Marylou Zuzarte

Dendritic cells derived from peripheral monocytes express endothelial markers and in the presence of angiogenic growth factors differentiate into endothelial-like cells.

CD14-positive monocytes obtained from human peripheral blood were cultured with GM-CSF and IL-4. During the early culture phase immature dendritic cells (DCs) developed which not only expressed CD1a, HLA-DR and CD86, but also expressed the endothelial cell markers von Willebrand factor (vWF), VE-cadherin and VEGF receptors Flt-1 and Flt-4. Further maturation of DCs was achieved by prolonged cultivation with TNFalpha. These cells showed typical DC morphology and like professional antigen-presenting cells (APCs) expressed CD83 and high levels of HLA-DR and CD86. However, if immature DCs were grown with VEGF, bFGF and IGF-1 on fibronectin/vitronectin-coated culture dishes, a marked change in morphology into caudated or oval cells occurred. In the presence of these angiogenic growth factors the cultured cells developed into endothelial-like cells (ELCs), characterized by increased expression of vWF, KDR and Flt-4 and a disappearance of CD1a and CD83. Addition of IL-4 and Oncostatin M also increased VE-cadherin expression, and the loosely adherent cells formed clusters, cobblestones and network-like structures. vWF- expressing ELCs mainly originated from CD1a-positive cells, and VEGF was responsible for the decrease in the expression of the DC markers CD1a and CD83. In mixed leukocyte cultures, mature DCs were more potent APCs than ELCs. Moreover, Ac-LDL uptake, and the formation of tubular structures on a plasma matrix was restricted to ELCs. These results suggest that in the presence of specific cytokines immature DCs have the potential to differentiate along different lineages, i.e. into a cell type resembling ELCs.

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 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.

Knock-out mice reveal the contributions of P2Y and P2X receptors to nucleotide-induced Ca2+ signaling in macrophages

Immune cell function is modulated by changes in extracellular nucleotide levels. Here we used reverse transcription-PCR analyses, single cell Ca2+ imaging, and knock-out mice to define the receptors mediating nucleotide-induced Ca2+ signaling in resident peritoneal macrophages. In Ca2+-free buffer, the potent (K0.5 <1 μm) stimulatory effect of UTP (or ATP) on endoplasmic reticulum (ER) Ca2+ release was abolished in cells isolated from P2Y2/P2Y4 double knock-out mice. Moreover, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{P}2\mathrm{Y}_{4}^{0{/}-}\) \end{document}, but not \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{P}2\mathrm{Y}_{2}^{-{/}-}\) \end{document}, macrophages responded to UTP. In \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{P}2\mathrm{Y}_{2}^{-{/}-}\) \end{document} macrophages, we could elicit Ca2+ responses to “pure” P2X receptor activation by applying ATP in buffer containing Ca2+. Purified UDP and ADP were ineffective agonists, although modest UDP-induced Ca2+ responses could be elicited in macrophages after “activation” with lipopolysaccharide and interferon-γ. Notably, in Ca2+-free buffer, UTP-induced Ca2+ transients decayed within 1 min, and there was no response to repeated agonist challenge. Measurements of ER [Ca2+] with mag-fluo-4 showed that ER Ca2+ stores were depleted under these conditions. When extracellular Ca2+ was available, ER Ca2+ stores refilled, but Ca2+ increased to only ∼40% of the initial value upon repeated UTP challenge. This apparent receptor desensitization persisted in GRK2+/- and GRK6-/- macrophages and after inhibition of candidate kinases protein kinase C and calmodulin-dependent kinase II. Initial challenge with UTP also reduced Ca2+ mobilization by complement component C5a (and vice versa). In conclusion, homologous receptor desensitization is not the major mechanism that rapidly dampens Ca2+ signaling mediated by P2Y2, the sole Gq-coupled receptor for UTP or ATP in macrophages. UDP responsiveness (P2Y6 receptor expression) increases following macrophage activation.

A splice variant of the two-pore domain potassium channel TREK-1 with only one pore domain reduces the surface expression of full-length TREK-1 channels.

We have identified a novel splice variant of the human and rat two-pore domain potassium (K2P) channel TREK-1. The splice variant TREK-1e results from skipping of exon 5, which causes a frame shift in exon 6. The frame shift produces a novel C-terminal amino acid sequence and a premature termination of translation, which leads to a loss of transmembrane domains M3 and M4 and of the second pore domain. RT-PCR experiments revealed a preferential expression of TREK-1e in kidney, adrenal gland, and amygdala. TREK-1e was nonfunctional when expressed in Xenopus oocytes. However, both the surface expression and the current density of full-length TREK-1 were reduced by co-expression of TREK-1e. Live cell imaging in COS-7 cells transfected with GFP-tagged TREK-1e showed that this splice variant was retained in the endoplasmic reticulum (ER). Attachment of the C-terminus of TREK-1e to two different reporter proteins (Kir2.1 and CD8) led to a strong reduction in the surface expression of these fusion proteins. Progressive truncation of the C-terminus of TREK-1e in these reporter constructs revealed a critical region (amino acids 198 to 205) responsible for the intracellular retention. Mutagenesis experiments indicated that amino acids I204 and W205 are key residues mediating the ER retention of TREK-1e. Our results suggest that the TREK-1e splice variant may interfere with the vesicular traffic of full-length TREK-1 channels from the ER to the plasma membrane. Thus, TREK-1e might modulate the copy number of functional TREK-1 channels at the cell surface, providing a novel mechanism for fine tuning of TREK-1 currents.

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

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 patients 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.—Seebohm, G., Strutz‐Seebohm, N., Ursu, O. N., Preisig‐Müller, R., Zuzarte, M., Hill, E. V., Kienitz, M.‐C., Bendahhou, S., Fauler, M., Tapken, D., Decher, N., Collins, A., Jurkat‐Rott, K., Steinmeyer, K., Lehmann‐Horn, F., Daut, J., Tavaré, J. M., Pott, L., Bloch,W., Lang, F. Altered stress stimulation of inward rectifier potassium channels in Andersen‐Tawil syndrome. FASEB J. 26, 513–522 (2012). www.fasebj.org

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.