Susanne Rinné

Popeye domain containing proteins are essential for stress-mediated modulation of cardiac pacemaking in mice

Cardiac pacemaker cells create rhythmic pulses that control heart rate; pacemaker dysfunction is a prevalent disorder in the elderly, but little is known about the underlying molecular causes. Popeye domain containing (Popdc) genes encode membrane proteins with high expression levels in cardiac myocytes and specifically in the cardiac pacemaking and conduction system. Here, we report the phenotypic analysis of mice deficient in Popdc1 or Popdc2. ECG analysis revealed severe sinus node dysfunction when freely roaming mutant animals were subjected to physical or mental stress. In both mutants, bradyarrhythmia developed in an age-dependent manner. Furthermore, we found that the conserved Popeye domain functioned as a high-affinity cAMP-binding site. Popdc proteins interacted with the potassium channel TREK-1, which led to increased cell surface expression and enhanced current density, both of which were negatively modulated by cAMP. These data indicate that Popdc proteins have an important regulatory function in heart rate dynamics that is mediated, at least in part, through cAMP binding. Mice with mutant Popdc1 and Popdc2 alleles are therefore useful models for the dissection of the mechanisms causing pacemaker dysfunction and could aid in the development of strategies for therapeutic intervention.

The Retention Factor p11 Confers an Endoplasmic Reticulum-Localization Signal to the Potassium Channel TASK-1

The interaction of the adaptor protein p11, also denoted S100A10, with the C‐terminus of the two‐pore‐domain K+ channel TASK‐1 was studied using yeast two‐hybrid analysis, glutathione S‐transferase pulldown, and co‐immunoprecipitation. We found that p11 interacts with a 40 amino‐acid region in the proximal C‐terminus of the channel. In heterologous expression systems, deletion of the p11‐interacting domain enhanced surface expression of TASK‐1. Attachment of the p11‐interacting domain to the cytosolic tail of the reporter protein CD8 caused retention/retrieval of the construct in the endoplasmic reticulum (ER). Attachment of the last 36 amino acids of p11 to CD8 also caused ER localization, which was abolished by removal or mutation of a putative retention motif (H/K)xKxxx, at the C‐terminal end of p11. Imaging of EGFP‐tagged TASK‐1 channels in COS cells suggested that wild‐type TASK‐1 was largely retained in the ER. Knockdown of p11 with siRNA enhanced trafficking of TASK‐1 to the surface membrane. Our results suggest that binding of p11 to TASK‐1 retards the surface expression of the channel, most likely by virtue of a di‐lysine retention signal at the C‐terminus of p11. Thus, the cytosolic protein p11 may represent a ‘retention factor’ that causes localization of the channel to the ER.

Intracellular traffic of the K+ channels TASK-1 and TASK-3: role of N- and C-terminal sorting signals and interaction with 14-3-3 proteins.

The two‐pore‐domain potassium channels TASK‐1 (KCNK3) and TASK‐3 (KCNK9) modulate the electrical activity of neurons and many other cell types. We expressed TASK‐1, TASK‐3 and related reporter constructs in Xenopus oocytes, mammalian cell lines and various yeast strains to study the mechanisms controlling their transport to the surface membrane and the role of 14‐3‐3 proteins. We measured potassium currents with the voltage‐clamp technique and fused N‐ and C‐terminal fragments of the channels to various reporter proteins to study changes in subcellular localisation and surface expression. Mutational analysis showed that binding of 14‐3‐3 proteins to the extreme C‐terminus of TASK‐1 and TASK‐3 masks a tri‐basic motif, KRR, which differs in several important aspects from canonical arginine‐based (RxR) or lysine‐based (KKxx) retention signals. Pulldown experiments with GST fusion proteins showed that the KRR motif in the C‐terminus of TASK‐3 channels was able to bind to COPI coatomer. Disabling the binding of 14‐3‐3, which exposes the KRR motif, caused localisation of the GFP‐tagged channel protein mainly to the Golgi complex. TASK‐1 and TASK‐3 also possess a di‐basic N‐terminal retention signal, KR, whose function was found to be independent of the binding of 14‐3‐3. Suppression of channel surface expression with dominant‐negative channel mutants revealed that interaction with 14‐3‐3 has no significant effect on the dimeric assembly of the channels. Our results give a comprehensive description of the mechanisms by which 14‐3‐3 proteins, together with N‐ and C‐terminal sorting signals, control the intracellular traffic of TASK‐1 and TASK‐3.

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.

A di-acidic sequence motif enhances the surface expression of the potassium channel TASK-3.

We have characterized a sequence motif, EDE, in the proximal C‐terminus of the acid‐sensitive potassium channel TASK‐3. Human TASK‐3 channels were expressed in Xenopus oocytes, and the density of the channels at the surface membrane was studied with two complementary techniques: a luminometric surface expression assay of hemagglutinin epitope‐tagged TASK‐3 channels and voltage‐clamp measurements of the acid‐sensitive potassium current. Both approaches showed that mutation of the two glutamate residues of the EDE motif to alanine (ADA mutant) markedly reduced the transport of TASK‐3 channels to the cell surface. Mutation of the central aspartate of the EDE motif had no effect on surface expression. The functional role of the EDE motif was further characterized in chimaeric constructs consisting of truncated Kir2.1 channels to which the C‐terminus of TASK‐3 was attached. In these constructs, too, replacement of the EDE motif by ADA strongly reduced surface expression. Live‐cell imaging of enhanced green fluorescent protein‐tagged channels expressed in COS‐7 cells showed that 24 h after transfection wild‐type TASK‐3 was mainly localized to the cell surface whereas the ADA mutant was largely retained in the endoplasmic reticulum (ER). Mutation of a second di‐acidic motif in the C‐terminus of TASK‐3 (DAE) had no effect on surface expression. Coexpression of TASK‐3 with a GTP‐restricted mutant of the coat recruitment GTPase Sar1 (Sar1H79G) resulted in ER retention of the channel. Our data suggest that the di‐acidic motif, EDE, in human TASK‐3 is a major determinant of the rate of ER export and is required for efficient surface expression of the channel.

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.

Side pockets provide the basis for a new mechanism of Kv channel-specific inhibition

Most known small-molecule inhibitors of voltage-gated ion channels have poor subtype specificity because they interact with a highly conserved binding site in the central cavity. Using alanine-scanning mutagenesis, electrophysiological recordings and molecular modeling, we have identified a new drug-binding site in Kv1.x channels. We report that Psora-4 can discriminate between related Kv channel subtypes because, in addition to binding the central pore cavity, it binds a second, less conserved site located in side pockets formed by the backsides of S5 and S6, the S4-S5 linker, part of the voltage sensor and the pore helix. Simultaneous drug occupation of both binding sites results in an extremely stable nonconducting state that confers high affinity, cooperativity, use-dependence and selectivity to Psora-4 inhibition of Kv1.x channels. This new mechanism of inhibition represents a molecular basis for the development of a new class of allosteric and selective voltage-gated channel inhibitors.

Soluble polysialylated NCAM: a novel player of the innate immune system in the lung

Posttranslational modification of the neural cell adhesion molecule (NCAM) by polysialic acid (polySia) is well studied in the nervous system and described as a dynamic modulator of plastic processes like precursor cell migration, axon fasciculation, and synaptic plasticity. Here, we describe a novel function of polysialylated NCAM (polySia-NCAM) in innate immunity of the lung. In mature lung tissue of healthy donors, polySia was exclusively attached to the transmembrane isoform NCAM-140 and located to intracellular compartments of epithelial cells. In patients with chronic obstructive pulmonary disease, however, increased polySia levels and processing of the NCAM carrier were observed. Processing of polysialylated NCAM was reproduced in a mouse model by bleomycin administration leading to an activation of the inflammasome and secretion of interleukin (IL)-1β. As shown in a cell culture model, polySia-NCAM-140 was kept in the late trans-Golgi apparatus of lung epithelial cells and stimulation by IL-1β or lipopolysaccharide induced metalloprotease-mediated ectodomain shedding, resulting in the secretion of soluble polySia-NCAM. Interestingly, polySia chains of secreted NCAM neutralized the cytotoxic activity of extracellular histones as well as DNA/histone-network-containing “neutrophil extracellular traps”, which are formed during invasion of microorganisms. Thus, shedding of polySia-NCAM by lung epithelial cells may provide a host-protective mechanism to reduce tissue damage during inflammatory processes.

Gain-of-function mutations in the calcium channel CACNA1C (Cav1.2) cause non-syndromic long-QT but not Timothy syndrome

Gain-of-function mutations in CACNA1C, encoding the L-type Ca(2+) channel Cav1.2, cause Timothy syndrome (TS), a multi-systemic disorder with dysmorphic features, long-QT syndrome (LQTS) and autism spectrum disorders. TS patients have heterozygous mutations (G402S and G406R) located in the alternatively spliced exon 8, causing a gain-of-function by reduced voltage-dependence of inactivation. Screening 540 unrelated patients with non-syndromic forms of LQTS, we identified six functional relevant CACNA1C mutations in different regions of the channel. All these mutations caused a gain-of-function combining different mechanisms, including changes in current amplitude, rate of inactivation and voltage-dependence of activation or inactivation, similar as in TS. Computer simulations support the theory that the novel CACNA1C mutations prolong action potential duration. We conclude that genotype-negative LQTS patients should be investigated for mutations in CACNA1C, as a gain-of-function in Cav1.2 is likely to cause LQTS and only specific and rare mutations, i.e. in exon 8, cause the multi-systemic TS.