N-Glycosylation of TREK-1/hK2P2.1 Two-Pore-Domain Potassium (K2P) Channels.

Mechanosensitive hTREK-1 two-pore-domain potassium (hK2P2.1) channels give rise to background currents that control cellular excitability. Recently, TREK-1 currents have been linked to the regulation of cardiac rhythm as well as to hypertrophy and fibrosis. Even though the pharmacological and biophysical characteristics of hTREK-1 channels have been widely studied, relatively little is known about their posttranslational modifications. This study aimed to evaluate whether hTREK-1 channels are N-glycosylated and whether glycosylation may affect channel functionality. Following pharmacological inhibition of N-glycosylation, enzymatic digestion or mutagenesis, immunoblots of Xenopus laevis oocytes and HEK-293T cell lysates were used to assess electrophoretic mobility. Two-electrode voltage clamp measurements were employed to study channel function. TREK-1 channel subunits undergo N-glycosylation at asparagine residues 110 and 134. The presence of sugar moieties at these two sites increases channel function. Detection of glycosylation-deficient mutant channels in surface fractions and recordings of macroscopic potassium currents mediated by these subunits demonstrated that nonglycosylated hTREK-1 channel subunits are able to reach the cell surface in general but with seemingly reduced efficiency compared to glycosylated subunits. These findings extend our understanding of the regulation of hTREK-1 currents by posttranslational modifications and provide novel insights into how altered ion channel glycosylation may promote arrhythmogenesis.

Genetic Ablation of TASK-1 (Tandem of P Domains in a Weak Inward Rectifying K+ Channel-Related Acid-Sensitive K+ Channel-1) (K2P3.1) K+ Channels Suppresses Atrial Fibrillation and Prevents Electrical Remodeling.

BACKGROUND: Despite an increasing understanding of atrial fibrillation (AF) pathophysiology, translation into mechanism-based treatment options is lacking. In atrial cardiomyocytes of patients with chronic AF, expression, and function of tandem of P domains in a weak inward rectifying TASK-1 (K+ channel-related acid-sensitive K+ channel-1) (K2P3.1) atrial-specific 2-pore domain potassium channels is enhanced, resulting in action potential duration shortening. TASK-1 channel inhibition prevents action potential duration shortening to maintain values observed among sinus rhythm subjects. The present preclinical study used a porcine AF model to evaluate the antiarrhythmic efficacy of TASK-1 inhibition by adeno-associated viral anti-TASK-1-siRNA (small interfering RNA) gene transfer. METHODS: AF was induced in domestic pigs by atrial burst stimulation via implanted pacemakers. Adeno-associated viral vectors carrying anti-TASK-1-siRNA were injected into both atria to suppress TASK-1 channel expression. After the 14-day follow-up period, porcine cardiomyocytes were isolated from right and left atrium, followed by electrophysiological and molecular characterization. RESULTS: AF was associated with increased TASK-1 transcript, protein and ion current levels leading to shortened action potential duration in atrial cardiomyocytes compared to sinus rhythm controls, similar to previous findings in humans. Anti-TASK-1 adeno-associated viral application significantly reduced AF burden in comparison to untreated AF pigs. Antiarrhythmic effects of anti-TASK-1-siRNA were associated with reduction of TASK-1 currents and prolongation of action potential durations in atrial cardiomyocytes to sinus rhythm values. Conclusions Adeno-associated viral-based anti-TASK-1 gene therapy suppressed AF and corrected cellular electrophysiological remodeling in a porcine model of AF. Suppression of AF through selective reduction of TASK-1 currents represents a new option for antiarrhythmic therapy.

N-glycosylation-dependent regulation of hK2P17.1 currents.

Two pore-domain potassium (K2P) channels mediate potassium background currents that stabilize the resting membrane potential and facilitate action potential repolarization. In the human heart, hK2P17.1 channels are predominantly expressed in the atria and Purkinje cells. Reduced atrial hK2P17.1 protein levels were described in patients with atrial fibrillation or heart failure. Genetic alterations in hK2P17.1 were associated with cardiac conduction disorders. Little is known about posttranslational modifications of hK2P17.1. Here, we characterized glycosylation of hK2P17.1 and investigated how glycosylation alters its surface expression and activity. Wild-type hK2P17.1 channels and channels lacking specific glycosylation sites were expressed in Xenopus laevis oocytes, HEK-293T cells, and HeLa cells. N-glycosylation was disrupted using N-glycosidase F and tunicamycin. hK2P17.1 expression and activity were assessed using immunoblot analysis and a two-electrode voltage clamp technique. Channel subunits of hK2P17.1 harbor two functional N-glycosylation sites at positions N65 and N94. In hemi-glycosylated hK2P17.1 channels, functionality and membrane trafficking remain preserved. Disruption of both N-glycosylation sites results in loss of hK2P17.1 currents, presumably caused by impaired surface expression. This study confirms diglycosylation of hK2P17.1 channel subunits and its pivotal role in cell-surface targeting. Our findings underline the functional relevance of N-glycosylation in biogenesis and membrane trafficking of ion channels.

Identification of the A293 (AVE1231) Binding Site in the Cardiac Two-Pore-Domain Potassium Channel TASK-1: a Common Low Affinity Antiarrhythmic Drug Binding Site.

BACKGROUND/AIMS: The two-pore-domain potassium channel TASK-1 regulates atrial action potential duration. Due to the atrium-specific expression of TASK-1 in the human heart and the functional upregulation of TASK-1 currents in atrial fibrillation (AF), TASK-1 represents a promising target for the treatment of AF. Therefore, detailed knowledge of the molecular determinants of TASK-1 inhibition may help to identify new drugs for the future therapy of AF. In the current study, the molecular determinants of TASK-1 inhibition by the potent and antiarrhythmic compound A293 (AVE1231) were studied in detail. METHODS: Alanine-scanning mutagenesis together with two-electrode voltage-clamp recordings were combined with in silico docking experiments. RESULTS: Here, we have identified Q126 located in the M2 segment together with L239 and N240 of the M4 segment as amino acids essential for the A293-mediated inhibition of TASK-1. These data indicate a binding site which is different to that of A1899 for which also residues of the pore signature sequence and the late M4 segments are essential. Using in silico docking experiments, we propose a binding site at the lower end of the cytosolic pore, located at the entry to lateral side fenestrations of TASK-1. Strikingly, TASK-1 inhibition by the low affinity antiarrhythmic TASK-1 blockers propafenone, amiodarone and carvedilol was also strongly diminished by mutations at this novel binding site. CONCLUSION: We have identified the A293 binding site in the central cavity of TASK-1 and propose that this site might represent a conserved site of action for many low affinity antiarrhythmic TASK-1 blockers.

Antiarrhythmic Properties of Ranolazine: Inhibition of Atrial Fibrillation Associated TASK-1 Potassium Channels.

Background: Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and one of the major causes of cardiovascular morbidity and mortality. Despite good progress within the past years, safe and effective treatment of AF remains an unmet clinical need. The anti-anginal agent ranolazine has been shown to exhibit antiarrhythmic properties via mainly late INa and IKr blockade. This results in prolongation of the atrial action potential duration (APD) and effective refractory period (ERP) with lower effect on ventricular electrophysiology. Furthermore, ranolazine has been shown to be effective in the treatment of AF. TASK-1 is a two-pore domain potassium (K2P) channel that shows nearly atrial specific expression within the human heart and has been found to be upregulated in AF, resulting in shortening the atrial APD in patients suffering from AF. We hypothesized that inhibition TASK-1 contributes to the observed electrophysiological and clinical effects of ranolazine. Methods: We used Xenopus laevis oocytes and CHO-cells as heterologous expression systems for the study of TASK-1 inhibition by ranolazine and molecular drug docking simulations to investigate the ranolazine binding site and binding characteristics. Results: Ranolazine acts as an inhibitor of TASK-1 potassium channels that inhibits TASK-1 currents with an IC50 of 30.6 ± 3.7 µM in mammalian cells and 198.4 ± 1.1 µM in X. laevis oocytes. TASK-1 inhibition by ranolazine is not frequency dependent but shows voltage dependency with a higher inhibitory potency at more depolarized membrane potentials. Ranolazine binds within the central cavity of the TASK-1 inner pore, at the bottom of the selectivity filter. Conclusions: In this study, we show that ranolazine inhibits TASK-1 channels. We suggest that inhibition of TASK-1 may contribute to the observed antiarrhythmic effects of Ranolazine. This puts forward ranolazine as a prototype drug for the treatment of atrial arrhythmia because of its combined efficacy on atrial electrophysiology and lower risk for ventricular side effects.

Atrial fibrillation and heart failure-associated remodeling of two-pore-domain potassium (K2P) channels in murine disease models: focus on TASK-1.

Understanding molecular mechanisms involved in atrial tissue remodeling and arrhythmogenesis in atrial fibrillation (AF) is essential for developing specific therapeutic approaches. Two-pore-domain potassium (K2P) channels modulate cellular excitability, and TASK-1 (K2P3.1) currents were recently shown to alter atrial action potential duration in AF and heart failure (HF). Finding animal models of AF that closely resemble pathophysiological alterations in human is a challenging task. This study aimed to analyze murine cardiac expression patterns of K2P channels and to assess modulation of K2P channel expression in murine models of AF and HF. Expression of cardiac K2P channels was quantified by real-time qPCR and immunoblot in mouse models of AF [cAMP-response element modulator (CREM)-IbΔC-X transgenic animals] or HF (cardiac dysfunction induced by transverse aortic constriction, TAC). Cloned murine, human, and porcine TASK-1 channels were heterologously expressed in Xenopus laevis oocytes. Two-electrode voltage clamp experiments were used for functional characterization. In murine models, among members of the K2P channel family, TASK-1 expression displayed highest levels in both atrial and ventricular tissue samples. Furthermore, K2P2.1, K2P5.1, and K2P6.1 showed significant expression levels. In CREM-transgenic mice, atrial expression of TASK-1 was significantly reduced in comparison with wild-type animals. In a murine model of TAC-induced pressure overload, ventricular TASK-1 expression remained unchanged, while atrial TASK-1 levels were significantly downregulated. When heterologously expressed in Xenopus oocytes, currents of murine, porcine, and human TASK-1 displayed similar characteristics. TASK-1 channels display robust cardiac expression in mice. Murine, porcine, and human TASK-1 channels share functional similarities. Dysregulation of atrial TASK-1 expression in murine AF and HF models suggests a mechanistic contribution to arrhythmogenesis.

The importance of scientific competencies in German medical curricula - the student perspective.

BACKGROUND: Scientific competencies are of great importance for physicians; not only for conducting reliable research, but also for patient care. However, there is growing concern that a lack of scientific competencies among physicians may lead to a deterioration in the quality on biomedical research. This study aims at assessing medical students' perspectives on the implementation of scientific competency training in German medical curricula. METHODS: An online survey was conducted in order to collect German medical students' opinions on the importance of acquiring scientific competencies during their medical studies and to provide us with an assessment of their current levels of basic scientific competencies by having them conduct a self-evaluation. Moreover, we wanted to understand their perceptions of current curricular content and to receive suggestions for improving scientific competency training. Participants were reached via the mailing lists of the German Medical Students' Association, as well as of local medical student committees, and the German Medical Students' Associations social media channel on Facebook. RESULTS: In total, 2380 medical students from across all 37 German medical faculties participated in the survey. The majority of students agreed that the ability to critically evaluate the relevant literature is an important competency for physicians, and that every student should conduct a research project during their medical studies. However, the students evaluated their scientific competencies as unsatisfactory, especially with regard to statistics and scientific writing. They were strongly in favor of receiving extended research training. CONCLUSION: Our study provides insight into German medical students' self-perception in relation to both patient care and biomedical research, and makes recommendations for potential improvements in scientific training. The study demonstrates that scientific competencies are of great importance to medical students in Germany. Students are not lacking motivation for scientific practice and have numerous ideas for enhancing scientific teaching opportunities. Scientific training should follow a holistic approach based on three pillars: (i) a scientific core curriculum, (ii) intracurricular research projects, and (iii) special research programs for students strongly interested in medical research.

New Targets for Old Drugs: Cardiac Glycosides Inhibit Atrial-Specific K2P3.1 (TASK-1) Channels.

Cardiac glycosides have been used in the treatment of arrhythmias for more than 200 years. Two-pore-domain (K2P) potassium channels regulate cardiac action potential repolarization. Recently, K2P3.1 [tandem of P domains in a weak inward rectifying K+ channel (TWIK)-related acid-sensitive K+ channel (TASK)-1] has been implicated in atrial fibrillation pathophysiology and was suggested as an atrial-selective antiarrhythmic drug target. We hypothesized that blockade of cardiac K2P channels contributes to the mechanism of action of digitoxin and digoxin. All functional human K2P channels were screened for interactions with cardiac glycosides. Human K2P channel subunits were expressed in Xenopus laevis oocytes, and voltage clamp electrophysiology was used to record K+ currents. Digitoxin significantly inhibited K2P3.1 and K2P16.1 channels. By contrast, digoxin displayed isolated inhibitory effects on K2P3.1. K2P3.1 outward currents were reduced by 80% (digitoxin, 1 Hz) and 78% (digoxin, 1 Hz). Digitoxin inhibited K2P3.1 currents with an IC50 value of 7.4 µM. Outward rectification properties of the channel were not affected. Mutagenesis studies revealed that amino acid residues located at the cytoplasmic site of the K2P3.1 channel pore form parts of a molecular binding site for cardiac glycosides. In conclusion, cardiac glycosides target human K2P channels. The antiarrhythmic significance of repolarizing atrial K2P3.1 current block by digoxin and digitoxin requires validation in translational and clinical studies.

Ion Channel Dysfunctions in Dilated Cardiomyopathy in Limb-Girdle Muscular Dystrophy.

BACKGROUND: Limb-Girdle muscular dystrophies (LGMD) are a heritable group of genetically determined disorders with a primary involvement of the pelvic or shoulder girdle musculature with partially cardiac manifestation, such as dilated cardiomyopathy (DCM) and life-threatening tachyarrhythmia. We report here that human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes from a patient with LGMD2I and DCM associated with recurrent ventricular tachycardia displayed ion channel dysfunction and abnormality of calcium homeostasis. METHODS: Dermal fibroblasts obtained from a patient with LGMD2I harboring a fukutin-related protein gene mutation (826C>A; Leu276Ile) and 3 healthy donors were reprogrammed to hiPSCs. The hiPSCs were differentiated into cardiomyocytes and used for biological and electrophysiological studies. RESULTS: Compared with hiPSC cardiomyocytes from the healthy donors, the hiPSC cardiomyocytes from the patient exhibited abnormal action potentials characterized by reduced amplitude and upstroke velocity. The peak and late Na channel currents (INa) as well as the peak L-type calcium channel currents were significantly reduced. The expression of SCN5A and CACNA1C was reduced in DCM cardiomyocytes, consistent with reduction of INa and L-type calcium channel currents. In addition, the rapidly activating delayed rectifier potassium current (IKr) was reduced, whereas the transient outward current (Ito) and slowly activating delayed rectifier potassium current (IKs) were similar in DCM and control cardiomyocytes. Finally, a significant reduction of systolic and diastolic intracellular Ca2+ concentrations was detected in DCM cardiomyocytes. CONCLUSIONS: This study demonstrates that patient-specific hiPSC cardiomyocytes can recapitulate some phenotypic properties of LGMD2I with DCM and provide a platform for studies on the cardiac events in LGMD.

Stretch-activated two-pore-domain (K2P) potassium channels in the heart: Focus on atrial fibrillation and heart failure.

Two-pore-domain potassium (K2P) channels modulate cellular excitability. The significance of stretch-activated cardiac K2P channels (K2P2.1, TREK-1, KCNK2; K2P4.1, TRAAK, KCNK4; K2P10.1, TREK-2, KCNK10) in heart disease has not been elucidated in detail. The aim of this work was to assess expression and remodeling of mechanosensitive K2P channels in atrial fibrillation (AF) and heart failure (HF) patients in comparison to murine models. Cardiac K2P channel levels were quantified in atrial (A) and ventricular (V) tissue obtained from patients undergoing open heart surgery. In addition, control mice and mouse models of AF (cAMP-response element modulator (CREM)-IbΔC-X transgenic animals) or HF (cardiac dysfunction induced by transverse aortic constriction, TAC) were employed. Human and murine KCNK2 displayed highest mRNA abundance among mechanosensitive members of the K2P channel family (V > A). Disease-associated K2P2.1 remodeling was studied in detail. In patients with impaired left ventricular function, atrial KCNK2 (K2P2.1) mRNA and protein expression was significantly reduced. In AF subjects, downregulation of atrial and ventricular KCNK2 (K2P2.1) mRNA and protein levels was observed. AF-associated suppression of atrial Kcnk2 (K2P2.1) mRNA and protein was recapitulated in CREM-transgenic mice. Ventricular Kcnk2 expression was not significantly altered in mouse models of disease. In conclusion, mechanosensitive K2P2.1 and K2P10.1 K+ channels are expressed throughout the heart. HF- and AF-associated downregulation of KCNK2 (K2P2.1) mRNA and protein levels suggest a mechanistic contribution to cardiac arrhythmogenesis.

Inverse remodelling of K2P3.1 K+ channel expression and action potential duration in left ventricular dysfunction and atrial fibrillation: implications for patient-specific antiarrhythmic drug therapy.

AIMS: Atrial fibrillation (AF) prevalence increases with advanced stages of left ventricular (LV) dysfunction. Remote proarrhythmic effects of ventricular dysfunction on atrial electrophysiology remain incompletely understood. We hypothesized that repolarizing K2P3.1 K+ channels, previously implicated in AF pathophysiology, may contribute to shaping the atrial action potential (AP), forming a specific electrical substrate with LV dysfunction that might represent a target for personalized antiarrhythmic therapy. METHODS AND RESULTS: A total of 175 patients exhibiting different stages of LV dysfunction were included. Ion channel expression was quantified by real-time polymerase chain reaction and Western blot. Membrane currents and APs were recorded from atrial cardiomyocytes using the patch-clamp technique. Severely reduced LV function was associated with decreased atrial K2P3.1 expression in sinus rhythm patients. In contrast, chronic (c)AF resulted in increased K2P3.1 levels, but paroxysmal (p)AF was not linked to significant K2P3.1 remodelling. LV dysfunction-related suppression of K2P3.1 currents prolonged atrial AP duration (APD) compared with patients with preserved LV function. In individuals with concomitant LV dysfunction and cAF, APD was determined by LV dysfunction-associated prolongation and by cAF-dependent shortening, respectively, consistent with changes in K2P3.1 abundance. K2P3.1 inhibition attenuated APD shortening in cAF patients irrespective of LV function, whereas in pAF subjects with severely reduced LV function, K2P3.1 blockade resulted in disproportionately high APD prolongation. CONCLUSION: LV dysfunction is associated with reduction of atrial K2P3.1 channel expression, while cAF leads to increased K2P3.1 abundance. Differential remodelling of K2P3.1 and APD provides a basis for patient-tailored antiarrhythmic strategies.