Sodium permeable and “hypersensitive” TREK ‐1 channels cause ventricular tachycardia
2017; Springer Nature; Volume: 9; Issue: 4 Linguagem: Inglês
10.15252/emmm.201606690
ISSN1757-4684
AutoresNiels Decher, Beatriz Ortiz‐Bonnin, Corinna Friedrich, Marcus Schewe, Aytuğ K. Kiper, Susanne Rinné, Gunnar Seemann, Rémi Peyronnet, Sven Zumhagen, Daniel Bustos, Jens Kockskämper, Peter Köhl, Steffen Just, Wendy González, Thomas Baukrowitz, Birgit Stallmeyer, Eric Schulze‐Bahr,
Tópico(s)Cardiac Arrhythmias and Treatments
ResumoResearch Article27 February 2017Open Access Transparent process Sodium permeable and "hypersensitive" TREK-1 channels cause ventricular tachycardia Niels Decher Corresponding Author Niels Decher [email protected] orcid.org/0000-0001-8892-1231 Institute of Physiology and Pathophysiology, Vegetative Physiology, Philipps-University of Marburg, Marburg, Germany Search for more papers by this author Beatriz Ortiz-Bonnin Beatriz Ortiz-Bonnin Institute of Physiology and Pathophysiology, Vegetative Physiology, Philipps-University of Marburg, Marburg, Germany Search for more papers by this author Corinna Friedrich Corinna Friedrich Department of Cardiovascular Medicine, Institute for Genetics of Heart Diseases (IfGH), University Hospital Münster, Münster, Germany Search for more papers by this author Marcus Schewe Marcus Schewe orcid.org/0000-0002-6192-5651 Institute of Physiology, Christian-Albrechts-University of Kiel, Kiel, Germany Search for more papers by this author Aytug K Kiper Aytug K Kiper Institute of Physiology and Pathophysiology, Vegetative Physiology, Philipps-University of Marburg, Marburg, Germany Search for more papers by this author Susanne Rinné Susanne Rinné Institute of Physiology and Pathophysiology, Vegetative Physiology, Philipps-University of Marburg, Marburg, Germany Search for more papers by this author Gunnar Seemann Gunnar Seemann Institute for Experimental Cardiovascular Medicine, University Heart Center Freiburg – Bad Krozingen, Medical Center – University of Freiburg, Freiburg, Germany Faculty of Medicine, University of Freiburg, Freiburg, Germany Search for more papers by this author Rémi Peyronnet Rémi Peyronnet Institute for Experimental Cardiovascular Medicine, University Heart Center Freiburg – Bad Krozingen, Medical Center – University of Freiburg, Freiburg, Germany Faculty of Medicine, University of Freiburg, Freiburg, Germany Search for more papers by this author Sven Zumhagen Sven Zumhagen Department of Cardiovascular Medicine, Institute for Genetics of Heart Diseases (IfGH), University Hospital Münster, Münster, Germany Search for more papers by this author Daniel Bustos Daniel Bustos Center for Bioinformatics and Molecular Simulation, University of Talca, Talca, Chile Search for more papers by this author Jens Kockskämper Jens Kockskämper Institute of Pharmacology and Clinical Pharmacy, Biochemical and Pharmacological Center (BPC), Philipps-University of Marburg, Marburg, Germany Search for more papers by this author Peter Kohl Peter Kohl Institute for Experimental Cardiovascular Medicine, University Heart Center Freiburg – Bad Krozingen, Medical Center – University of Freiburg, Freiburg, Germany Faculty of Medicine, University of Freiburg, Freiburg, Germany Search for more papers by this author Steffen Just Steffen Just Molecular Cardiology, University Hospital Ulm, Ulm, Germany Search for more papers by this author Wendy González Wendy González Center for Bioinformatics and Molecular Simulation, University of Talca, Talca, Chile Search for more papers by this author Thomas Baukrowitz Thomas Baukrowitz Institute of Physiology, Christian-Albrechts-University of Kiel, Kiel, Germany Search for more papers by this author Birgit Stallmeyer Birgit Stallmeyer Department of Cardiovascular Medicine, Institute for Genetics of Heart Diseases (IfGH), University Hospital Münster, Münster, Germany Search for more papers by this author Eric Schulze-Bahr Eric Schulze-Bahr Department of Cardiovascular Medicine, Institute for Genetics of Heart Diseases (IfGH), University Hospital Münster, Münster, Germany Search for more papers by this author Niels Decher Corresponding Author Niels Decher [email protected] orcid.org/0000-0001-8892-1231 Institute of Physiology and Pathophysiology, Vegetative Physiology, Philipps-University of Marburg, Marburg, Germany Search for more papers by this author Beatriz Ortiz-Bonnin Beatriz Ortiz-Bonnin Institute of Physiology and Pathophysiology, Vegetative Physiology, Philipps-University of Marburg, Marburg, Germany Search for more papers by this author Corinna Friedrich Corinna Friedrich Department of Cardiovascular Medicine, Institute for Genetics of Heart Diseases (IfGH), University Hospital Münster, Münster, Germany Search for more papers by this author Marcus Schewe Marcus Schewe orcid.org/0000-0002-6192-5651 Institute of Physiology, Christian-Albrechts-University of Kiel, Kiel, Germany Search for more papers by this author Aytug K Kiper Aytug K Kiper Institute of Physiology and Pathophysiology, Vegetative Physiology, Philipps-University of Marburg, Marburg, Germany Search for more papers by this author Susanne Rinné Susanne Rinné Institute of Physiology and Pathophysiology, Vegetative Physiology, Philipps-University of Marburg, Marburg, Germany Search for more papers by this author Gunnar Seemann Gunnar Seemann Institute for Experimental Cardiovascular Medicine, University Heart Center Freiburg – Bad Krozingen, Medical Center – University of Freiburg, Freiburg, Germany Faculty of Medicine, University of Freiburg, Freiburg, Germany Search for more papers by this author Rémi Peyronnet Rémi Peyronnet Institute for Experimental Cardiovascular Medicine, University Heart Center Freiburg – Bad Krozingen, Medical Center – University of Freiburg, Freiburg, Germany Faculty of Medicine, University of Freiburg, Freiburg, Germany Search for more papers by this author Sven Zumhagen Sven Zumhagen Department of Cardiovascular Medicine, Institute for Genetics of Heart Diseases (IfGH), University Hospital Münster, Münster, Germany Search for more papers by this author Daniel Bustos Daniel Bustos Center for Bioinformatics and Molecular Simulation, University of Talca, Talca, Chile Search for more papers by this author Jens Kockskämper Jens Kockskämper Institute of Pharmacology and Clinical Pharmacy, Biochemical and Pharmacological Center (BPC), Philipps-University of Marburg, Marburg, Germany Search for more papers by this author Peter Kohl Peter Kohl Institute for Experimental Cardiovascular Medicine, University Heart Center Freiburg – Bad Krozingen, Medical Center – University of Freiburg, Freiburg, Germany Faculty of Medicine, University of Freiburg, Freiburg, Germany Search for more papers by this author Steffen Just Steffen Just Molecular Cardiology, University Hospital Ulm, Ulm, Germany Search for more papers by this author Wendy González Wendy González Center for Bioinformatics and Molecular Simulation, University of Talca, Talca, Chile Search for more papers by this author Thomas Baukrowitz Thomas Baukrowitz Institute of Physiology, Christian-Albrechts-University of Kiel, Kiel, Germany Search for more papers by this author Birgit Stallmeyer Birgit Stallmeyer Department of Cardiovascular Medicine, Institute for Genetics of Heart Diseases (IfGH), University Hospital Münster, Münster, Germany Search for more papers by this author Eric Schulze-Bahr Eric Schulze-Bahr Department of Cardiovascular Medicine, Institute for Genetics of Heart Diseases (IfGH), University Hospital Münster, Münster, Germany Search for more papers by this author Author Information Niels Decher *,1,‡, Beatriz Ortiz-Bonnin1,‡, Corinna Friedrich3, Marcus Schewe4, Aytug K Kiper1, Susanne Rinné1, Gunnar Seemann5,6, Rémi Peyronnet5,6, Sven Zumhagen3, Daniel Bustos7, Jens Kockskämper2, Peter Kohl5,6, Steffen Just8, Wendy González7, Thomas Baukrowitz4, Birgit Stallmeyer3 and Eric Schulze-Bahr3 1Institute of Physiology and Pathophysiology, Vegetative Physiology, Philipps-University of Marburg, Marburg, Germany 2Institute of Pharmacology and Clinical Pharmacy, Biochemical and Pharmacological Center (BPC), Philipps-University of Marburg, Marburg, Germany 3Department of Cardiovascular Medicine, Institute for Genetics of Heart Diseases (IfGH), University Hospital Münster, Münster, Germany 4Institute of Physiology, Christian-Albrechts-University of Kiel, Kiel, Germany 5Institute for Experimental Cardiovascular Medicine, University Heart Center Freiburg – Bad Krozingen, Medical Center – University of Freiburg, Freiburg, Germany 6Faculty of Medicine, University of Freiburg, Freiburg, Germany 7Center for Bioinformatics and Molecular Simulation, University of Talca, Talca, Chile 8Molecular Cardiology, University Hospital Ulm, Ulm, Germany ‡These authors contributed equally to this work *Corresponding author. Tel: +49 6421 2862148; E-mail: [email protected] EMBO Mol Med (2017)9:403-414https://doi.org/10.15252/emmm.201606690 See also: SAN Goldstein (April 2017) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract In a patient with right ventricular outflow tract (RVOT) tachycardia, we identified a heterozygous point mutation in the selectivity filter of the stretch-activated K2P potassium channel TREK-1 (KCNK2 or K2P2.1). This mutation introduces abnormal sodium permeability to TREK-1. In addition, mutant channels exhibit a hypersensitivity to stretch-activation, suggesting that the selectivity filter is directly involved in stretch-induced activation and desensitization. Increased sodium permeability and stretch-sensitivity of mutant TREK-1 channels may trigger arrhythmias in areas of the heart with high physical strain such as the RVOT. We present a pharmacological strategy to rescue the selectivity defect of the TREK-1 pore. Our findings provide important insights for future studies of K2P channel stretch-activation and the role of TREK-1 in mechano-electrical feedback in the heart. Synopsis A point mutation in the selectivity filter of the stretch-activated K2P potassium channel TREK-1 was identified in a patient with right ventricular outflow tract tachycardia. The mutation most likely causes arrhythmias through abnormal sodium permeability and hypersensitivity to stretch-activation. Analysis of a patient with right ventricular outflow tract tachycardia (RVOT-VT) led to the identification of a heterozygous mutation, resulting in an Ile to Thr exchange directly preceding the selectivity filter of the K2P potassium channel TREK-1. The mutation introduces an abnormal sodium permeability and a hypersensitivity to stretch-activation to TREK-1 channels. The study suggests that the selectivity filter is directly involved in stretch-induced activation and desensitization of stretch-sensitive K2P potassium channels. Increased sodium permeability and stretch-sensitivity of mutant TREK-1 channels may trigger arrhythmias in areas of the heart with high physical strain. The findings provide important insights for future studies of K2P channel stretch-activation and the role of TREK-1 in mechano-electrical feedback in the heart. Introduction Right ventricular outflow tract ventricular tachycardia (RVOT-VT) is a common form of monomorphic ventricular tachycardia (VT) characterized by the absence of structural heart disease and a mostly unknown etiology (Srivathsan et al, 2005). Some evidence suggests that RVOT-VT can be triggered by delayed afterdepolarizations (DADs) during β-adrenergic stimulation since a rise in intracellular cAMP will lead to a PKA-dependent phosphorylation of different Ca2+ handling proteins, resulting in overloading of intracellular Ca2+ in cardiomyocytes. Following the repolarization phase of the cardiac action potential, the Na+/Ca2+ exchanger transports excessive Ca2+ out of the cell, producing a transient inward current that generates DADs (Lerman, 2015) which in turn can trigger VT. Despite advances in the understanding of the molecular mechanisms that trigger VT, the genetic basis of RVOT-VT is largely unknown. Recently, gene mutations in two-pore domain potassium (K2P) channels have been discovered as a cause for familial or sporadic forms of migraine (Lafreniere et al, 2010) (TRESK), Birk-Barel syndrome (Barel et al, 2008) (TASK-3), and a progressive cardiac conduction disorder (Friedrich et al, 2014) (TASK-4). These discoveries provide direct proof for the pathophysiological relevance of K2P "leak" channels. The stretch-activated TREK-1 (K2P2.1) channel is regulated by a plethora of different physiological stimuli (Feliciangeli et al, 2015) and has been proposed to be involved in multiple cellular and pathophysiological processes. In the heart, it has been suggested that TREK-1 channels play a major role in mechano-electrical feedback, since the stretch-activated K+ current (SAK) shortens action potential duration (APD) and decreases the heterogeneity of repolarization, a potentially anti-arrhythmic activity (Kelly et al, 2006). Although TREK-1 is one of the most studied cardiac K2P channels, its physiological role in the human heart and cardiac arrhythmias remained elusive. Results A heterozygous KCNK2 (TREK-1) mutation in a patient with RVOT-VT We systematically investigated the role of K2P channels for inherited forms of cardiac arrhythmias (Friedrich et al, 2014). In a large patient cohort comprising 438 probands with different genetically unresolved arrhythmia syndromes [RVOT-VT (40), AFib (10), AVB (16), BrS (188), CPVT (32), iVF (68), PCCD (49), PMVT (13), and SND (22)], all coding exons and adjacent intronic sites of KCNK2, encoding the K2P channel TREK-1, were sequenced (4 and Appendix Table S1). In one of the RVOT-VT patients (Fig 1A), a heterozygous single nucleotide variant (SNV) (c.800T > C) was identified in exon 5 (Fig 1B), resulting in an amino acid exchange of a highly conserved Ile (p.Ile267Thr) in the selectivity filter (SF, Fig 1B and C) of the second pore domain of TREK-1. In a control cohort of the same ethnicity (n = 379), this SNV was absent and consistent with a putative disease-causing mutation, it was only found rarely in the Exome Variant Server (EVS) database (3/13,003 alleles). Figure 1. Identification of a heterozygous KCNK2 (TREK-1) mutation in a patient with RVOT-VT A. 12-lead ECG during exercise of the proband (10772-3) presenting with LBBB (with inferior axis) tachycardia being typical for its origin from the RVOT. B, C. (B) Electropherogram and nucleotide sequence of KCNK2 illustrating a heterozygous c.800T > C single nucleotide exchange with a predicted non-synonymous amino acid exchange (p. Ile267Thr; shortly: I276T). Localization of the TREK-1I267T residue (highlighted in blue) within a highly conserved signature sequence (red) upon partial sequence alignment with TREK-1 orthologues. The I276T mutation is located in the second pore domain of the TREK-1 channel (indicated in a cartoon in B), or in a pore homology model based on the crystal structure of TREK-2 (C). D. Prioritization scheme for filtering nucleotide variants obtained after WES. Download figure Download PowerPoint Starting at an age of 45 years, the affected patient suffered from recurrent and sudden onset VTs that were triggered by physical exercise. The maximum duration of the VT was > 10 min, but did not result in cardiac syncope or arrest. During sinus rhythm, time intervals measured from a 12-lead surface ECG were normal (Appendix Fig S1). Ischemic heart disease was ruled out by coronary angiography when the patient was 49 years old. Additionally, transthoracic echocardiography and MRI together with contrast imaging were unremarkable for the presence of structural heart disease or cardiomyopathy. Programmed electrophysiological stimulation was unable to induce any supraventricular or VT. Two years later, a fast, broad complex tachycardia with a rate of 240 beats/min was noted during exercise (left bundle branch block (LBBB) with inferior axis), indicating RVOT-VT (Fig 1A). Hereafter, surface ECG showed preterminal negative T-waves in the right precordial leads. There was no family history of tachycardia or sudden cardiac death, and other family members were not available for clinical or genetic investigations. We therefore performed whole exome sequencing (WES) (4 and Appendix Table S2) in this proband to identify or exclude additional relevant variants that may be responsible for the arrhythmia phenotype. Following a prioritization scheme (Fig 1D), only SNVs with a minimum sequencing coverage of 20× and that were present in an in-house cardiovascular priority gene list (CARDIO panel; Friedrich et al, 2014), harboring 388 relevant cardiovascular or ion channel genes, were further evaluated. Considering only SNVs with potentially serious consequences to the protein, only two were absent or very rare in genomic databases (Appendix Table S3), whereas 58 were already known and not further considered as causative (Appendix Table S4). The two remaining SNVs were independently confirmed by Sanger sequencing and were predicted to cause amino acid exchanges (KCNK2: c.800T > C, p.Ile267Thr, and RYR3: c.1753A > G, p.Ile585Val). RYR3 encodes the ryanodine receptor type 3 which is preferentially expressed in the brain and only faintly expressed in the heart. The pathogenic impact of both amino acid exchanges was determined using five different in silico pathogenicity prediction tools (PPTs) (Fig 1D and Appendix Table S3). The identified Ile585Val variant in the RYR3 gene had discordant, but mainly non-deleterious pathogenicity predictions (4/5: "neutral") and was not further considered (Fig 1D). Confirming our initial candidate gene approach, WES and the prioritization scheme predicted the I267T exchange in TREK-1 as a likely disease-related variant. All PPTs concordantly evaluated the TREK-1I267T exchange as "damaging" (Appendix Table S3). Reduced outward currents of homomeric TREK-1I267T and heteromeric TREK-1/TREK-1I267T Heterologous expression of wild-type TREK-1 channels in Xenopus oocytes resulted in large outward currents with a reversal potential (Erev) of −90 mV (Fig 2A). In contrast, TREK-1I267T currents were much smaller in magnitude (Fig 2A and B) with an Erev near −50 mV (Fig 2C and D). TREK-1I267T expressing oocytes appeared as leaky cells. However, one can note an unusual "hook" in the most negative voltage range of the current–voltage relationship (Fig 2D). This unusual feature that was not observed in uninjected oocytes, results from the rate of activation of mutant channels after the voltage is stepped to −120 mV (Appendix Fig S2). Co-injection of oocytes with equal amounts of wild-type TREK-1 plus TREK-1I267T cRNA (to mimic the heterozygous state) results in reduced current compared to that observed in oocytes injected with an equal amount of wild-type TREK-1 cRNA alone (Fig 2E and F). Therefore, the mutation might act in a dominant-negative manner. Intriguingly, also for the co-expression, mimicking heterozygosity, the membrane potential (Em) was strongly depolarized (Fig 2G). Generally, depolarized membrane potentials could result from reduced outward K+ currents or altered ion selectivity. In ventricular myocytes, the resting membrane potential is primarily determined by Kir2.x channels. Kir2.1 co-expressed with TREK-1 showed typical Kir2.1-like currents (Fig 2H), while co-expression with TREK-1I267T results in a dramatic depolarization of the Erev and the Em (Fig 2H and I). Blocking TREK-1 channels under these conditions only mildly depolarizes cells (Em is already set by Kir2.1), while it hyperpolarizes cells expressing TREK-1I267T (Appendix Fig S3), indicating that the TREK-1 mutation has the inherent potential to depolarize cells. Figure 2. TREK-1I267T causes a dominant-negative reduction in outward currents and depolarizes membrane potentials Representative voltage-clamp measurements in Xenopus oocytes injected with wild-type TREK-1 (black) or TREK-1I267T (blue) cRNA (1 ng/oocyte). Currents were recorded 48 h after cRNA injection, applying a ramp protocol, for 3.5 s rising from −120 mV to +40 mV, from a holding potential of −80 mV. Mean current amplitudes of TREK-1- and TREK-1I267T-mediated currents analyzed at +40 mV. Numbers of experiments are indicated within the bar graph. Mean membrane potential (Em) of TREK-1 (black) and TREK-1I267T (blue). Numbers of experiments are indicated within the bar graph. Representative measurement of the current–voltage relationship of TREK-1I267T. Representative current–voltage relationship measurements in oocytes injected with TREK-1 cRNA (2.5 ng/oocyte), TREK-1I267T (2.5 ng/oocyte), wild-type 1.25 ng TREK-1 (mimicking haploinsufficiency), or with wild-type TREK-1 and TREK-1I267T cRNA (1.25 ng each/oocyte, mimicking a heterozygous state). Current–voltage relationship was recorded from a holding potential of −80 mV, applying 10 mV steps from −130 to +50 mV with a duration of 200 ms. Analysis of the relative current amplitude of (E) at +40 mV normalized to TREK-1 wild-type currents (2.5 ng). Numbers of experiments are indicated within the bar graph. Analysis of the Em of (E and F). Representative current traces of Kir2.1 co-expressed with TREK-1(WT) or the I267T mutant. Em analysis from (H) (n = 13) and of Kir2.1 expressed alone. Data information: Data are presented as mean ± SEM. Data in (B, G, and I) are analyzed by non-parametric Mann–Whitney U-test. Data in (C and F) are analyzed by unpaired Student's t-test. P-values are indicated. Download figure Download PowerPoint Na+ permeability in TREK-1I267T The membrane depolarization associated with expression of TREK-1I267T mutant channels might be achieved via an altered ion selectivity. Thus, the relative permeability of mutant channels was assessed by gradually replacing extracellular Na+ by K+ (Fig 3A and B). While for TREK-1, a linear relationship between the extracellular K+ and the Erev was observed, reflecting the Nernst equation, the TREK-1I267T mutant showed a loss of K+ selectivity (13.2 mV/decade) (Fig 3B). In contrast, when extracellular Na+ was gradually replaced by NMDG+, the cells expressing homomeric or heteromeric TREK-1I267T hyperpolarized near to the K+ equilibrium potential (Fig 3C), indicating that the mutant channel is permeable for Na+. Figure 3. The TREK-1I267T mutation alters the potassium selectivity and introduces a sodium permeability A, B. (A) Representative voltage-clamp recordings of wild-type TREK-1 (black) and TREK-1I267T (blue) using different extracellular K+ concentrations, by replacing extracellular NaCl to KCl, and (B) the respective analysis of the Erev of TREK-1 (black) or TREK-1I267T (blue) as a function of the external K+ concentration (n = 10). The slope of the TREK-1I267T mutant was only 13.2 mV/decade. C. Analysis of the Erev of TREK-1 (black), mutant TREK-1I267T (blue), and TREK-1 co-expressed with TREK-1I267T (dark blue) while gradually removing the extracellular Na+, by replacing NaCl to NMDG-Cl (n = 12). The K+ concentration was maintained at 2 mM. D. Representative current traces of TREK-1 (black) and TREK-1I267T (blue) recorded in a bath solution containing 96 mM Na+ or 0 Na+ (NaCl to NMDG-Cl with Ko+ maintained at 2 mM). E. Percentage of current change when Na+ was removed from the extracellular recording solution, as in (D). Numbers of experiments are indicated within the bar graph. F. Average current amplitude of TREK-1 or TREK-1I267T in extracellular solution containing 98 mM K+ but no Na+ (all NaCl replaced to KCl). Numbers of experiments are indicated within the bar graph. G. Erev of wild-type TREK-1 (black) and TREK-1I267T (blue) as a function of the external K+ concentration (w/o Na+, 0 mM Na+) (n = 9). The slope was about 52 mV/decade for both constructs. H. Normalized current–voltage relationship of TREK-1 (black) (n = 7) and TREK-1I267T (blue) (n = 6) at pHo 8.5 and 6.0. The inset shows a magnification, highlighting the different Erev. Panels at the bottom illustrate the average of the Erev at pHo 8.5 and 6.0 of TREK-1 (black) and TREK-1I267T (blue). Data information: Data are presented as mean ± SEM. Data in (E) are analyzed by non-parametric Mann–Whitney U-test. Data in (H) are analyzed by unpaired Student's t-test. P-values are indicated. Download figure Download PowerPoint TREK-1 is "activated" by extracellular sodium (Fink et al, 1996), accordingly, replacing extracellular Na+ by NMDG+ strongly reduced wild-type TREK-1 outward currents (Fig 3D). In contrast, for TREK-1I267T the outward currents were increased, the leak with its unique current kinetics was removed and the Erev was hyperpolarized to a value close to EK (Fig 3D). Thus, extracellular Na+ activates the outward conductance of wild-type TREK-1, but causes Na+ inward currents and a partial reduction in the outward currents recorded for the TREK-1I267T mutant (Fig 3D and E). In the absence of Na+, wild-type and mutant TREK-1 channels conduct outward currents with similar amplitudes (Fig 3F), indicating that the reduced outward currents of TREK-1I267T are caused by their greatly enhanced Na+ permeability, while the surface expression of the mutant is most likely not affected. As expected, determining the selectivity in Na+-free solution (NMDG+ by K+) the TREK-1I267T mutant obeys the Nernst equation for a selective K+ channel (Fig 3G). In addition, we have performed molecular dynamic simulations which are also in agreement with the loss of selectivity effect of TREK-1I267T (Appendix Fig S4). TREK-1I267T has a TWIK-like sodium permeable selectivity filter TREK-1 has two pore loops containing the amino acid sequence "TIGFG". In TWIK-1, the pore signature sequence of the first pore loop differs from that of other K2P channels. Here, the conserved isoleucine preceding the first signature sequence is substituted by a threonine ("TTGYG"; Ma et al, 2011; Chatelain et al, 2012), which results in an enhanced sodium permeability of these channels under hypokalemic (Ma et al, 2011) or acidic conditions (Chatelain et al, 2012; Ma et al, 2012). The TREK-1I267T mutation in the second pore loop creates a TWIK-like selectivity filter, potentially explaining the high Na+ ion permeability observed in the mutant channel which, however, already occurs under baseline conditions. Similar to TWIK-1 (Chatelain et al, 2012; Ma et al, 2012) and unlike wild-type TREK-1, the Erev of TREK-1I267T expressing cells is depolarized upon extracellular acidification (Fig 3H and Appendix Fig S5). TREK-1I267T depolarizes HL-1 cardiomyocytes and slows upstroke velocity Consistent with the overexpression of a K+ channel, transfection of wild-type TREK-1 (Fig 4A) slowed the spontaneous beating frequency of HL-1 cardiomyocytes (Fig 4B). In contrast, the action potential (AP) frequency of HL-1 cells overexpressing TREK-1I267T was significantly increased compared to wild-type transfected cells (Fig 4B). Patch clamp recordings showed that the overexpression of TREK-1I267T did not significantly alter the AP repolarization (Fig 4C and D). The shape of the afterhyperpolarization (AHP) was not altered (Fig 4E and F). As expected for a Na+ permeable channel, HL-1 cells expression TREK-1I267T were more depolarized (Fig 4C, E and G), an effect that would be pro-arrhythmic in ventricular cardiomyocytes, as the membrane potential is closer to the AP threshold. In addition, a slowing of the upstroke velocity was observed (Fig 4H and I), a factor that is known to contribute to ventricular re-entry arrhythmias due to conduction velocity reduction and thus a shorter wavelength. Next we utilized a computational model of the human ventricular action potential (Appendix Supplementary Methods and Appendix Fig S6) and assigned a background potassium current with a relative sodium permeability of 20%, as observed for TREK-1I267T. Consistent with our experimental observations, a mild depolarization and a slowing of the upstroke velocity were present (Appendix Fig S6H and I). Figure 4. TREK-1I267T speeds early repolarization and depolarizes the membrane potential of spontaneously beating HL-1 cells Fluorescence imaging of HL-1 cells transfected with EGFP-tagged TREK-1 or TREK-1I267T. Action potential (AP) frequency, as beats per minute (bpm), counted in HL-1 cells (HL-1) bathed in Claycomb medium containing 100 μM norepinephrine or HL-1 cells transfected with TREK-1-EGFP (WT) or TREK-1I267T-EGFP (I267T). The number of experiments are indicated in the bar graphs. Patch clamp measurements in the current-clamp mode of HL-1 cells transfected with TREK-1-EGFP or mutant TREK-1I267T-EGFP. Boxes indicate the zoom area for panel (E). Analyses of the AP duration, APD50, and APD90 of HL-1 cells transfected with TREK-1-EGFP or TREK-1I267T-EGFP. The number of experiments are indicated in the bar graphs. Illustration of the hyperpolarization observed following an AP of HL-1 cells transfected with TREK-1-EGFP or TREK-1I267T-EGFP. Analysis of the afterhyperpolarization (AHP) observed in HL-1 cells transfected with TREK-1-EGFP (n = 5) or TREK-1I267T-EGFP (n = 6). Analyses of the maximum diastolic hyperpolarization (max. HP) of HL-1 cells transfected with TREK-1-EGFP (n = 5) or TREK-1I267T-EGFP (n = 6). Illustration of the upstroke velocity of HL-1 cells transfected with TREK-1-EGFP or TREK-1I267T-EGFP. The threshold for the action potentials was aligned in order to compare the upstroke phase and velocity. Analyses of the upstroke velocity (mV/ms) of HL-1 cells transfected with TREK-1-EGFP (n = 5) or TREK-1I267T-EGFP (n = 6). Data information: Data are presented as mean ± SEM. Data in (B, G, and I) are analyzed by non-parametric Mann–Whitney U-test. P-values are indicated. Download figure Download PowerPoint BL-1249 restores TREK-1I267T K+ selectivity Based on our observation that inhibition of TREK-1 in cells co-expressing TREK-1I267T and Kir2.1 results in hyperpolarization of cells (Appendix Fig S3), blocking TREK-1I267T-mediated c
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