Publications by authors named "Nathalie Strutz-Seebohm"

64 Publications

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

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

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

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

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

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

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

Design, Synthesis, Pharmacological Evaluation and Docking Studies of GluN2B-Selective NMDA Receptor Antagonists with a Benzo[7]annulen-7-amine Scaffold.

ChemMedChem 2017 08 27;12(15):1212-1222. Epub 2017 Jul 27.

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

Antagonists that selectively target GluN2B-subunit-containing N-methyl-d-aspartate (NMDA) receptors are of major interest for the treatment of various neurological disorders. In this study, relationships between variously substituted benzo[7]annulen-7-amines and their GluN2B affinity were investigated. 2-Nitro-5,6,8,9-tetrahydrobenzo[7]annulen-7-one (8) represents the central building block for the introduction of various substituents at the 2-position and various 7-amino moieties. N-(3-Phenylpropyl)-6,7,8,9-tetrahydro-5H-benzo[7]annulen-7-amines with a 2-NO (7 c), 2-Cl (15 c), or 2-OBn group (22 c) show very high GluN2B affinity (K =1.6-3.6 nm). Docking studies revealed the same binding poses for benzo[7]annulen-7-amines and ifenprodil at the interface of GluN1b and GluN2B subunits. The large 2-OBn moiety of 22 c occupies a previously unrecognized subpocket, which explains its high GluN2B affinity (K =3.6 nm). In two-electrode voltage clamp experiments and cytoprotection assays, the high-affinity GluN2B ligands 7 c, 15 c, and 22 c could not inhibit the glutamate-/glycine-evoked current and cytotoxic effects. However, the analogous phenols 16 c ((3-phenylpropyl)amino moiety) and 16 d ((4-phenylbutyl)amino moiety) with 10-fold lower GluN2B affinity (K =28 and 21 nm, respectively) showed promising inhibition of glutamate-/glycine-evoked effects in both assays. The presence of a phenolic hydroxy group seems to be essential for inducing conformational changes of the receptor protein, which finally results in closure of the ion conduction pathway.
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http://dx.doi.org/10.1002/cmdc.201700311DOI Listing
August 2017

Deconstruction - reconstruction approach to analyze the essential structural elements of tetrahydro-3-benzazepine-based antagonists of GluN2B subunit containing NMDA receptors.

Eur J Med Chem 2017 Sep 1;138:552-564. Epub 2017 Jul 1.

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, Germany. Electronic address:

The role of the phenolic and benzylic OH moieties for the interaction of tetrahydro-3-benzazepine-1,7-diol 3d with GluN2B subunit containing NMDA receptors was analyzed by their stepwise removal. Elimination of trifluormethanesulfinate from 10 and 13 represent the key steps in the synthesis. Removal of phenolic OH moiety led to 5-fold reduced GluN2B affinity of 4d compared with 3d. Additional removal of the benzylic OH moiety (5d) resulted in further reduced GluN2B affinity but increased σ and σ affinities. Introduction of a NO (6d) or NH moiety (7d) decreased the GluN2B affinity. 3-Benzazepin-1-ol 4i with the N-phenylcyclohexyl side chain showed the highest GluN2B affinity of this series of compounds (K = 2.2 nM) and, moreover, high selectivity over the PCP binding site, σ and σ receptors. In docking studies 3-benzazepines (S)-4-7 adopt the same binding poses as ifenprodil and display the same crucial interactions. Unexpectedly, the high-affinity ligands (S)-4i, (S)-4j, and (S)-6i were not able to inhibit the glutamate/glycine evoked current in two-electrode voltage clamp measurements and the cytotoxic effects of glutamate/glycine on transfected cell lines.
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http://dx.doi.org/10.1016/j.ejmech.2017.06.068DOI Listing
September 2017

From iPSC towards cardiac tissue-a road under construction.

Pflugers Arch 2017 10 1;469(10):1233-1243. Epub 2017 Jun 1.

Myocellular Electrophysiology and Molecular Biology, IfGH, Department of Cardiovascular Medicine, University Hospital Muenster, 48149, Münster, Germany.

The possibility to generate induced pluripotent stem cells (iPSC) opens the way to generate virtually all cell types of our human body. In combination with modern gene editing techniques like CRISPR/CAS, a new set of powerful tools becomes available for life science. Scientific fields like genotype and cell type-specific pharmacology, disease modeling, stem cell biology, and developmental biology have been dramatically fostered and their faces have been changed. However, as golden as the age of iPSC-derived cells and their manipulation has started, the shine begins to tarnish. Researchers face more and more practical problems intrinsic to the system. These problems are related to the specific culturing conditions which are not yet sufficient to mimic the natural environment of native stem cells differentiating towards adult cells. However, researchers work hard to uncover these factors. Here, we review a common standard approach to generate iPSCs and transduce these to iPSC cardiomyocytes. Further, we review recent achievements and discuss their current limitations and future perspectives. We are on track, but the road is still under construction.
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http://dx.doi.org/10.1007/s00424-017-2003-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5590027PMC
October 2017

Structural interplay of K7.1 and KCNE1 is essential for normal repolarization and is compromised in short QT syndrome 2 (K7.1-A287T).

HeartRhythm Case Rep 2016 Nov 12;2(6):521-529. Epub 2016 Sep 12.

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

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http://dx.doi.org/10.1016/j.hrcr.2016.08.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5420010PMC
November 2016

Systematic variation of the benzenesulfonamide part of the GluN2A selective NMDA receptor antagonist TCN-201.

Eur J Med Chem 2017 Mar 14;129:124-134. Epub 2017 Feb 14.

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

GluN2A subunit containing N-methyl-d-aspartate receptors (NMDARs) are highly involved in various physiological processes in the central nervous system, but also in some diseases, such as anxiety, depression and schizophrenia. However, the role of GluN2A subunit containing NMDARs in pathological processes is not exactly elucidated. In order to obtain potent and selective inhibitors of GluN2A subunit containing NMDARs, the selective negative allosteric modulator 2 was systematically modified at the benzenesulfonamide part. The activity of the test compounds was recorded in two electrode voltage clamp experiments using Xenopus laevis oocytes expressing exclusively NMDARs with GluN1a and GluN2A subunits. It was found that halogen atoms in 3-position of the benzenesulfonamide part result in high GluN2A antagonistic activity. With an IC value of 204 nM the 3-bromo derivative 5i (N-{4-[(2-benzoylhydrazino)carbonyl]benzyl}-3-bromobenzenesulfonamide) has 2.5-fold higher antagonistic activity than the lead compound 2 and represents our new lead compound.
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http://dx.doi.org/10.1016/j.ejmech.2017.02.018DOI Listing
March 2017

The Natural Plant Product Rottlerin Activates Kv7.1/KCNE1 Channels.

Cell Physiol Biochem 2016 21;40(6):1549-1558. Epub 2016 Dec 21.

Department of Cytology, Institute of Anatomy, Ruhr-University Bochum, Bochum, Germany.

Background/aims: Acquired as well as inherited channelopathies are disorders that are caused by altered ion channel function. A family of channels whose malfunction is associated with different channelopathies is the Kv7 K+ channel family; and restoration of normal Kv7 channel function by small molecule modulators is a promising approach for treatment of these often fatal diseases.

Methods: Here, we show the modulation of Kv7 channels by the natural compound Rottlerin heterologously expressed in Xenopus laevis oocytes and on iPSC cardiomyocytes overexpressing Kv7.1 channels.

Results: We show that currents carried by Kv7.1 (EC50 = 1.48 μM), Kv7.1/KCNE1 (EC50 = 4.9 μM), and Kv7.4 (EC50 = 0.148 μM) are strongly enhanced by the compound, whereas Kv7.2, Kv7.2/Kv7.3, and Kv7.5 are not sensitive to Rottlerin. Studies on Kv7.1/KCNE1 mutants and in silico modelling indicate that Rottlerin binds to the R-L3-activator site. Rottlerin mediated activation of Kv7.1/KCNE1 channels might be a promising approach in long QT syndrome. As a proof of concept, we show that Rottlerin shortens cardiac repolarisation in iPSC-derived cardiomyocytes expressing Kv7.1.

Conclusion: Rottlerin or an optimized derivative holds a potential as QT interval correcting drug.
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http://dx.doi.org/10.1159/000453205DOI Listing
February 2017

KCNE1 induces fenestration in the Kv7.1/KCNE1 channel complex that allows for highly specific pharmacological targeting.

Nat Commun 2016 10 12;7:12795. Epub 2016 Oct 12.

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

Most small-molecule inhibitors of voltage-gated ion channels display poor subtype specificity because they bind to highly conserved residues located in the channel's central cavity. Using a combined approach of scanning mutagenesis, electrophysiology, chemical ligand modification, chemical cross-linking, MS/MS-analyses and molecular modelling, we provide evidence for the binding site for adamantane derivatives and their putative access pathway in Kv7.1/KCNE1 channels. The adamantane compounds, exemplified by JNJ303, are highly potent gating modifiers that bind to fenestrations that become available when KCNE1 accessory subunits are bound to Kv7.1 channels. This mode of regulation by auxiliary subunits may facilitate the future development of potent and highly subtype-specific Kv channel inhibitors.
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http://dx.doi.org/10.1038/ncomms12795DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5064022PMC
October 2016

Tau Tubulin Kinase TTBK2 Sensitivity of Glutamate Receptor GluK2.

Cell Physiol Biochem 2016 9;39(4):1444-52. Epub 2016 Sep 9.

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

Background/aims: Inherited, autosomal dominant spinocerebellar ataxia type 11 (SCA11) is caused by loss of function mutations of TTBK2 (tau tubulin kinase 2). Mutations observed in patients with SCA11 include truncated TTBK2(450). The present study explored the possibility that TTBK2 influences the function of the glutamate receptor GluK2.

Methods: GluK2 was expressed in Xenopus oocytes without and with additional expression of wild type TTBK2, the truncated mutant TTBK2(450), or the kinase dead mutants TTBK2(KD) and TTBK2(450/KD). GluK2 current was determined by dual electrode voltage clamp and GluK2 protein abundance in the cell membrane utilizing confocal microscopy.

Results: Glutamate exposure of GluK2 expressing oocytes generated a current, which was significantly lower in oocytes expressing GluK2 together with TTBK2 wt or TTBK2(KD) than in oocytes expressing GluK2 alone or together with either TTBK2(450) or TTBK2(450/KD). According to confocal microscopy of EGFP-tagged GluK2, TTBK2 wt decreased the GluK2 protein abundance in the cell membrane. Overexpression of an inactive RAB5(N133I) mutant but not RAB5wt could reverse the TTBK2 effect on GluK2 suggesting that RAB5 function is required for the effect.

Conclusions: TTBK2 down-regulates GluK2 activity by decreasing the receptor protein abundance in the cell membrane via RAB5-dependent endocytosis, an effect that may protect against neuroexcitotoxicity.
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http://dx.doi.org/10.1159/000447847DOI Listing
January 2017

NDRG2 phosphorylation provides negative feedback for SGK1-dependent regulation of a kainate receptor in astrocytes.

Front Cell Neurosci 2015 6;9:387. Epub 2015 Oct 6.

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

Glutamate receptors play an important role in the function of astrocytes. Among their tasks is the regulation of gliotransmission, gene expression and exocytosis of the tissue-type plasminogen activator (tPA), which has an enhancing effect on N-methyl-D-aspartate (NMDA) receptors and thus prevent over-excitation of neighboring neurons. The kainate receptor GluK2, which is expressed in neurons and astrocytes, is under tight regulation of the PI3-kinase SGK pathway as shown in neurons. SGK1 targets include N-myc downstream-regulated genes (NDRGs) 1 and 2 (NDRG1, NDRG2), proteins with elusive function. In the present study, we analyzed the effects of SGK1, NDRG1, and NDRG2 on GluK2 current amplitude and plasma membrane localization in astrocytes and heterologous expression. We demonstrate that NDRG1 and NDRG2 themselves have no effect on GluK2 current amplitudes in heterologous expressed ion channels. However, when NDRG2 is coexpressed with GluK2 and SGK1, the stimulating effect of SGK1 on GluK2 is suppressed both in heterologous expression and in astrocytes. Here, we reveal a new negative feedback mechanism, whereby GluK2 stimulation by SGK1 is regulated by parallel phosphorylation of NDRG2. This regulation of GluK2 by SGK1 and NDRG2 in astrocytes may play an important role in gliotransmission, modulation of gene expression and regulation of exocytosis of tPA.
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http://dx.doi.org/10.3389/fncel.2015.00387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4594022PMC
October 2015

Paving the Way to Understand Autoantibody-Mediated Epilepsy on the Molecular Level.

Front Neurol 2015 6;6:149. Epub 2015 Jul 6.

Receptor Structure and Function Group, Department of Cardiovascular Medicine, Institute for Genetics of Heart Diseases (IfGH), University Hospital Münster , Münster , Germany.

Correct function of neuronal networks is enabled by a delicate interplay among neurons communicating with each other. One of the keys is the communication at chemical synapses where neurotransmitters like glutamate, GABA, and glycine enable signal transfer over the synaptic cleft. Thereby, the neurotransmitters are released from the presynapse and bind as ligands to specific receptors at the postsynaptic side to allow for modulation of the postsynaptic membrane potentials. The postsynaptic electrical signal, which is highly modulated by voltage-gated ion channels, spreads over the dendritic tree and is thus integrated to allow for generation of action potentials at the axon hillock. This concert of receptors and voltage-gated ion channels depends on correct function of all its components. Misfunction of receptors and/or voltage-gated potassium channels (VGKC) leads to diverse adverse effects in patients. Such malfunctions can be the result of inherited genetic alterations or pharmacological side effects by drugs. Recently, autoantibodies targeting receptor or channel complexes like NMDAR, AMPAR, GABA-receptors, glycine receptors, LGI1 or CASPR2 (previously termed as VGKC-complex antibodies) have been discovered. The presence of specific autoantibodies against these targets associates with severe forms of antibody-mediated encephalitis. Understanding the molecular details of autoantibody actions on receptor and VGKC complexes is highly desirable and may open the path to develop specific therapies to treat humoral autoimmune encephalitis. Here, we summarize the current knowledge and discuss technical approaches to fill the gap of knowledge. These techniques include electrophysiology, biochemical approaches for epitope mapping, and in silico modeling to simulate molecular interactions between autoantibody and its molecular target.
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http://dx.doi.org/10.3389/fneur.2015.00149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4491625PMC
July 2015

Novel Kv7.1-phosphatidylinositol 4,5-bisphosphate interaction sites uncovered by charge neutralization scanning.

J Biol Chem 2014 Aug 19;289(33):22749-22758. Epub 2014 Jun 19.

International Graduate School of Neuroscience, Ruhr University Bochum, 44801 Bochum, Germany; Ruhr University Bochum Research School, and Ruhr University Bochum, 44801 Bochum, Germany; IfGH-Myocellular Electrophysiology, Department of Cardiovascular Medicine, University Hospital of Münster, 48149 Münster, Germany, and. Electronic address:

Kv7.1 to Kv7.5 α-subunits belong to the family of voltage-gated potassium channels (Kv). Assembled with the β-subunit KCNE1, Kv7.1 conducts the slowly activating potassium current IKs, which is one of the major currents underlying repolarization of the cardiac action potential. A known regulator of Kv7 channels is the lipid phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 increases the macroscopic current amplitude by stabilizing the open conformation of 7.1/KCNE1 channels. However, knowledge about the exact nature of the interaction is incomplete. The aim of this study was the identification of the amino acids responsible for the interaction between Kv7.1 and PIP2. We generated 13 charge neutralizing point mutations at the intracellular membrane border and characterized them electrophysiologically in complex with KCNE1 under the influence of diC8-PIP2. Electrophysiological analysis of corresponding long QT syndrome mutants suggested impaired PIP2 regulation as the cause for channel dysfunction. To clarify the underlying structural mechanism of PIP2 binding, molecular dynamics simulations of Kv7.1/KCNE1 complexes containing two PIP2 molecules in each subunit at specific sites were performed. Here, we identified a subset of nine residues participating in the interaction of PIP2 and Kv7.1/KCNE1. These residues may form at least two binding pockets per subunit, leading to the stabilization of channel conformations upon PIP2 binding.
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http://dx.doi.org/10.1074/jbc.M114.589796DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4132781PMC
August 2014

Structural basis of PI(4,5)P2-dependent regulation of GluA1 by phosphatidylinositol-5-phosphate 4-kinase, type II, alpha (PIP5K2A).

Pflugers Arch 2014 Oct 5;466(10):1885-97. Epub 2014 Jan 5.

Institute for Genetics of Heart Diseases (IfGH), -Myocellular Electrophysiology, Department of Cardiovascular Medicine, University Hospital Muenster, 48149, Muenster, Germany.

Ionotropic glutamate receptors are the most important excitatory receptors in the central nervous system, and their impairment can lead to multiple neuronal diseases. Here, we show that glutamate-induced currents in oocytes expressing GluA1 are increased by coexpression of the schizophrenia-associated phosphoinositide kinase PIP5K2A. This effect was due to enhanced membrane abundance and was blunted by a point mutation (N251S) in PIP5K2A. An increase in GluA1 currents was also observed upon acute injection of PI(4,5)P2, the main product of PIP5K2A. By expression of wild-type and mutant PIP5K2A in human embryonic kidney cells, we were able to provide evidence of impaired kinase activity of the mutant PIP5K2A. We defined the region K813-K823 of GluA1 as critical for the PI(4,5)P2 effect by performing an alanine scan that suggested PI(4,5)P2 binding to this area. A PIP strip assay revealed PI(4,5)P2 binding to the C-terminal GluA1 peptide. The present observations disclose a novel mechanism in the regulation of GluA1.
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http://dx.doi.org/10.1007/s00424-013-1424-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4159565PMC
October 2014

A common structural component for β-subunit mediated modulation of slow inactivation in different KV channels.

Cell Physiol Biochem 2013 26;31(6):968-80. Epub 2013 Jun 26.

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

Background/aims: Potassium channels are tetrameric proteins providing potassium selective passage through lipid embedded proteinaceous pores with highest fidelity. The selectivity results from binding to discrete potassium binding sites and stabilization of a hydrated potassium ion in a central internal cavity. The four potassium binding sites, generated by the conserved TTxGYGD signature sequence are formed by the backbone carbonyls of the amino acids TXGYG. Residues KV1.5-Val481, KV4.3-Leu368 and KV7.1- Ile 313 represent the amino acids in the X position of the respective channels.

Methods: Here, we study the impact of these residues on ion selectivity, permeation and inactivation kinetics as well as the modulation by β-subunits using site-specific mutagenesis, electrophysiological analyses and molecular dynamics simulations.

Results: We identify this position as key in modulation of slow inactivation by structurally dissimilar β-subunits in different KV channels.

Conclusion: We propose a model in which structural changes accompanying activation and β-subunit modulation allosterically constrain the backbone carbonyl oxygen atoms via the side chain of the respective X-residue in the signature sequence to reduce conductance during slow inactivation.
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http://dx.doi.org/10.1159/000350115DOI Listing
February 2014

Coxsackievirus B3 modulates cardiac ion channels.

FASEB J 2013 Oct 27;27(10):4108-21. Epub 2013 Jun 27.

2Institut für Genetik von Herzerkrankungen (IfGH), Abteilung Myozelluläre Elektrophysiologie, Albert-Schweitzer-Campus 1 (Gebäude D3), D-48149 Münster.

Infections with coxsackieviruses of type B (CVBs), which are known to induce severe forms of acute and chronic myocarditis, are often accompanied by ventricular arrhythmias and sudden cardiac death. The mechanisms underlying the development of virus-induced, life-threatening arrhythmias, which are phenotypically similar to those observed in patients having functionally impaired cardiac ion channels, remain, however, enigmatic. In the present study, we show, for the first time, modulating time-dependent effects of CVB3 on the cardiac ion channels KCNQ1, hERG1, and Cav1.2 in heterologous expression. Channel protein abundance in cellular plasma membrane and patterns of their subcellular distribution were altered in infected murine hearts. The antiviral compound AG7088 did not prevent these effects on channels. In silico analyses of infected human myocytes suggest pronounced alterations of electrical and calcium signaling and increased risk of arrhythmogenesis. These modifications are attenuated by the common Asian polymorphism KCNQ1 P448R, a genetic determinant preventing coxsackievirus-induced effects in vitro. This study provides a previously unknown explanation for the development of arrhythmias in enteroviral myocarditis, which will help to develop therapeutic strategies for arrhythmia treatment.
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http://dx.doi.org/10.1096/fj.13-230193DOI Listing
October 2013

Overlapping cardiac phenotype associated with a familial mutation in the voltage sensor of the KCNQ1 channel.

Cell Physiol Biochem 2012 11;29(5-6):809-18. Epub 2012 May 11.

Biochemistry I - Cation Channel Group, Ruhr University Bochum, Bochum, Germany.

Background: Cardiac action potential repolarisation is determined by K(+) currents including I(Ks). I(Ks) channels are heteromeric channels composed of KCNQ1 and KCNE E-subunits. Mutations in KCNQ1 are associated with sinus bradycardia, familial atrial fibrillation (fAF) and/or short QT syndrome as a result of gain-of-function, and long QT syndrome (LQTS) due to loss-of-function in the ventricles. Here, we report that the missense mutation R231C located in S4 voltage sensor domain is associated with a combined clinical phenotype of sinus bradycardia, fAF and LQTS. We aim to understand the molecular basis of the complex clinical phenotype.

Methods: We expressed and functionally analyzed the respective channels kinetics in Xenopus laevis oocytes. The molecular nature of the residue R231 was studied by homology modeling and molecular dynamics simulation.

Results: As a result, the mutation reduced voltage sensitivity of channels, possibly due to neutralization of the positive charge of the arginine side chain substituted by cysteine. Modeling suggested that the charge carrying side chain of R231 is positioned suitably to transfer transmembrane voltages into conformational energy. Further, the mutation altered the functional interactions with KCNE subunits.

Conclusion: The mutation acted in a E-subunit dependent manner, suggesting I(Ks) function altered by the presence of different KCNE subunits in sinus node, atria and ventricles as the molecular basis of sinus bradycardia, fAF and LQTS in mutation carriers.
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http://dx.doi.org/10.1159/000178470DOI Listing
November 2012

Identification of a novel signaling pathway and its relevance for GluA1 recycling.

PLoS One 2012 21;7(3):e33889. Epub 2012 Mar 21.

Department of Biochemistry I-Cation Channel Group, Ruhr University Bochum, Bochum, Germany.

We previously showed that the serum- and glucocorticoid-inducible kinase 3 (SGK3) increases the AMPA-type glutamate receptor GluA1 protein in the plasma membrane. The activation of AMPA receptors by NMDA-type glutamate receptors eventually leads to postsynaptic neuronal plasticity. Here, we show that SGK3 mRNA is upregulated in the hippocampus of new-born wild type Wistar rats after NMDA receptor activation. We further demonstrate in the Xenopus oocyte expression system that delivery of GluA1 protein to the plasma membrane depends on the small GTPase RAB11. This RAB-dependent GluA1 trafficking requires phosphorylation and activation of phosphoinositol-3-phosphate-5-kinase (PIKfyve) and the generation of PI(3,5)P(2). In line with this mechanism we could show PIKfyve mRNA expression in the hippocampus of wild type C57/BL6 mice and phosphorylation of PIKfyve by SGK3. Incubation of hippocampal slices with the PIKfyve inhibitor YM201636 revealed reduced CA1 basal synaptic activity. Furthermore, treatment of primary hippocampal neurons with YM201636 altered the GluA1 expression pattern towards reduced synaptic expression of GluA1. Our findings demonstrate for the first time an involvement of PIKfyve and PI(3,5)P(2) in NMDA receptor-triggered synaptic GluA1 trafficking. This new regulatory pathway of GluA1 may contribute to synaptic plasticity and memory.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0033889PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3309939PMC
August 2012

GluN3 subunit-containing NMDA receptors: not just one-trick ponies.

Trends Neurosci 2012 Apr 10;35(4):240-9. Epub 2012 Jan 10.

Department of Biochemistry I - Receptor Biochemistry, Ruhr University Bochum, 44780 Bochum, Germany.

The two GluN3 subunits were the last NMDA receptor subunits to be cloned some 15 years ago. Strikingly, despite the steadily growing interest in their function, their physiological role remains elusive. The original billing as dominant-negative modulators of classical NMDA receptors composed of GluN1 and GluN2 subunits has given way to proposals of much more complex functions, including roles in synaptogenesis and synaptic plasticity. In addition, GluN3 subunits in the absence of GluN2 surprisingly assemble with GluN1 into excitatory glycine receptors. This review provides an overview of the unique spatial and temporal expression patterns of the GluN3 subunits, discusses proposed functions and physiological roles for receptors comprising these subunits, and briefly summarizes their putative involvement in several neural diseases.
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http://dx.doi.org/10.1016/j.tins.2011.11.010DOI Listing
April 2012

Altered stress stimulation of inward rectifier potassium channels in Andersen-Tawil syndrome.

FASEB J 2012 Feb 14;26(2):513-22. Epub 2011 Oct 14.

Department of Biochemistry I-Cation Channel Group, Ruhr University Bochum, Bochum, Germany.

Inward rectifier potassium channels of the Kir2 subfamily are important determinants of the electrical activity of brain and muscle cells. Genetic mutations in Kir2.1 associate with Andersen-Tawil syndrome (ATS), a familial disorder leading to stress-triggered periodic paralysis and ventricular arrhythmia. To identify the molecular mechanisms of this stress trigger, we analyze Kir channel function and localization electrophysiologically and by time-resolved confocal microscopy. Furthermore, we employ a mathematical model of muscular membrane potential. We identify a novel corticoid signaling pathway that, when activated by glucocorticoids, leads to enrichment of Kir2 channels in the plasma membranes of mammalian cell lines and isolated cardiac and skeletal muscle cells. We further demonstrate that activation of this pathway can either partly restore (40% of cases) or further impair (20% of cases) the function of mutant ATS channels, depending on the particular Kir2.1 mutation. This means that glucocorticoid treatment might either alleviate or deteriorate symptoms of ATS depending on the patient's individual Kir2.1 genotype. Thus, our findings provide a possible explanation for the contradictory effects of glucocorticoid treatment on symptoms in patients with ATS and may open new pathways for the design of personalized medicines in ATS therapy.
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http://dx.doi.org/10.1096/fj.11-189126DOI Listing
February 2012

Structural basis of slow activation gating in the cardiac I Ks channel complex.

Cell Physiol Biochem 2011 15;27(5):443-52. Epub 2011 Jun 15.

Department of Biochemistry I--Cation Channel Group, Ruhr University, Bochum, Germany.

Accessory β-subunits of the KCNE gene family modulate the function of various cation channel α-subunits by the formation of heteromultimers. Among the most dramatic changes of biophysical properties of a voltage-gated channel by KCNEs are the effects of KCNE1 on KCNQ1 channels. KCNQ1 and KCNE1 are believed to form nativeI(Ks) channels. Here, we characterize molecular determinants of KCNE1 interaction with KCNQ1 channels by scanning mutagenesis, double mutant cycle analysis, and molecular dynamics simulations. Our findings suggest that KCNE1 binds to the outer face of the KCNQ1 channel pore domain, modifies interactions between voltage sensor, S4-S5 linker and the pore domain, leading to structural modifications of the selectivity filter and voltage sensor domain. Molecular dynamics simulations suggest a stable interaction of the KCNE1 transmembrane α-helix with the pore domain S5/S6 and part of the voltage sensor domain S4 of KCNQ1 in a putative pre-open channel state. Formation of this state may induce slow activation gating, the pivotal characteristic of native cardiac I(Ks) channels. This new KCNQ1-KCNE1 model may become useful for dynamic modeling of disease-associated mutant I(Ks) channels.
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http://dx.doi.org/10.1159/000329965DOI Listing
October 2011