Michael F. Netter

Knock-Out of the Potassium Channel TASK-1 Leads to a Prolonged QT Interval and a Disturbed QRS Complex

Background/Aims: The aim of the study was to characterize the whole cell current of the two-pore domain potassium channel TASK-1 (K2P3) in mouse ventricular cardiomyocytes (ITASK-1) and to analyze the cardiac phenotype of the TASK-1-/- mice. Methods and Results: We have quantified the ventricular ITASK-1 current using the blocker A293 and TASK-1-/- mice. Surface electrocardiogram recordings of TASK-1-/- mice showed a prolonged QTc interval and a broadened QRS complex. The differences in electrocardiograms between wild type and TASK-1-/- mice disappeared during sympathetic stimulation of the animals. Quantitative RT-PCR, patch clamp recordings and measurements of hemodynamic performance of TASK-1-/- mice revealed no major compensatory changes in ion channel transcription. Action potential recordings of TASK-1-/- mouse cardiomyocytes indicated that ITASK-1 modulates action potential duration. Our in vivo electrophysiological studies showed that isoflurane, which activates TASK-1, slowed heart rate and atrioventricular conduction of wild-type but not of TASK-1-/- mice. Conclusion: The results of an invasive electrophysiological catheter protocol in combination with the observed QRS time prolongation in the surface electrocardiogram point towards a regulatory role of TASK-1 in the cardiac conduction system.

A Specific Two-pore Domain Potassium Channel Blocker Defines the Structure of the TASK-1 Open Pore

Two-pore domain potassium (K2P) channels play a key role in setting the membrane potential of excitable cells. Despite their role as putative targets for drugs and general anesthetics, little is known about the structure and the drug binding site of K2P channels. We describe A1899 as a potent and highly selective blocker of the K2P channel TASK-1. As A1899 acts as an open-channel blocker and binds to residues forming the wall of the central cavity, the drug was used to further our understanding of the channel pore. Using alanine mutagenesis screens, we have identified residues in both pore loops, the M2 and M4 segments, and the halothane response element to form the drug binding site of TASK-1. Our experimental data were used to validate a K2P open-pore homology model of TASK-1, providing structural insights for future rational design of drugs targeting K2P channels.

TASK-1 Channels May Modulate Action Potential Duration of Human Atrial Cardiomyocytes

Background/Aims: Atrial fibrillation is the most common arrhythmia in the elderly, and potassium channels with atrium-specific expression have been discussed as targets to treat atrial fibrillation. Our aim was to characterize TASK-1 channels in human heart and to functionally describe the role of the atrial whole cell current ITASK-1. Methods and Results: Using quantitative PCR, we show that TASK-1 is predominantly expressed in the atria, auricles and atrio-ventricular node of the human heart. Single channel recordings show the functional expression of TASK-1 in right human auricles. In addition, we describe for the first time the whole cell current carried by TASK-1 channels (ITASK-1) in human atrial tissue. We show that ITASK-1 contributes to the sustained outward current IKsus and that ITASK-1 is a major component of the background conductance in human atrial cardiomyocytes. Using patch clamp recordings and mathematical modeling of action potentials, we demonstrate that modulation of ITASK-1 can alter human atrial action potential duration. Conclusion: Due to the lack of ventricular expression and the ability to alter human atrial action potential duration, TASK-1 might be a drug target for the treatment of atrial fibrillation.

RNA editing modulates the binding of drugs and highly unsaturated fatty acids to the open pore of Kv potassium channels

The time course of inactivation of voltage‐activated potassium (Kv) channels is an important determinant of the firing rate of neurons. In many Kv channels highly unsaturated lipids as arachidonic acid, docosahexaenoic acid and anandamide can induce fast inactivation. We found that these lipids interact with hydrophobic residues lining the inner cavity of the pore. We analysed the effects of these lipids on Kv1.1 current kinetics and their competition with intracellular tetraethylammonium and Kvβ subunits. Our data suggest that inactivation most likely represents occlusion of the permeation pathway, similar to drugs that produce ‘open‐channel block’. Open‐channel block by drugs and lipids was strongly reduced in Kv1.1 channels whose amino acid sequence was altered by RNA editing in the pore cavity, and in Kv1.x heteromeric channels containing edited Kv1.1 subunits. We show that differential editing of Kv1.1 channels in different regions of the brain can profoundly alter the pharmacology of Kv1.x channels. Our findings provide a mechanistic understanding of lipid‐induced inactivation and establish RNA editing as a mechanism to induce drug and lipid resistance in Kv channels.

Mitochondrial small conductance SK2 channels prevent glutamate-induced oxytosis and mitochondrial dysfunction

Background: SK2 channels modulate NMDA-dependent neuronal excitability and provide neuroprotection against excitotoxicity. Results: We identify mitoSK2/KCa2.2 channels in neuronal mitochondria and demonstrate their protective function in cells lacking NMDAR. Conclusion: SK2 channels prevent mitochondrial dysfunction and completely restore cell viability independently of NMDAR modulation. Significance: Understanding how mitochondrial SK2 channels operate is crucial to develop novel therapeutic strategies for diseases caused by mitochondrial demise. Small conductance calcium-activated potassium (SK2/KCa2.2) channels are known to be located in the neuronal plasma membrane where they provide feedback control of NMDA receptor activity. Here, we provide evidence that SK2 channels are also located in the inner mitochondrial membrane of neuronal mitochondria. Patch clamp recordings in isolated mitoplasts suggest insertion into the inner mitochondrial membrane with the C and N termini facing the intermembrane space. Activation of SK channels increased mitochondrial K+ currents, whereas channel inhibition attenuated these currents. In a model of glutamate toxicity, activation of SK2 channels attenuated the loss of the mitochondrial transmembrane potential, blocked mitochondrial fission, prevented the release of proapoptotic mitochondrial proteins, and reduced cell death. Neuroprotection was blocked by specific SK2 inhibitory peptides and siRNA targeting SK2 channels. Activation of mitochondrial SK2 channels may therefore represent promising targets for neuroprotective strategies in conditions of mitochondrial dysfunction.

Gain-of-function mutation in TASK-4 channels and severe cardiac conduction disorder

Analyzing a patient with progressive and severe cardiac conduction disorder combined with idiopathic ventricular fibrillation (IVF), we identified a splice site mutation in the sodium channel gene SCN5A. Due to the severe phenotype, we performed whole‐exome sequencing (WES) and identified an additional mutation in the KCNK17 gene encoding the K2P potassium channel TASK‐4. The heterozygous change (c.262G>A) resulted in the p.Gly88Arg mutation in the first extracellular pore loop. Mutant TASK‐4 channels generated threefold increased currents, while surface expression was unchanged, indicating enhanced conductivity. When co‐expressed with wild‐type channels, the gain‐of‐function by G88R was conferred in a dominant‐active manner. We demonstrate that KCNK17 is strongly expressed in human Purkinje cells and that overexpression of G88R leads to a hyperpolarization and strong slowing of the upstroke velocity of spontaneously beating HL‐1 cells. Thus, we propose that a gain‐of‐function by TASK‐4 in the conduction system might aggravate slowed conductivity by the loss of sodium channel function. Moreover, WES supports a second hit‐hypothesis in severe arrhythmia cases and identified KCNK17 as a novel arrhythmia gene.

Kv1.5 blockers preferentially inhibit TASK-1 channels: TASK-1 as a target against atrial fibrillation and obstructive sleep apnea?

Atrial fibrillation and obstructive sleep apnea are responsible for significant morbidity and mortality in the industrialized world. There is a high medical need for novel drugs against both diseases, and here, Kv1.5 channels have emerged as promising drug targets. In humans, TASK-1 has an atrium-specific expression and TASK-1 is also abundantly expressed in the hypoglossal motor nucleus. We asked whether known Kv1.5 channel blockers, effective against atrial fibrillation and/or obstructive sleep apnea, modulate TASK-1 channels. Therefore, we tested Kv1.5 blockers with different chemical structures for their TASK-1 affinity, utilizing two-electrode voltage clamp (TEVC) recordings in Xenopus oocytes. Despite the low structural conservation of Kv1.5 and TASK-1 channels, we found all Kv1.5 blockers analyzed to be even more effective on TASK-1 than on Kv1.5. For instance, the half-maximal inhibitory concentration (IC50) values of AVE0118 and AVE1231 (A293) were 10- and 43-fold lower on TASK-1. Also for MSD-D, ICAGEN-4, S20951 (A1899), and S9947, the IC50 values were 1.4- to 70-fold lower than for Kv1.5. To describe this phenomenon on a molecular level, we used in silico models and identified unexpected structural similarities between the two drug binding sites. Kv1.5 blockers, like AVE0118 and AVE1231, which are promising drugs against atrial fibrillation or obstructive sleep apnea, are in fact potent TASK-1 blockers. Accordingly, block of TASK-1 channels by these compounds might contribute to the clinical effectiveness of these drugs. The higher affinity of these blockers for TASK-1 channels suggests that TASK-1 might be an unrecognized molecular target of Kv1.5 blockers effective in atrial fibrillation or obstructive sleep apnea.

TASK-1 and TASK-3 may form heterodimers in human atrial cardiomyocytes

TASK-1 channels have emerged as promising drug targets against atrial fibrillation, the most common arrhythmia in the elderly. While TASK-3, the closest relative of TASK-1, was previously not described in cardiac tissue, we found a very prominent expression of TASK-3 in right human auricles. Immunocytochemistry experiments of human right auricular cardiomyocytes showed that TASK-3 is primarily localized at the plasma membrane. Single-channel recordings of right human auricles in the cell-attached mode, using divalent-cation-free solutions, revealed a TASK-1-like channel with a single-channel conductance of about 30pS. While homomeric TASK-3 channels were not found, we observed an intermediate single-channel conductance of about 55pS, possibly reflecting the heteromeric channel formed by TASK-1 and TASK-3. Subsequent experiments with TASK-1/TASK-3 tandem channels or with co-expressed TASK-1 and TASK-3 channels in HEK293 cells or Xenopus oocytes, supported that the 55pS channels observed in right auricles have electrophysiological characteristics of TASK-1/TASK-3 heteromers. In addition, co-expression experiments and single-channel recordings suggest that heteromeric TASK-1/TASK-3 channels have a predominant surface expression and a reduced affinity for TASK-1 blockers. In summary, the evidence for heteromeric TASK-1/TASK-3 channel complexes together with an altered pharmacologic response to TASK-1 blockers in vitro is likely to have further impact for studies isolating ITASK-1 from cardiomyocytes and for the development of drugs specifically targeting TASK-1 in atrial fibrillation treatment.

Putative Impact of RNA Editing on Drug Discovery

Virtually all organisms use RNA editing as a powerful post‐transcriptional mechanism to recode genomic information and to increase functional protein diversity. The enzymatic editing of pre‐mRNA by ADARs and CDARs is known to change the functional properties of neuronal receptors and ion channels regulating cellular excitability. However, RNA editing is also an important mechanism for genes expressed outside the brain. The fact that RNA editing breaks the ‘one gene encodes one protein’ hypothesis is daunting for scientists and a probable drawback for drug development, as scientists might search for drugs targeting the ‘wrong’ protein. This possible difficulty for drug discovery and development became more evident from recent publications, describing that RNA editing events have profound impact on the pharmacology of some common drug targets. These recent studies highlight that RNA editing can cause massive discrepancies between the in vitro and in vivo pharmacology. Here, we review the putative impact of RNA editing on drug discovery, as RNA editing has to be considered before using high‐throughput screens, rational drug design or choosing the right model organism for target validation.

The HCN4 Channel Mutation D553N Associated With Bradycardia Has a C-linker Mediated Gating Defect

Background/Aims: The D553N mutation located in the C-linker of the cardiac pacemaker channel HCN4 is thought to cause sino-atrial dysfunction via a pronounced dominant-negative trafficking defect. Since HCN4 mutations usually have a minor defect in channel gating, it was our aim to further characterize the disease causing mechanism of D553N. Methods: Fluorescence microscopy, FACS, TEVC and patch-clamp recordings were performed to characterize D553N. Results: Surprisingly, we found that D553N channels reach the plasma membrane and have no apparent trafficking defect. Co-expression of D553N with HCN4 also revealed no dominant-negative effect on wild-type channels. Consistent with the normal cell surface expression of D553N, it was possible to extensively characterize D553N mutants in Xenopus oocytes and mammalian cells. D553N channels generate currents with reduced amplitude, while the kinetics of activation and deactivation are not altered. While the regulation of D553N by tyrosine kinases is normal, we observed a change in the cAMP regulation which however cannot account for the strong loss-of-function of the mutant. Conclusion: The pronounced current reduction and the regular surface expression indicate a major gating defect of the C-linker gate. We hypothesize that the D553N mutation stabilizes a previously reported salt bridge important for the gating of the channel.