 10 8;10(1):16804. Epub 2020 Oct 8.

Institute for Genetics of Heart Diseases (IfGH), Department of Cardiovascular Medicine, University Hospital Münster, 48149, Münster, Germany.

A detailed description of pathophysiological effects that viruses exert on their host is still challenging. For the first time, we report a highly controllable viral expression model based on an iPS-cell line from a healthy human donor. The established viral model system enables a dose-dependent and highly localized RNA-virus expression in a fully controllable environment, giving rise for new applications for the scientific community.
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http://dx.doi.org/10.1038/s41598-020-72966-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7546621PMC
October 2020

Discrete spatio-temporal regulation of tyrosine phosphorylation directs influenza A virus M1 protein towards its function in virion assembly.

PLoS Pathog 2020 08 31;16(8):e1008775. Epub 2020 Aug 31.

Institute of Virology Muenster, University of Muenster, Muenster, Germany.

Small RNA viruses only have a very limited coding capacity, thus most viral proteins have evolved to fulfill multiple functions. The highly conserved matrix protein 1 (M1) of influenza A viruses is a prime example for such a multifunctional protein, as it acts as a master regulator of virus replication whose different functions have to be tightly regulated. The underlying mechanisms, however, are still incompletely understood. Increasing evidence points towards an involvement of posttranslational modifications in the spatio-temporal regulation of M1 functions. Here, we analyzed the role of M1 tyrosine phosphorylation in genuine infection by using recombinant viruses expressing M1 phosphomutants. Presence of M1 Y132A led to significantly decreased viral replication compared to wildtype and M1 Y10F. Characterization of phosphorylation dynamics by mass spectrometry revealed the presence of Y132 phosphorylation in M1 incorporated into virions that is most likely mediated by membrane-associated Janus kinases late upon infection. Molecular dynamics simulations unraveled a potential phosphorylation-induced exposure of the positively charged linker domain between helices 4 and 5, supposably acting as interaction platform during viral assembly. Consistently, M1 Y132A showed a defect in lipid raft localization due to reduced interaction with viral HA protein resulting in a diminished structural stability of viral progeny and the formation of filamentous particles. Importantly, reduced M1-RNA binding affinity resulted in an inefficient viral genome incorporation and the production of non-infectious virions that interferes with virus pathogenicity in mice. This study advances our understanding of the importance of dynamic phosphorylation as a so far underestimated level of regulation of multifunctional viral proteins and emphasizes the potential feasibility of targeting posttranslational modifications of M1 as a novel antiviral intervention.
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http://dx.doi.org/10.1371/journal.ppat.1008775DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7485975PMC
August 2020

Structure of the TSC2 GAP Domain: Mechanistic Insight into Catalysis and Pathogenic Mutations.

Structure 2020 08 4;28(8):933-942.e4. Epub 2020 Jun 4.

Westfälische Wilhelms-Universität, Institute of Biochemistry, Wilhelm Klemm-Str. 2, 48149 Münster, Germany. Electronic address:

The TSC complex is the cognate GTPase-activating protein (GAP) for the small GTPase Rheb and a crucial regulator of the mechanistic target of rapamycin complex 1 (mTORC1). Mutations in the TSC1 and TSC2 subunits of the complex cause tuberous sclerosis complex (TSC). We present the crystal structure of the catalytic asparagine-thumb GAP domain of TSC2. A model of the TSC2-Rheb complex and molecular dynamics simulations suggest that TSC2 Asn1643 and Rheb Tyr35 are key active site residues, while Rheb Arg15 and Asp65, previously proposed as catalytic residues, contribute to the TSC2-Rheb interface and indirectly aid catalysis. The TSC2 GAP domain is further stabilized by interactions with other TSC2 domains. We characterize TSC2 variants that partially affect TSC2 functionality and are associated with atypical symptoms in patients, suggesting that mutations in TSC1 and TSC2 might predispose to neurological and vascular disorders without fulfilling the clinical criteria for TSC.
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http://dx.doi.org/10.1016/j.str.2020.05.008DOI Listing
August 2020

Rottlerin: Structure Modifications and KCNQ1/KCNE1 Ion Channel Activity.

ChemMedChem 2020 06 5;15(12):1078-1088. Epub 2020 May 5.

Faculty of Mathematics and Natural Sciences, University Wuppertal, 42119, Wuppertal, Germany.

The slow delayed rectifier potassium current (I ) is formed by the KCNQ1 (K 7.1) channel, an ion channel of four α-subunits that modulates KCNE1 β-subunits. I is central to the repolarization of the cardiac action potential. Loss of function mutation reducing ventricular cardiac I cause the long-QT syndrome (LQTS), a disorder that predisposes patients to arrhythmia and sudden death. Current therapy for LQTS is inadequate. Rottlerin, a natural product of the kamala tree, activates I and has the potential to provide a new strategy for rational drug therapy. In this study, we show that simple modifications such as penta-acetylation or penta-methylation of rottlerin blunts activation activity. Total synthesis was used to prepare side-chain-modified derivatives that slowed down KCNQ1/KCNE1 channel deactivation to different degrees. A binding hypothesis of rottlerin is provided that opens the way to improved I activators as novel therapeutics for the treatment of LQTS.
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http://dx.doi.org/10.1002/cmdc.202000083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7318133PMC
June 2020

4,4'-Diisothiocyanato-2,2'-Stilbenedisulfonic Acid (DIDS) Modulates the Activity of KCNQ1/KCNE1 Channels by an Interaction with the Central Pore Region.

Cell Physiol Biochem 2020 Apr;54(2):321-332

Cellular Electrophysiology and Molecular Biology, Institute for Genetics of Heart Diseases, Department of Cardiovascular Medicine, University Hospital Münster, Münster, Germany,

Background/aims: The cardiac current IKs is carried by the KCNQ1/KCNE1-channel complex. Genetic aberrations that affect the activity of KCNQ1/KCNE1 can lead to the Long QT Syndrome 1 and 5 and, thereby, to a predisposition to sudden cardiac death. This might be prevented by pharmacological modulation of KCNQ1/KCNE1. The prototypic KCNQ1/KCNE1 activator 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) represents a candidate drug. Here, we study the mechanism of DIDS action on KCNQ1/KCNE1.

Methods: Channels were expressed in Xenopus oocytes and iPSC cardiomyocytes. The role of the central S6 region was investigated by alanin-screening of KCNQ1 residues 333-338. DIDS effects were measured by TEVC and MEA.

Results: DIDS-action is influenced by the presence of KCNE1 but not by KCNQ1/KCNE1 stochiometry. V334A produces a significant higher increase in current amplitude, whereas deactivation (slowdown) DIDS-sensitivity is affected by residues 334-338.

Conclusion: We show that the central S6 region serves as a hub for allosteric channel activation by the drug and that DIDS shortens the pseudo QT interval in iPSC cardiomyocytes. The elucidation of the structural and mechanistic underpinnings of the DIDS action on KCNQ1/KCNE1 might allow for a targeted design of DIDS derivatives with improved potency and selectivity.
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http://dx.doi.org/10.33594/000000222DOI Listing
April 2020

Negative allosteric modulators of the GluN2B NMDA receptor with phenylethylamine structure embedded in ring-expanded and ring-contracted scaffolds.

Eur J Med Chem 2020 Mar 10;190:112138. Epub 2020 Feb 10.

Institut für Pharmazeutische und Medizinische Chemie der Universität Münster, Corrensstraße 48, D-48149, Münster, Germany; Cells-in-Motion Cluster of Excellence (EXC 1003 - CiM), Westfälische Wilhelms-Universität, Münster, Germany. Electronic address:

A set of GluN2B NMDA receptor antagonists with conformationally restricted phenylethylamine substructure was prepared and pharmacologically evaluated. The phenylethylamine substructure was embedded in ring expanded 3-benzazocines 4 as well as ring-contracted tetralinamines 6 and indanamines 7. The ligands 4, 6 and 7 were synthesized by reductive alkylation of secondary amine 11, reductive amination of ketones 12 and 16 and nucleophilic substitution of nosylates 14 and 17. The moderate GluN2B affinity of 3-benzazocine 4d (K = 32 nM) translated into moderate cytoprotective activity (IC = 890 nM) and moderate ion channel inhibition (60% at 10 μM) in two-electrode voltage clamp experiments with GluN1a/GluN2B expressing oocytes. Although some of the tetralinamines 6 and indanamines 7 showed very high GluN2B affinity (e.g. K (7f) = 3.2 nM), they could not inhibit glutamate/glycine inducted cytotoxicity. The low cytoprotective activity of 3-benzazocines 4, tetralinamines 6 and indanamines 7 was attributed to the missing OH moiety at the benzene ring and/or in benzylic position. Docking studies showed that the novel GluN2B ligands adopt similar binding poses as Ro 25-6981 with the central H-bond interaction between the protonated amino moiety of the ligands and the carbamoyl moiety of Gln110. However, due to the lack of a second H-bond forming group, the ligands can adopt two binding poses within the ifenprodil binding pocket.
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http://dx.doi.org/10.1016/j.ejmech.2020.112138DOI Listing
March 2020

Two-stage electro-mechanical coupling of a K channel in voltage-dependent activation.

Nat Commun 2020 02 3;11(1):676. Epub 2020 Feb 3.

Department of Biomedical Engineering, Center for the Investigation of Membrane Excitability Disorders, Cardiac Bioelectricity and Arrhythmia Center, Washington University in St. Louis, St. Louis, MO, 63130, USA.

In voltage-gated potassium (K) channels, the voltage-sensing domain (VSD) undergoes sequential activation from the resting state to the intermediate state and activated state to trigger pore opening via electro-mechanical (E-M) coupling. However, the spatial and temporal details underlying E-M coupling remain elusive. Here, utilizing K7.1's unique two open states, we report a two-stage E-M coupling mechanism in voltage-dependent gating of K7.1 as triggered by VSD activations to the intermediate and then activated state. When the S4 segment transitions to the intermediate state, the hand-like C-terminus of the VSD-pore linker (S4-S5L) interacts with the pore in the same subunit. When S4 then proceeds to the fully-activated state, the elbow-like hinge between S4 and S4-S5L engages with the pore of the neighboring subunit to activate conductance. This two-stage hand-and-elbow gating mechanism elucidates distinct tissue-specific modulations, pharmacology, and disease pathogenesis of K7.1, and likely applies to numerous domain-swapped K channels.
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http://dx.doi.org/10.1038/s41467-020-14406-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6997178PMC
February 2020

A common mechanism allows selective targeting of GluN2B subunit-containing -methyl-D-aspartate receptors.

Commun Biol 2019 15;2:420. Epub 2019 Nov 15.

5Cellular Electrophysiology and Molecular Biology, Institute for Genetics of Heart Diseases (IfGH), Department of Cardiovascular Medicine, University Hospital Münster, Robert-Koch-Str. 45, D-48149 Münster, Germany.

-methyl-D-aspartate receptors (NMDARs), especially GluN2B-containing NMDARs, are associated with neurodegenerative diseases like Parkinson, Alzheimer and Huntington based on their high Ca conductivity. Overactivation leads to high intracellular Ca concentrations and cell death rendering GluN2B-selective inhibitors as promising drug candidates. Ifenprodil represents the first highly potent prototypical, subtype-selective inhibitor of GluN2B-containing NMDARs. However, activity of ifenprodil on serotonergic, adrenergic and sigma receptors limits its therapeutic use. Structural reorganization of the ifenprodil scaffold to obtain 3-benzazepines retained inhibitory GluN2B activity but decreased the affinity at the mentioned non-NMDARs. While scaffold optimization improves the selectivity, the molecular inhibitory mechanism of these compounds is still not known. Here, we show a common inhibitory mechanism of ifenprodil and the related 3-benzazepines by mutational modifications of the receptor binding site, chemical modifications of the 3-benzazepine scaffold and subsequent in silico simulation of the inhibitory mechanism.
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http://dx.doi.org/10.1038/s42003-019-0645-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6858350PMC
May 2020

Cardiac α-Actin (ACTC1) Gene Mutation Causes Atrial-Septal Defects Associated With Late-Onset Dilated Cardiomyopathy.

Circ Genom Precis Med 2019 08 20;12(8):e002491. Epub 2019 Aug 20.

Institute for Genetics of Heart Diseases (IfGH), Department of Cardiovascular Medicine (C.F., S.D., B.S., Ellen Schulze-Bahr, S.P., G.S., A.U., Eric Schulze-Bahr), University Hospital Münster.

Background: Familial atrial septal defect (ASD) has previously been attributed primarily to mutations in cardiac transcription factors. Here, we report a large, multi-generational family (78 members) with ASD combined with a late-onset dilated cardiomyopathy and further characterize the consequences of mutant α-actin.

Methods: We combined a genome-wide linkage analysis with cell biology, microscopy, and molecular biology tools to characterize a novel ACTC1 (cardiac α-actin) mutation identified in association with ASD and late-onset dilated cardiomyopathy in a large, multi-generational family.

Results: Using a genome-wide linkage analysis, the ASD disease locus was mapped to chromosome 15q14 harboring the ACTC1 gene. In 15 affected family members, a heterozygous, nonsynonymous, and fully penetrant mutation (p. Gly247Asp) was identified in exon 5 of ACTC1 that was absent in all healthy family members (n=63). In silico tools predicted deleterious consequences of this variant that was found absent in control databases. Ultrastructural analysis of myocardial tissue of one of the mutation carriers showed sarcomeric disarray, myofibrillar degeneration, and increased apoptosis, while cardiac proteomics revealed a significant increase in extracellular matrix proteins. Consistently, structural defects and increased apoptosis were also observed in neonatal rat ventricular cardiomyocytes overexpressing the mutant, but not native human ACTC1. Molecular dynamics studies and additional mechanistic analyses in cardiomyocytes confirmed actin polymerization/turnover defects, thereby affecting contractility.

Conclusions: A combined phenotype of ASD and late-onset heart failure was caused by a heterozygous, nonsynonymous ACTC1 mutation. Mechanistically, we found a shared molecular mechanism of defective actin signaling and polymerization in both cardiac development and contractile function. Detection of ACTC1 mutations in patients with ASD may thus have further clinical implications with regard to monitoring for (late-onset) dilated cardiomyopathy.
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http://dx.doi.org/10.1161/CIRCGEN.119.002491DOI Listing
August 2019

A Kidnapping Story: How Coxsackievirus B3 and Its Host Cell Interact.

Cell Physiol Biochem 2019 ;53(1):121-140

Cellular Electrophysiology and Molecular Biology, Institute for Genetics of Heart Diseases (IfGH), University Hospital Münster, Münster, Germany,

Infections with Coxsackievirus B3 and other members of the enterovirus genus are a common reason for myocarditis and sudden cardiac death in modern society. Despite intensive scientific efforts to cure enterovirus infections, there is still no standardized treatment option. The complexity of Coxsackievirus B3´s effects on the host cell make well defined studies on this topic very challenging. However, recent publications report newly found effects of CVB3´s structural and non-structural proteins on infected cells. For the first time, the viral capsid protein VP1 was shown to have direct influence on the viral life-cycle. By shortening the G0 and the G2 phase and simultaneously prolonging the G1 and G1-S phase, the translation of viral proteins is enhanced and the production of viable CVB3 particles is promoted. Coxsackievirus B3´s viroporin, protein 2B, was recently studied in more detail as well. Structural and physiological analyses identified two hydrophilic α-helices in the structure of 2B, enabling it to insert into cellular membranes of host cells. As main target of 2B the endoplasmatic reticulum was identified. The insertion of 2B into the ER membranes leads to an uncontrolled calcium outflow into the cytoplasm. Additional insertion of 2B into the cell membrane leads to host cell destabilization and in the end to release of viral progeny. The importance of the Coxsackievirus B3´s proteases 2A and 3C in pathogenicity is observed since years. Recently, DAP5 and eIf4G were identified as new cleavage targets for protease 2A. Cleavage of DAP-5 into DAP5-N and DAP5-C changes the gene expression of the host cell and promotes cell death. Additionally, protease 3C targets and cleaves procaspase 8 promoting the mitochondrial apoptosis pathway and cell death. Recent studies identified significant effects of CVB3 on mitochondria of infected cells. Mouse cardiomyocytes showed decreased activities of respiratory chain complexes I-III and changed transcription of important subunits of the complexes I-IV. A disrupted energy metabolism may be one of the main causes of cardiac insufficiency and death in CVB3 infected patients. In addition to a modified energy metabolism, CVB3 affects cardiac ion channels, KCNQ1 in particular. SGK1, which is an important mediator in KCNQ1 membrane insertions, is highly upregulated during CVB3 infections. This results in an increased insertion of KCNQ1 into the cell membrane of cardiac cells. Under stress conditions, this KCNQ1 overshoot may lead to a disturbed cardiac action potential and therefore to sudden cardiac death, as it is often observed in CVB3 infected persons.
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http://dx.doi.org/10.33594/000000125DOI Listing
June 2019

An Assay to Determine Mechanisms of Rapid Autoantibody-Induced Neurotransmitter Receptor Endocytosis and Vesicular Trafficking in Autoimmune Encephalitis.

Front Neurol 2019 1;10:178. Epub 2019 Mar 1.

Department of Neurology, University of Muenster, Muenster, Germany.

N-Methyl-D-aspartate (NMDA) receptors (NMDARs) are among the most important excitatory neurotransmitter receptors in the human brain. Autoantibodies to the human NMDAR cause the most frequent form of autoimmune encephalitis involving autoantibody-mediated receptor cross-linking and subsequent internalization of the antibody-receptor complex. This has been deemed to represent the predominant antibody effector mechanism depleting the NMDAR from the synaptic and extra-synaptic neuronal cell membrane. To assess in detail the molecular mechanisms of autoantibody-induced NMDAR endocytosis, vesicular trafficking, and exocytosis we transiently co-expressed rat GluN1-1a-EGFP and GluN2B-ECFP alone or together with scaffolding postsynaptic density protein 95 (PSD-95), wild-type (WT), or dominant-negative (DN) mutant Ras-related in brain (RAB) proteins (RAB5WT, RAB5DN, RAB11WT, RAB11DN) in HEK 293T cells. The cells were incubated with a pH-rhodamine-labeled human recombinant monoclonal GluN1 IgG1 autoantibody (GluN1-aAb) genetically engineered from clonally expanded intrathecal plasma cells from a patient with anti-NMDAR encephalitis, and the pH-rhodamine fluorescence was tracked over time. We show that due to the acidic luminal pH, internalization of the NMDAR-autoantibody complex into endosomes and lysosomes increases the pH-rhodamine fluorescence. The increase in fluorescence allows for mechanistic assessment of endocytosis, vesicular trafficking in these vesicular compartments, and exocytosis of the NMDAR-autoantibody complex under steady state conditions. Using this method, we demonstrate a role for PSD-95 in stabilization of NMDARs in the cell membrane in the presence of GluN1-aAb, while RAB proteins did not exert a significant effect on vertical trafficking of the internalized NMDAR autoantibody complex in this heterologous expression system. This novel assay allows to unravel molecular mechanisms of autoantibody-induced receptor internalization and to study novel small-scale specific molecular-based therapies for autoimmune encephalitis syndromes.
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http://dx.doi.org/10.3389/fneur.2019.00178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6405626PMC
March 2019

Familial Sinus Node Disease Caused by a Gain of GIRK (G-Protein Activated Inwardly Rectifying K Channel) Channel Function.

Circ Genom Precis Med 2019 01;12(1):e002238

Department of Cardiovascular Medicine, Institute for Genetics of Heart Diseases (IfGH), University Hospital Münster, Germany (J.K., B.S., S.Z., G.S., E.S.-B.).

Background: 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 K channel). Thus, human cardiac GIRK channels are important for heart rate regulation and subsequently, genes encoding their subunits Kir3.1 and Kir3.4 ( KCNJ3 and KCNJ5) are potential candidates for inherited SND in human.

Methods: We 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).

Results: 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 ( I) and a reduced inward rectification which was not compensated by intracellular spermidine. Moreover, in silico modeling of heterotetrameric mutant GIRK channels indicates a structurally altered binding site for spermine.

Conclusions: For 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.
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http://dx.doi.org/10.1161/CIRCGEN.118.002238DOI Listing
January 2019

Systematic variation of the benzoylhydrazine moiety of the GluN2A selective NMDA receptor antagonist TCN-201.

Eur J Med Chem 2018 Oct 6;158:259-269. Epub 2018 Sep 6.

Institute of Pharmaceutical and Medicinal Chemistry, University of Münster, Corrensstr. 48, Münster, D-48149, Germany; Cells-in-Motion Cluster of Excellence (EXC 1003 - CiM), University Münster, Germany. Electronic address:

GluN2A containing N-methyl-D-aspartate receptors (NMDARs) are important ion channels in the central nervous system and highly involved in several different neurophysiological but also neuropathophysiological processes. However, current understanding of the contribution of GluN2A containing NMDARs in these processes is incomplete. Therefore, highly selective compounds are required to further investigate these ion channels. In 2010, TCN-201 (2), one of the first selective negative allosteric modulators was reported. While the binding site of 2 and the influence of the substitution pattern of the benzenesulfonamide part has been reported recently, detailed structure-activity-relationships of the diacylhydrazine part and the linked phenyl moiety are still missing. In order to examine the critical interactions between these moieties and the binding site, several TCN-201 analogs with modified diacylhydrazine part were synthesized. The negative allosteric effect was recorded by two-electrode voltage clamp (TEVC) experiments using GluN1a/GluN2A expressing Xenopus laevis oocytes. Our data led to the conclusion, that the terminal phenyl moiety is involved in a cation-π-interaction with the guanidinium moiety of Arg755 of the GluN1a subunit, which plays a crucial role for high activity. Additionally, structure optimization by replacing the phenyl moiety with a thiophen-2-yl (10c), indol-2-yl (10g) or indol-3-yl (10h) moiety significantly increased the activity of 2 by the factor 2.5. At a test compound concentration of 200 nM, the negative allosteric effect of the most potent ligands 10c, 10h and 17 was significantly influenced by the glycine concentration. Although glycine dependency is higher than those of the lead compound 4, 10c and 17 showed significantly higher negative allosteric effects than 4 at glycine concentrations from 1 μM up to 10 μM. The potent GluN2A-NMDA receptor inhibitors 10c, 10h and 17 did not influence the ion current of GluN2B-NMDA receptors.
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http://dx.doi.org/10.1016/j.ejmech.2018.09.006DOI Listing
October 2018

In Vitro Analyses of Novel HCN4 Gene Mutations.

Cell Physiol Biochem 2018 7;49(3):1197-1207. Epub 2018 Sep 7.

Myocellular Electrophysiology and Molecular Biology, Institute for Genetics of Heart Diseases (IfGH), Department of Cardiovascular Medicine, University Hospital Muenster, Muenster, Germany.

Background/aims: The hyperpolarization-activated cyclic nucleotide-gated cation channel HCN4 contributes significantly to the generation of basic cardiac electrical activity in the sinus node and is a mediator of modulation by β-adrenergic stimulation. Heterologous expression of sick sinus syndrome (SSS) and bradycardia associated mutations within the human HCN4 gene results in altered channel function. The main aim was to describe the functional characterization of three (two novel and one known) missense mutations of HCN4 identified in families with SSS.

Methods: Here, the two-electrode voltage clamp technique on Xenopus laevis oocytes and confocal imaging on transfected COS7 cells respectively, were used to analyze the functional effects of three HCN4 mutations; R378C, R550H, and E1193Q. Membrane surface expressions of wild type and the mutant channels were assessed by confocal microscopy, chemiluminescence assay, and Western blot in COS7 and HeLa cells.

Results: The homomeric mutant channels R550H and E1193Q showed loss of function through increased rates of deactivation and distinctly reduced surface expression in all three homomeric mutant channels. HCN4 channels containing R550H and E1193Q mutant subunits only showed minor effects on the voltage dependence and rates of activation/deactivation. In contrast, homomeric R378C exerted a left-shifted activation curve and slowed activation kinetics. These effects were reduced in heteromeric co-expression of R378C with wild-type (WT) channels.

Conclusion: Dysfunction of homomeric/heteromeric mutant HCN4-R378C, R550H, and E1193Q channels in the present study was primarily caused by loss of function due to decreased channel surface expression.
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http://dx.doi.org/10.1159/000493301DOI Listing
October 2018

Bioelectric-calcineurin signaling module regulates allometric growth and size of the zebrafish fin.

Sci Rep 2018 Jul 10;8(1):10391. Epub 2018 Jul 10.

Department of Genetics, Harvard Medical School; Department of Orthopedic Research, Boston Children's Hospital, 300 Longwood Avenue, Boston, MA, 02115, USA.

The establishment of relative size of organs and structures is paramount for attaining final form and function of an organism. Importantly, variation in the proportions of structures frequently underlies adaptive change in morphology in evolution and maybe a common mechanism underlying selection. However, the mechanism by which growth is integrated within tissues during development to achieve proper proportionality is poorly understood. We have shown that signaling by potassium channels mediates coordinated size regulation in zebrafish fins. Recently, calcineurin inhibitors were shown to elicit changes in zebrafish fin allometry as well. Here, we identify the potassium channel kcnk5b as a key player in integrating calcineurin's growth effects, in part through regulation of the cytoplasmic C-terminus of the channel. We propose that the interaction between Kcnk5b and calcineurin acts as a signaling node to regulate allometric growth. Importantly, we find that this regulation is epistatic to inherent mechanisms instructing overall size as inhibition of calcineurin is able to bypass genetic instruction of size as seen in sof and wild-type fins, however, it is not sufficient to re-specify positional memory of size of the fin. These findings integrate classic signaling mediators such as calcineurin with ion channel function in the regulation of size and proportion during growth.
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http://dx.doi.org/10.1038/s41598-018-28450-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6039437PMC
July 2018

Synthesis and Pharmacological Evaluation of Enantiomerically Pure GluN2B Selective NMDA Receptor Antagonists.

ChemMedChem 2018 08 4;13(15):1580-1587. Epub 2018 Jul 4.

Institut für Pharmazeutische und Medizinische Chemie der, Universität Münster, Corrensstraße 48, 48149, Münster, Germany.

To determine the eutomers of potent GluN2B-selective N-methyl-d-aspartate (NMDA) receptor antagonists with a 3-benzazepine scaffold, 7-benzyloxy-3-(4-phenylbutyl)-2,3,4,5-tetrahydro-1H-3-benzazepin-1-ols (S)-2 and (R)-2 were separated by chiral HPLC. Hydrogenolysis and subsequent methylation of the enantiomerically pure benzyl ethers of (S)-2 and (R)-2 provided the enantiomeric phenols (S)-3 and (R)-3 [3-(4-phenylbutyl)-2,3,4,5-tetrahydro-1H-3-benzazepine-1,7-diol] and methyl ethers (S)-4 and (R)-4. All enantiomers were obtained with high enantiomeric purity (≥99.7 % ee). The absolute configurations were determined by CD spectroscopy. R-configured enantiomers turned out to be the eutomers in receptor binding studies and two-electrode voltage clamp experiments. The most promising ligand of this compound series is the R-configured phenol (R)-3, displaying high GluN2B affinity (K =30 nm), high inhibition of ion flux (IC =61 nm), and high cytoprotective activity (IC =93 nm). Whereas the eudismic ratio in the receptor binding assay is 25, the eudismic ratio in the electrophysiological experiment is 3.
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http://dx.doi.org/10.1002/cmdc.201800214DOI Listing
August 2018

Cardiogenic programming of human pluripotent stem cells by dose-controlled activation of EOMES.

Nat Commun 2018 01 30;9(1):440. Epub 2018 Jan 30.

Human Stem Cell Pluripotency Laboratory, Max Planck Institute for Molecular Biomedicine, 48149, Münster, Germany.

Master cell fate determinants are thought to induce specific cell lineages in gastrulation by orchestrating entire gene programs. The T-box transcription factor EOMES (eomesodermin) is crucially required for the development of the heart-yet it is equally important for endoderm specification suggesting that it may act in a context-dependent manner. Here, we define an unrecognized interplay between EOMES and the WNT signaling pathway in controlling cardiac induction by using loss and gain-of-function approaches in human embryonic stem cells. Dose-dependent EOMES induction alone can fully replace a cocktail of signaling molecules otherwise essential for the specification of cardiogenic mesoderm. Highly efficient cardiomyocyte programming by EOMES mechanistically involves autocrine activation of canonical WNT signaling via the WNT3 ligand, which necessitates a shutdown of this axis at a subsequent stage. Our findings provide insights into human germ layer induction and bear biotechnological potential for the robust production of cardiomyocytes from engineered stem cells.
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http://dx.doi.org/10.1038/s41467-017-02812-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5789885PMC
January 2018

Stress-Kinase Regulation of TASK-1 and TASK-3.

Cell Physiol Biochem 2017 27;44(3):1024-1037. Epub 2017 Nov 27.

Institute of Physiology and Pathophysiology, Vegetative Physiology, University of Marburg, Marburg, Germany.

Background/aims: TASK channels belong to the two-pore-domain potassium (K2P) channel family. TASK-1 is discussed to contribute to chronic atrial fibrillation (AFib) and has been together with uncoupling protein 1 found as a marker protein of brown adipose tissue (BAT) fat. In addition, TASK-1 was linked in a genome-wide association study to an increased body mass index. A recent study showed that TASK-1 inhibition is causing obesity in mice by a BAT whitening and that these effects are linked to the mineralocorticoid receptor pathway, albeit the mechanism remained elusive. Therefore, we aimed to probe whether K2P channels are regulated by serum- and glucocorticoid-inducible kinases (SGKs) which are known to modify many cellular functions by modulating ion channels.

Methods: To this end we used functional co-expression studies and chemiluminescence-assays in Xenopus oocytes, together with fluorescence imaging and quantitative PCR experiments.

Results: SGKs and proteinkinase B (PKB) induced a strong, dose- and time-dependent current reduction of TASK-1 and TASK-3. SGK co-expression reduced the surface expression of TASK-1/3, leading to a predominant localization of the channels into late endosomes. The down regulation of TASK-3 channels was abrogated by the dynamin inhibitor dynasore, confirming a role of SGKs in TASK-1/3 channel endocytosis.

Conclusion: Stress-mediated changes in SGK expression pattern or activation is likely to alter TASK-1/3 expression at the surface membrane. The observed TASK-1 regulation might contribute to the pathogenesis of chronic AFib and provide a mechanistic link between increased mineralocorticoid levels and TASK-1 reduction, both linked to BAT whitening.
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http://dx.doi.org/10.1159/000485402DOI Listing
January 2018

NMDAR encephalitis: passive transfer from man to mouse by a recombinant antibody.

Ann Clin Transl Neurol 2017 11 3;4(11):768-783. Epub 2017 Oct 3.

Department of Neurology Medical Faculty Heinrich Heine University Düsseldorf Düsseldorf Germany.

Objective: Autoimmune encephalitis is most frequently associated with anti-NMDAR autoantibodies. Their pathogenic relevance has been suggested by passive transfer of patients' cerebrospinal fluid (CSF) in mice in vivo. We aimed to analyze the intrathecal plasma cell repertoire, identify autoantibody-producing clones, and characterize their antibody signatures in recombinant form.

Methods: Patients with recent onset typical anti-NMDAR encephalitis were subjected to flow cytometry analysis of the peripheral and intrathecal immune response before, during, and after immunotherapy. Recombinant human monoclonal antibodies (rhuMab) were cloned and expressed from matching immunoglobulin heavy- (IgH) and light-chain (IgL) amplicons of clonally expanded intrathecal plasma cells (cePc) and tested for their pathogenic relevance.

Results: Intrathecal accumulation of B and plasma cells corresponded to the clinical course. The presence of cePc with hypermutated antigen receptors indicated an antigen-driven intrathecal immune response. Consistently, a single recombinant human GluN1-specific monoclonal antibody, rebuilt from intrathecal cePc, was sufficient to reproduce NMDAR epitope specificity in vitro. After intraventricular infusion in mice, it accumulated in the hippocampus, decreased synaptic NMDAR density, and caused severe reversible memory impairment, a key pathogenic feature of the human disease, in vivo.

Interpretation: A CNS-specific humoral immune response is present in anti-NMDAR encephalitis specifically targeting the GluN1 subunit of the NMDAR. Using reverse genetics, we recovered the typical intrathecal antibody signature in recombinant form, and proved its pathogenic relevance by passive transfer of disease symptoms from man to mouse, providing the critical link between intrathecal immune response and the pathogenesis of anti-NMDAR encephalitis as a humorally mediated autoimmune disease.
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http://dx.doi.org/10.1002/acn3.444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5682115PMC
November 2017

Adrenergic Stress Protection of Human iPS Cell-Derived Cardiomyocytes by Fast K7.1 Recycling.

Front Physiol 2017 14;8:705. Epub 2017 Sep 14.

Department of Cardiovascular Medicine, Institute of Genetics of Heart Diseases, University of Münster Medical SchoolMünster, Germany.

The fight-or-flight response (FFR), a physiological acute stress reaction, involves positive chronotropic and inotropic effects on heart muscle cells mediated through β-adrenoceptor activation. Increased systolic calcium is required to enable stronger heart contractions whereas elevated potassium currents are to limit the duration of the action potentials and prevent arrhythmia. The latter effect is accomplished by an increased functional activity of the K7.1 channel encoded by . Current knowledge, however, does not sufficiently explain the full extent of rapid K7.1 activation and may hence be incomplete. Using inducible genetic complementation in -deficient human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we here reinvestigate the functional role of K7.1 in adapting human CMs to adrenergic stress. Under baseline conditions, K7.1 was barely detectable at the plasma membrane of hiPSC-CMs, yet it fully protected these from adrenergic stress-induced beat-to-beat variability of repolarization and -like arrhythmia. Furthermore, isoprenaline treatment increased field potential durations specifically in KCNQ1-deficient CMs to cause these adverse macroscopic effects. Mechanistically, we find that the protective action by K7.1 resides in a rapid translocation of channel proteins from intracellular stores to the plasma membrane, induced by adrenergic signaling. Gene silencing experiments targeting RAB GTPases, mediators of intracellular vesicle trafficking, showed that fast K7.1 recycling under acute stress conditions is RAB4A-dependent.Our data reveal a key mechanism underlying the rapid adaptation of human cardiomyocytes to adrenergic stress. These findings moreover aid to the understanding of disease pathology in long QT syndrome and bear important implications for safety pharmacological screening.
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http://dx.doi.org/10.3389/fphys.2017.00705DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5603700PMC
September 2017

Impact of presynaptic sympathetic imbalance in long-QT syndrome by positron emission tomography.

Heart 2018 02 1;104(4):332-339. Epub 2017 Sep 1.

Department of Cardiovascular Medicine, Institute for Genetics of Heart Diseases (IfGH), University Hospital Münster, Münster, Germany.

Objective: We investigated the impact of cardiac presynaptic norepinephrine recycling in patients with long-QT syndrome (LQTS) using positron emission tomography (PET) with C-meta-hydroxyephedrine ([C]mHED-PET).

Methods: [C]mHED-PET was performed in 25 patients with LQTS (LQT1: n=14; LQT2: n=11) and 20 healthy controls and correlated with clinical parameters. [C]mHED-PET images were analysed for global and regional retention indices (RI) and washout rates (WO) reflecting dynamic parameters of the tracer activity.

Results: Global and regional RI values were similar between patients with LQTS and controls. Although the global WO rates were similar between these groups, regional WO rates were on average higher in the lateral left ventricle (LV) wall in patients with LQTS (dose, mean ±SD; 0.08±0.14 vs 0.00%±0.09% min; p=0.033). In addition, patients with LQTS with a longer QTc interval showed a higher global WO rate. Clinical symptoms correlated with higher global WO rates. In the presence of normal global WO rates, asymptomatic LQTS patients showed higher global RI values.

Conclusion: The increased regional WO rate of [C]mHED in the lateral LV suggests an imbalance of presynaptic catecholamine reuptake and release, resulting in a higher synaptic catecholamine concentration, in particular in LQT1 patients. This might enhance β-adrenoceptor signalling and thereby aggravate inherited ion channel dysfunction and may facilitate occurrence of ventricular tachyarrhythmias. Detection of regional differences in LV sympathetic nervous function may modify disease expression and potentially serve as a non-invasive risk marker in congenital LQTS.

Trial Registration Number: 2006-002767-41;Results.
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http://dx.doi.org/10.1136/heartjnl-2017-311667DOI Listing
February 2018
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