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TWIK-related acid sensitive K+ channel 1 (TASK1) and TASK3 critically influence T lymphocyte effector functions

Sven G. Meuth*1, Stefan Bittner*1, Patrick Meuth3, Ole J. Simon1, Thomas Budde2, Heinz Wiendl1

* both authors contributed equally 1 University of Wuerzburg, Department of Neurology, Josef-Schneider-Str. 11, 97080 Wuerzburg, Germany. 2 Westfaelische Wilhelms-University Muenster, Institute for Experimental Epilepsy Research, Huefferstr. 68, 48149 Muenster, Germany. 3 Westfaelische Wilhelms-University Muenster, Institute of Physiology I, Robert-Koch-Str. 27a, 48149 Muenster, Germany.

Running head: TASK on T cells Address correspondence to: Sven G. Meuth (MD, PhD), University of Wuerzburg, Department of Neurology, Josef-Schneider-Str. 11, 97080 Wuerzburg, Germany; Tel: +49 931 201 23453; Fax: +49 931 201 23489; email: meuth_s@klinik.uni-wuerzburg.de

Two major K+ channels are expressed in T cells, (i) the voltage-dependent KV1.3 channel and (ii) the Ca2+-activated K+ channel KCa 3.1 (IKCa channel). Both critically influence T cell effector functions in vitro and animal models in vivo. We here identify and characterize TWIK-related acid-sensitive potassium channel 1 (TASK1) and TASK3 as an important third K+ conductance on T lymphocytes. T lymphocytes constitutively express TASK1 and 3 protein. Application of semi-selective TASK blockers resulted in a significant reduction of cytokine production and cell proliferation. Interference with TASK channels on CD3+ T cells revealed a dose-dependent reduction (∼∼∼∼ 40%) of an outward current in patch-clamp recordings indicative of TASK channels, a finding confirmed by computational modeling. In vivo relevance of our findings was addressed in an experimental model of multiple sclerosis, adoptive transfer experimental autoimmune encephalomyelitis. Pre-treatment of myelin basic protein specific encephalitogenic T lymphocytes with TASK modulators was associated with significant amelioration of the disease course in Lewis rats. These data introduce K2P channels as novel potassium conductance on T lymphocytes critically influencing T effector function and identify a possible molecular target for immunomodulation in T cell-mediated autoimmune disorders.

The last decade has revealed much knowledge about the intracellular events accompanied by T lymphocyte activation following recognition of antigens bound to

major histocompatibility complexes (MHC). K+ selective ion channels in T cells and their role in immune responses have been discussed for decades, since the discovery that non-selective K+ blockers could inhibit T cell proliferation in vitro (1-3). The role of K+ channels in the activation of T cells is pivotal, since opening of the channels hyperpolarize the membrane potential, which in turn increases the influx of Ca2+ via Ca2+ release-activated Ca2+ channels (CRAC channels; (4)). Signaling cascades after T cell receptor stimulation involve phospholipase C-γ (PLC-γ, (5)) mediated cleavage of phosphotidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and 1,4,5-inositol trisphosphate (IP3), which results in a transient elevation of intracellular calcium concentration ([Ca2+] i) triggered by IP3 binding to IP3-receptors (IP3R, (5)).

Intracellular Ca2+ release triggers activation of CRAC channels resulting in longer-lasting (∼ 1 hour) elevated [Ca2+] i mandatory for further transcription dependent steps of T cell activation (4,6). With the combined use of patch-clamp electrophysiology and molecular biology, the voltage-dependent K+ channel 1.3 (KV1.3 channel) and the intermediate-conductance Ca2+-activated channel (IKCa channel; IKCa1 or KCa3.1; which acquires its Ca2+ dependency from constitutively bound calmodulin) were found to be the dominating K+ channel types expressed in T cells (7-10). KV1.3 is activated by membrane depolarization, whereas KCa3.1 channel opening is triggered by a rise in Ca2+ ions (5). Selective blockade of KV1.3 leads to suppression of T cell effector function, e.g. decreased cytokine release and suppression of proliferation (11,12). Underlining this finding, in vivo blockade of

http://www.jbc.org/cgi/doi/10.1074/jbc.M800637200The latest version is at JBC Papers in Press. Published on March 28, 2008 as Manuscript M800637200

Copyright 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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KV1.3 has been shown to mediate beneficial effects in experimental autoimmune encephalomyelitis (EAE), a rodent model for multiple sclerosis (12,13). The importance of K+ channels has been further validated by research on human T lymphocytes, which revealed a differential expression pattern of KV1.3 and KCa3.1 on specific T cell subtypes (14). Interestingly, “chronically activated” T cells induced by repeated antigenic challenges of the cells selectively and stably up-regulate KV1.3 channels (15). Furthermore these cells could be defined as CCR7

-CD45RA- effector memory T lymphocytes (TEM; (16)) which are implicated in different autoimmune disorders (15). In agreement with that KV1.3 has been shown to be highly expressed in human myelin-reactive T cells of patients with multiple sclerosis (17) as well as in autoreactive T cells from patients with other autoimmune diseases such as type-1 diabetes or rheumatoid arthritis (18). Thus K+ channels, especially KV1.3, have emerged as attractive targets for highly selective immunosuppressive strategies applicable e.g. in the therapy of T cell mediated autoimmune disorders (19).

Two-pore-domain K+ channels (K2P, KCNK) – often described as “background” or “leak” channels – play a pivotal role in setting the resting membrane potential and modulating neuronal excitability (20-23). K2P channels consist of four transmembrane domains arranged in tandem building two pores. Importantly, their action is mostly time- and voltage independent (21). TASK1 and TASK3 channels - two functional members of the K2P channel family - can be regulated by a diversity of stimuli (extracellular acidification, Gq proteins, muscarine; (20,22-24), and exhibit insensitivity to “classical” potassium channel blockers (e.g. tetraethylammonium, 4-aminopyridine). Based on their specific electrophysiological properties, pharmacological profile and functional impact on the resting membrane potential they might represent a relevant ionic conductance influencing basic T cell function. We therefore questioned whether K2P channels are expressed on T lymphocytes and dissect their functional role on the level of electrophysiological properties and T cell effector function, both in vitro and in an animal model of MS in vivo.

Experimental Procedures

Materials and reagents Charybdotoxin, bupivacaine, spermine and ruthenium red (diluted in H2O; Sigma, Deisenhofen, Germany), Psora-4 (DMSO; Carl Roth GmbH, Germany) and anandamide (EtOH; Tocris, Germany) were frozen as aliquots for further use. CD3/CD28 dynabeads for cell stimulation were obtained from Dynal Biotech (Karlsruhe, Germany). Annexin-FLUOS (Roche, Mannheim, Germany), propidium iodide (PI, Calbiochem, Darmstadt, Germany), DAPI (Merck, Darmstadt, Germany) and carboxyfluorescein diacetate succinimidyl ester (CFSE, Molecular Probes, Invitrogen, Karlsruhe, Germany) were used for cell labelling. Isolation and culture of human T cells Human T cells were purified from peripheral blood samples of healthy donors (n = 49). Peripheral blood mononuclear cells (PBMCs) were prepared by density centrifugation (Lymphoprep, Axis-Shield, Oslo, Norway) according to manufacturer’s instructions. This was followed by magnetic cell sorting (MACS®, CD3 Microbeads, Miltenyi, Biotec, Karlsruhe, Germany) for CD3. Purity of CD3+ T lymphocytes was >98% as assessed by flow cytometry. Cells were maintained in RPMI 1640 containing 10% human AB-serum, 25 mM HEPES, 1% glutamine and 1% antibiotics. Assessment of T cell function – pharmacological blockades 4*106 freshly isolated CD3+ T lymphocytes were seeded in 1 ml T cell medium per well. CD3/CD28 dynabeads (Dynal Biotech) were added in a T cell to bead ratio of 1:1. Channel blockers in different concentrations were applied in parallel to bead stimulation (KV1.3 blockers: 10 nM and 100 nM charybdotoxin and 1 nM, 10 nM and 100 nM Psora-4; TASK1/3 blocker: 30 µM, 100 µM and 250 µM bupivacaine; TASK1 inhibitor: 3 µM, 30 µM and 100 µM anandamide. TASK3 blockers: 50 µM, 500 µM and 1 mM spermine and 100 nM, 1 µM, 10 µM ruthenium red). The solvent solution in the final experimental solution did not exceed 1‰. Application of the solvent alone (1‰) did not influence the analyzed parameters. Stimulations were all done in duplicates. After 24 h incubation at 37°C and 5% CO2, cells were centrifuged and subjected to further analysis by flow cytometry (stainings for annexin V and

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propidium iodide). In parallel, supernatants were assessed for IFNγ or IL2 protein levels by ELISA (R&D Systems, Wiesbaden, Germany) according to the manufacturer’s instructions. Flow cytometry for TASK channel expression on T-lymphocytes Flow cytometry acquisition was done by standard methods. For antibody staining cells were resuspended in FACS® buffer (PBS containing 0.1% BSA and 0.1% NaN3), directly labelled antibodies were added. For annexin V / propidium iodide (PI) assays, cells were resuspended in annexin binding buffer (10 mM HEPES, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2 and 0.18 mM CaCl2) and stained with annexin-FLUOS (Roche) and PI (Calbiochem). For evaluation of TASK1/3 antibody specificity following antibodies were used: rabbit-anti-TASK1 (No. P0981, Sigma, Deisenhofen, Germany), goat anti-TASK1 (No. SC-32067), rabbit anti-TASK3 (No. AB5721, Chemicon, Ochsenhausen, Germany) and goat anti-TASK3 (No. SC-11320, Santa Cruz, Santa Cruz, CA, USA) followed by the appropriate secondary antibodies (goat anti-rabbit FITC or donkey anti-goat Cy3; Dianova, Hamburg, Germany) using standard protocols for extracellular staining (see above) and intracellular staining (permeabilization buffer and fixation buffer from ebioscience, San Diego, USA). Data acquisition was done using a FACS-Calibur system (BD Bioscience, Heidelberg, Germany). Results were analyzed using CellQuest Pro Software (BD Bioscience). T cell proliferation assay in the presence and absence of TASK channel modulators Human T cells were labeled with 4 µM carboxyfluorescein diacetate succinimidyl ester (CFSE, Molecular Probes) for 10 min in dark. RPMI containing 15% FCS was added for 20 min to stop labeling and was followed by three washing steps with RPMI / 10% FCS. CFSE-labeled cells were suspended in T cell medium and 1*105 cells per well were cultured at 37° and 5% CO2 in the presence of CD3/CD28 beads and ion channel modulators as described above. After three days of proliferation CSFE-labeled cells were washed and resuspended in FACS buffer. Proliferation assays were performed in duplicate and analyzed by flow cytometry calculating the responder frequency (dividing

the number of dividing cells by total number of cells). Immunocytochemistry of human and rat T cells Immunocytochemical stainings were performed on human T cells and rat MBP-specific T-cells. Cells were placed on coverslips coated with poly-L-Lysin (Sigma, Germany) and fixed with 4% paraformaldehyde. Subsequently, cells were blocked with PBS containing 10% horse serum (PAA Laboratories, Cölbe, Germany), 2% BSA and 0.3% Triton X-100 overnight. Next, the primary antibodies (rabbit anti-TASK1, Sigma; rabbit anti-TASK3, Chemicon; rabbit anti-KV1.3, Chemicon) were added and incubated for 1 h. Cells were washed with PBS containing 0.3% Triton X-100 and incubated with secondary antibodies (Cy3-conjugated rabbit anti-goat, 1:100, Dianova) for another hour. Counterstaining of cell nuclei was performed using DAPI (0.5 µg/ml, Merck). Pictures were collected by immunofluorescence microscopy (Axiophot, Zeiss, Jena, Germany). Negative controls without the primary antibody revealed no positive signals (data not shown). Western Blot of T cell lysates Whole cell lysates from isolated purified T cells were used for western blot analysis. In brief, cells were washed with ice-cold PBS, resuspended in lysis buffer (PBS containing 1% Triton X-100 and protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) and solubilized by sonification on ice. Cell lysates were centrifuged and protein content in the clarified supernatant was measured by Bradford reaction. Samples (50 µg / lane for TASK1 and 100 µg / lane for TASK3) were subjected to 10% SDS-PAGE, followed by transfer to nitrocellulose membranes. Whole mouse brain (C57/Bl6) was used as positive control for TASK channel expression (23). Protein transfer was visualized by Ponceau S staining and membranes were then blocked with PBS containing 0.05% Tween 20 and 5% dry milk. The membranes were probed with rabbit anti-TASK1 (raised against the C-terminal part of the channel, 1:200; Sigma) or rabbit anti-TASK3 (polyclonal antibody against the C-terminal part of TASK3, 1:200; Chemicon), respectively. The secondary antibody was horse radish peroxidase (HRP) - conjugated donkey anti-rabbit (1:3,000; Amersham, Freiburg, Germany). The antibody

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reaction was detected by enhanced chemiluminescence reaction (ECL, Amersham). Electrophysiological recording of whole-cell outward currents in T cells All experiments were conducted in whole-cell configuration of the patch-clamp technique. Individual human T lymphocytes were visually identified by infrared differential interference contrast video-microscopy (25). Starting from a holding potential of -80 mV membrane currents were recorded with pipettes pulled from borosilicate glass (GC150TF-10, Clark Electromedical Instruments, Kent, UK), connected to an EPC-10 amplifier (HEKA Elektronic, Lamprecht, Germany), and filled with (in mM): K-gluconate, 95; K3-citrate, 20; NaCl, 10; HEPES, 10; MgCl2, 1; Ca Cl2, 0.5; BAPTA, 1; Mg-ATP, 3; Na-GTP, 0.5. The internal solution was set to a pH of 7.25 with KOH and an osmolarity of 295 mOsm / kg. Extracellular solution contained (in mM): NaCl, 120; KCl, 2.5; NaH2PO4, 1.25; HEPES, 30; MgSO4, 2; CaCl2, 2; dextrose, 10; pH 7.2 was adjusted with NaOH and osmolarity was set to 305 mOsm/kg. Outward currents were elicited by repeated 500 ms pulses from -80 mV to 40 mV, applied at 30 s intervals. Typical electrode resistance was 3-6 MΩ with an access resistance of 6 – 15 MΩ. Series resistance compensation of more than 40% was routinely used. Voltage clamp experiments were governed by Pulse software (HEKA Elektronic) operating on an IBM-compatible PC. A liquid junction potential of 6 ± 2 mV (n = 6) was measured and taken into account according to Neher (26). Adoptive transfer experimental autoimmune encephalomyelitis (AT-EAE) Female Lewis rats (150 - 160 g) were kept at standard conditions with free access to food and water. All animal experiments were approved by local authorities and conducted according to the German law of animal protection. Adoptive transfer experimental allergic encephalomyelitis (AT-EAE) was induced using a specific rat myelin basic protein (MBP) T-cell line (MBP-TC) which was generated as described previously (27). The MBP-TCs used for this experiment were freshly restimulated for 3 days with antigen presenting cells (APCs) and guinea pig (gp) MBP protein (in one petri dish: 3*106

MBP-TCs + 150*106 APCs + gpMBP 10 µg/ml in 10 ml restimulation medium which consists of RPMI 1640 containing 1% glutamine, 1% antibiotics, 1% rat serum and 0.4% β-mercaptoethanol). After 24 and 48h cells were splitted and feeded with RPMI 1640 containing 1% glutamine, 1% antibiotics, 5% FCS and 7.5% supernatants of ConA activated spleen cells. 24 hours before cell transfer, channel blockers (10 nM Psora-4, 30 µM anandamide and 100 nM charybdotoxin) were added directly into the cell cultures. After separation from the APCs by Ficoll gradients, 6*106 MBP-TCs were transferred into four groups of Lewis rats intravenously (each n = 8-9) or plated for another 24 h for evaluation of IFNγ production. The animals were weighed and scored every day by a blinded investigator. Disease severity was assessed following a well established 6-grade scoring system: 0 = healthy; 1 = weight loss, limp tip of tail; 2 = limp tail, mild paresis; 3 = moderate paraparesis, ataxia; 4 = tetraparesis; 5 = moribund; 6 = death (28). Numeric model of T cell outward currents A single compartment T cell model including the currents IKv1.3, ITASK and IC was developed within the NEURON Simulation Environment (29). The compartment’s length and diameter were set to 10 µm each, resulting in a total membrane area of 314 µm². By assuming the following ion distribution [mM]: K+

in = 135, K+

out = 3.5, Ca2+in = 6.5*10-4 (30), Ca2+

out = 0.13 we derived a potassium reversal potential (Ek) of about -94 mV and a calcium reversal potential (Eca) of about 98 mV following the Nernst equation. A specific membrane capacitance of 1 µF/cm² was applied and all simulations were executed at 25°C. The delayed rectifier current IKv1.3 was realized by adapting the membrane mechanism IKD from a Purkinje cell model (31). Shifting the half maximum activation and inactivation to -28.8 mV and -57.8 mV, respectively, led to a current that matches the slowly inactivating current component seen in human T-cells (32,33). The maximum specific conductance gkbar was set to 0.0023 S/cm² in order to reach a maximum current of about 200 pA at a membrane potential of +40 mV. The current’s amplitude Ik was calculated according to the following equation.

Ik = gkbar * m * h * (VM-Ek) m: activation state variable

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h: inactivation state variable VM: membrane voltage

Including the potassium leak current ITASK into the model was accomplished by using the corresponding membrane mechanism as described earlier (34). The described kinetic of that current thereby remained unaffected, whereas the maximum specific conductance was adapted to reach a maximum current of about 200 pA at a membrane potential of +40 mV. The calcium-dependent potassium current IC originates from Arthur Houweling’s NEURON demo. Kinetics of the current were previously published (35) and remained unchanged during all simulations. Setting the maximum specific conductance gkbar to 0.00034 S/cm² resulted in a total of about 115 pA at a membrane potential of +40 mV. The following equation describes this non-inactivating current.

Ik = gkbar * m * (VM - Ek) Ik: current amplitude m: activation state variable VM: membrane voltage

Statistical analysis All results are presented as mean ± SEM. Statistical analysis was performed using the student’s t-test modified for small samples (36). Statistical significance was set at p < 0.05 which is indicated by (**).

RESULTS

TASK1 and TASK3 channels are expressed on human T lymphocytes. In a first set of experiments purified human CD3+ T lymphocytes (n = 6 donors) were assessed by immunocytochemistry for the expression of the K2P channels TASK1 and TASK3. Voltage-gated KV1.3 channel was stained as a positive control (18). Immunoreactivity for TASK1, TASK3 and Kv1.3 was clearly detectable (Fig. 1 A, B, C). Counterstaining of cell nuclei with DAPI indicated a membrane-bound distribution pattern for all channels, while incubation with the secondary antibody alone showed no signals (data not shown). Protein expression of TASK1 and TASK3 by human T cells was corroborated by western blot analysis (n = 6 donors). The band recognized by the anti-TASK1 antibody was approximately 50 kDa (Fig. 1 D, lane 4-6) as published earlier (37). In human lymphocytes

as well as in our positive control (whole mouse brain, lane 3) a second band was visible at 65 kDa (Fig. 1 D, second arrow), not visible in specificity controls (Fig. 1 D, lane 1, 2). Using an anti-TASK3 antibody a distinct band at 60 kDa was visible, both in mouse brain and human T lymphocytes (Fig. 1 E, lane 2-5). To verify antibody specificity, different antibodies for TASK1 and TASK3 were also assessed by flow cytometry. Antibodies from Sigma (TASK1) and Santa Cruz (TASK3) recognizing intracellular epitopes of these channels showed specific binding only in intracellular FACS staining with permeablized cells (Fig. 1 F, right side) and not in extracellular staining protocols (Fig. 1 F, left side). The same specificity was found for an anti-TASK1 antibody (intracellular target) by Santa Cruz, while anti-TASK3 (extracellular; Chemicon) revealed positive signals in intracellular and extracellular staining protocols (data not shown). Taken together, these results point to expression of the K2P channels TASK1 and 3 on human CD3+ T lymphocytes.

TASK1 and TASK3 modulate T cell effector function: pharmacological blockade inhibits IFN γ secretion, IL2 secretion and T cell proliferation. To correlate K2P channel expression to T cell effector functions we next studied the influence of pharmacological blocking reagents for K+ channels on cytokine production and T cell proliferation. Human CD3+ T cells were stimulated with CD3/CD28 beads. Levels of IFNγ and IL2 secreted in the supernatants were assessed in response to the application of different ion channel modulators. To exclude unspecific effects of the compounds on cell viability, each inhibitor was titrated and tested in an annexin V / PI staining to estimate T cell apoptosis and necrosis. Gating procedure, results under control conditions and after treatment with H2O2 (100 µM, positive control for cell death) are depicted in Fig. 2 A. Charybdotoxin (C, 10 nM, 100 nM) and Psora-4 (P, 1 nM, 10 nM, 100 nM), both inhibitors of KV1.3 channels, the TASK channel inhibitor bupivacaine (B, 30 µM, 100 µM, 250 µM) and anandamide (A, 3 µM, 30 µM, 100 µM) as well as the TASK3 modulating compounds spermine (S, 50 µM, 500 µM, 1 mM) and ruthenium red (R, 100 nM, 1 µM, 10 µM) had no significant direct effect on cell survival (Fig. 2 B, C, D; upper panel) as

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compared to control conditions (con). Charybdotoxin and Psora-4, both modulators of Kv1.3 were used as positive controls (38). Application of 100 nM charybdotoxin significantly reduced IFNγ production to 56 ± 3% (n=5, p = 0.0002; 10nM: no effect, n=5, p = 0.76; Fig. 2 B; C1, C2). KV1.3 channel modulation by Psora-4 significantly lowered IFNγ amounts in the supernatant at all used concentrations (Fig. 2 B, Tab. 1). TASK channel modulation using bupivacaine (23) significantly decreased IFNγ levels at concentration ≥ 100 µM (Fig. 2 C, Tab. 1). TASK channel inhibition by the endogenous cannabinoide anandamide significantly reduced IFNγ secretion in a dose dependent manner (e.g. 30 µM: 68 ± 8%, n=5, p = 0.02; Fig. 2 C, Tab. 1). Similarly, the TASK3 channel modulator spermine suppressed IFNγ secrection at concentrations ≥500 µM (Fig. 2 D, Tab. 1). Ruthenium red, a second inhibitor of TASK3, similarly suppressed IFNγ production by human T cells (e.g. 100 nM: 47 ± 6%, n=5, p = 0.0007; Fig. 2 D, Tab 1). Analysis for IL2, a second important cytokine for T lymphocyte effector functions, revealed similar results for KV1.3 and TASK blockade (C1: 56 ± 7%, n=5, p = 0.03; P2: 74 ± 3%, n=5, p = 0.05; A2: 45 ± 5%, n=5, p = 0.001; R2: 53 ± 7%, n=5, p = 0.02; Fig. 2 E). In addition we assessed the effect of TASK modulation on T cell proliferation. CFSE dilution was measured after three days of CD3/CD28 stimulation (positive control, normalized to 100%) and compared to control values (Fig. 3 A, Tab. 2). Unstimulated T cells displayed a responder frequency of 6 ± 1% (Tab. 2). In accordance with the literature, KV1.3 modulation (charybdotoxin, Psora-4) significantly reduced responder frequency after bead stimulation (n=5 each; Fig. 3 B, Tab. 2). TASK channel modulation by bupivacaine (n=5; Tab. 2), anandamide (n=5; Fig. 3 C, Tab. 2), spermine (TASK3, n=5; Tab. 2) and ruthenium red (TASK3, n=5; Fig. 3D, Tab. 2) significantly suppressed T cell proliferation (30-70%; Tab. 2).

Electrophysiological recordings reveal the contribution of TASK to the potassium outward current in human T cells. In a next set of experiments whole-cell patch-clamp recordings of purified CD3+ human T lymphocytes were used to evaluate the

contribution of TASK channels to the potassium outward current of T cells (n=31 donors; multiple single cell recordings of each donor). Stepping the membrane potential from -80 mV to +40 mV (Fig. 4 A, left panel, inset) under voltage-clamp conditions evoked an outward current of 424 pA (range: 213 – 944 pA). Application of the voltage protocol at 30 s intervals indicated a stable outward current over time and current run-down could by analyzed as 6 ± 2% (Fig. 4 A, left panel; C, left and middle panel; n = 6). Calculation of the underlying time constant (τ) under control conditions revealed a single exponential decay with 195 ± 15 ms (Fig. 4 A, left panel; C, right panel and inset; n = 6). However, addition of the well established KV1.3 channel inhibitor Psora-4 (100 nM) immediately reduced the outward current by 54.8 ± 8.8% (Fig. 4 A, right panel, grey trace; n = 4, p = 0.003) although the time constant was not significantly altered (τ: 217 ± 17.3 ms). In contrast, applying the TASK specific modulator anandamide resulted in a rapid reduction of the K+ outward current (30 µM: 42 ± 10%, n = 4, p = 0.01; 100 µM: 44 ± 8%, n = 4, p = 0.0017; Fig. 4 B, C, left and middle panel) and an accelerated time constant (30 µM: τ = 80 ± 17 ms; n = 4, p = 0.0002; 100 µM: τ = 69 ± 10 ms; n = 4, p = 0.00001; Fig. 4 C, right panel). These results for the first time demonstrate a contribution of currents through K2P channels to the outward currents in human T cells. Next, blocker sensitive currents were analyzed after graphical subtraction of currents in the presence and absence of channel modulators. Expectedly, the Psora-4 sensitive current component (the current after application of Psora-4 was subtracted from the control current, Fig. 4 A, right panel, black trace minus grey trace) revealed a rapid current onset upon stimulation and a slow inactivation kinetic indicative of the delayed rectifying current KV1.3 (Fig. 4 D, left panel). However, the anandamide sensitive current component (the current after application of anandamide was subtracted from the control current, Fig. 4 B, left panel, black trace minus grey trace) displayed a fast current onset with nearly no inactivation over the stimulation protocol, a typical feature of voltage-independent TASK channels (Fig. 4 D, right panel). These findings were further corroborated by the electrophysiological fingerprint of

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charybdotoxin (50 nM) - a second blocker of KV1.3 (and KCa3.1) - on human T lymphocytes (64.5 ± 6.4% reduction, n = 4, p = 0.0002; Fig. 5 A, inset and right panel). The charybdotoxin-resistant currents (Fig. 5 A, left panel) strongly resemble the properties of pure TASK currents which provides further support for the coexistence of voltage-dependent and leak potassium currents on T cells. TASK3 modulation by ruthenium red (1 and 10 µM) induced a significant current reduction of 40.9 ± 8.5% and 52.6 ± 4%, respectively (n = 4, p = 0.04/0.001; Fig. 5 B). Taken together this data therefore clearly indicate the functional relevance of TASK channels on outward currents in human T lymphocytes and demonstrate the functional coexpression of KV1.3 and TASK channels on these cells.

A computational model to dissect the contribution of three K channels (KV1.3, KCa, TASK) for K+ outward currents. In the next experimental step we used a numeric model of a human T cells to analyze the current components contributing to the resting membrane potential in human T cells. The model included the voltage-dependent KV1.3 (IKV1.3) channel (Fig. 6 A) whereas inactivation and activation parameters were calculated according to the literature (31,39). Based on these settings the currents evoked by a depolarizing voltage protocol (500 ms, from -50 mV to +50 mV, decrement 10 mV; Fig. 6 A, inset) showed slow inactivation kinetics and characteristics as described for the net outward current of human T cells (16,33). Next, the model was extended by the inclusion of a TASK current (ITASK) as described earlier (34). TASK current alone resulted in a nearly voltage-independent model response to depolarizing voltage steps (Fig. 6 B). Finally, we supplemented the model by including a Ca2+-dependent K+ current (IC, Fig. 6 C; (35)). Based on our pharmacological results we assumed the contribution of all three conductances to the net outward current in human T cells and calculated 40% IKv1.3, 40% ITASK and 20% IC. The composed current evoked by a depolarizing voltage step to +40 mV displayed typical features (e.g. slow inactivation) as recorded for human T lymphocytes (Fig. 6 D, left panel). Mimicking Psora-4 actions (inhibitor of KV1.3 channels as demonstrated by elctrophysiological recordings) in the model we reduced the KV1.3

component to 0% resulting in a nearly voltage independent current response evoked by a depolarizing voltage step (Fig. 4 A, right panel, gray trace; Fig. 6 D, middle panel). This finding further corroborates our electrophysiological data above, suggesting a marked contribution of TASK channels to the outward current of human T cells. In a last step we modeled anandamide actions (TASK channel inhibitor as demonstrated by electrophysiological recordings) by reducing ITASK to 0%. The remaining current component represents Kv1.3 channels (Fig. 4 B, left panel, gray trace; Fig. 6 D, right panel). Recapitulating these findings from a numerical cell model support the results from electrophysiological recordings showing the functional coexpression of TASK and KV1.3 channels in human T lymphocytes.

Selective blockade of T lymphocyte TASK channels ameliorates experimental autoimmune encephalomyelitis, a model of multiple sclerosis. Guided by our data on the functional impact of TASK on T cell activation in vitro and our patch clamp results, we challenged the question whether selective modulation of T lymphocyte TASK channels modulates T cell mediated inflammatory disorders in vivo. We therefore chose adoptive transfer experimental autoimmune encephalomyelitis (AT-EAE), a well established animal model of MS in Lewis rats. In order to evaluate the effect of TASK modulation on T cells, we incubated encephalitogenic MBP-specific T lymphocytes with the K+ channel modulators (10 nM Psora-4, 100 nM charybdotoxin and 30 µM anandamide) prior to adoptive transfer in rats. Expression of KV1.3 as well as TASK1 could be demonstrated on MBP-specific rat T lymphocytes by immunocytochemistry, similar to our results observed in human T lymphocytes (Fig. 7 A). Intravenous transfer of restimulated MBP-specific (6*106 cells/animal) T cells elicited a typical EAE with symptom onset at day 2-3, disease maximum around day 5 and recovery from day 6-10 (onset: 3.3 ± 0.3 days, n = 8; disease maximum: score 4.7 ± 0.5, n = 8; decline to baseline: 10.4 ± 0.5 days, n = 8; Fig. 7 C-E). Incubation of encephalitogenic T cells with the Kv1.3 blocker Psora-4 24 h prior to transfer led to a slightly delayed onset of disease (not significant, ns, p = 0.09), amelioration of the

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disease maximum (score 3.0 ± 0.6, n = 8, p = 0.03) and faster recovery of symptoms (p = 0.03; Fig. 7 C). Different results were seen when using charybdotoxin which showed no significant effect on the investigated parameters (onset: p = 0.25; disease maximum: score 4.8 ± 0.8, p = 0.74, n = 8; decline to baseline: p = 0.57; Fig. 7 D). Selective blockade of TASK channels with anandamide ameliorated AT-EAE. While disease onset was delayed in the presence of anandamide (ns, p = 0.39), maximum disease score was reduced signficantly (2.8 ± 0.3, n = 9, p = 0.02). Furthermore, animals recovered earlier (p = 0.03; Fig. 7 E). In agreement with these results from EAE, MBP-specific T cells showed a significantly reduced IFNγ production in vitro when pretreated with Psora-4 (0.71 ± 0.08, p = 0.02, n = 5) and anandamide (0.66 ± 0.09, p = 0.01, n = 5) while charybdotoxin failed to have lasting effect on T cells (0.84 ± 0.15, p = 0.32, n = 5). This could be due to different binding affinities of the used substances (Fig. 7 B). Of note, the application of channel modulators in vivo was not associated with obvious side effects. Taken together, results obtained from the animal model demonstrate the pathophysiological relevance of TASK channel modulation in a T cell mediated disorder.

DISCUSSION

Potassium-selective ion channels play a key role in modulating effector functions of T lymphocytes. Based on convincing data in vitro and in animal models of autoimmune disorders in vivo, their selective pharmacological intervention has been proposed as a potential therapeutic strategy for T cell mediated autoimmune disorders (12,16). Thus far, research reports have focused on 2 major K+ channels expressed in T cells, (i) the voltage-dependent KV1.3 channel and (ii) the Ca2+-activated K channel KCa3.1. Pharmacological interventions of both types inhibit T effector functions in vitro and ameliorate disease in animal models of T cell-mediated autoimmunity in vivo (17). TASK channel expression on human T lymphocytes: implications for T cell effector functions

Our report identifies and characterizes the K2P channels TASK1 and 3 as a third K+ conductance relevant for T lymphocyte activation and function. TASK channel expression was demonstrated by immunocytochemical stainings as well as western blotting techniques. Functionally TASK channels contribute to the membrane resting potential of T cells and critically affect T cell receptor mediated effector functions in vitro. This was evidenced by experiments measuring cytokine secretion and cell proliferation in the presence of two pharmacological TASK inhibitors and whole cell patch clamp recordings of human CD3+ T cells. Computational modeling corroborated and extended electrophysiological dissection of the role of TASK channels among three relevant K+ conductances on T cells (Kv1.3, KCa3.1, TASK) and support a hypothetical model, how TASK channels contribute to Ca2+ entry and stabilization of the membrane potential. Finally, pathophysiological relevance of our findings was demonstrated by testing T cell selective pharmacological blockade of TASK channels in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis: TASK inhibition was associated with slightly delayed disease onset, significant amelioration of disease severity and significant earlier recovery after adoptive transfer of pretreated encephalitogenic cells.

Role of potassium currents in Ca2+ dependent T cell activation. Interaction of the T-cell receptor-CD3 complex with the antigen-loaded MHC molecules initiates intracellular signaling via the phospholipase C-γ (PLC γ) pathway, resulting in initial release of Ca2+ from intracellular stores, increased intracellular Ca2+, activation of protein kinase C (PKC), and augmented expression of Kv1.3 and IK channels in the cell membrane. Ca2+ re-enters the cell through CRAC channels, causing membrane depolarization and further raising intracellular Ca2+ levels. The resulting K+ efflux hyperpolarizes the membrane and maximizes the driving force for continued Ca2+ influx via CRAC channels. T cell response to antigenic stimulation is highly modulated by the shape and nature of this calcium signal indicating the importance of differences in this signaling pattern (16).

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The ionic conductances thus far described to be responsible for the calcium signaling in T lymphocytes are the voltage-gated potassium channel (KV1.3), the calcium-activated potassium channel (IKCa1) and the calcium release-activated calcium channel (CRAC) (3,7,11,14,40,41). Interestingly, a broad spectrum of antigenic and mitogenic stimuli of T cells up-regulate expression of IK channels and also Kv1.3 channels in vitro (8,42). The increased expression of the K+ channels significantly augments the hyperpolarizing capacity of the activated T cells (compared with naïve cells), accelerates the Ca2+ influx, and secures a sustained high level of intracellular Ca2+, which is necessary for full-blown T cell proliferation and cytokine production. Accordingly, in the presence of K+ blockers, the capability of the T cells to maintain a negative membrane potential and a long-lasting Ca2+ signal is reduced, which is the rationale for their putative efficacy in attenuating T cell mediated immune responses.

Role of K2P channels in T cell physiology: electrophysiology and computational modeling. What might be the role of our newly describe K2P channels in T cells in this complex interplay? K2P represent a unique family of potassium channels and are mainly responsible for the setting of the membrane potential in a number of different cell types (20,34,43-46). Given the insensitivity of TASK channels against classical potassium channel blockers, these channels show a response against a unique panel of inhibitors including bupivacaine (23,47-51), anandamide (52,53), spermine (54) and ruthenium red (54,55). Pharmacological interference with TASK channels attenuates T cell proliferation and cytokine secretion, similar to the interference with Kv1.3 using well established channel blocking compounds such as Psora-4 (38) and charybdotoxin (56). Notably, the inhibitory effect on T cell activity was comparable between the KV1.3 silencing substances Psora-4 and charybdotoxin (note here that charybdotoxin also acts on IKCa1 channels) and the TASK channel inhibitors strongly suggesting an important functional impact of the leak channels on T-cell function.

Contribution of TASK currents to the net outward current of human T lymphocytes was assessed using whole-cell patch-clamp recordings. A well established protocol stepping

the membrane potential from -80 mV to +40 mV over 500 ms every 30 seconds elicited a marked outward current with a rapid onset and a slow inactivation (single exponential decay, τ: ∼195 ms) as described earlier (16). Expectedly, application of KV1.3 channel modulators resulted in a marked current reduction while current kinetics remained unchanged (16). The endocannabinoid anandamide however, a well known inhibitor of TASK channels (52,53), induced a comparable inhibition of the outward current, but in contrast to KV1.3 blockers anandamide significantly altered current kinetics. Blocker sensitive currents obtained by graphical subtraction of currents after application of the inhibitor from currents under control conditions showed nearly no voltage-dependency in the anandamide sensitive component, therefore suggesting the contribution of the TASK leak current (23,34,53). These findings were corroborated by results from a single-compartment computational model including the currents IKv1.3, ITASK and IC. Furthermore, functional expression of K2P channels could be shown on isolated human CD3+ T lymphocytes and all of the recorded CD3+ T cells showed currents indicative of TASK channels. It is currently unknown how (or if) expression of these channels is regulated (e.g. under inflammatory conditions). Furthermore the investigation of expression levels in different T cell subtypes (naïve, Th17 encephalitogenic Tcells, regulatory T cells, CD4+, CD8+, T central memory cells (Tcm), T effector memory cells (Tem)) is warranted to determine whether TASK channels correlate to specific T cell functions (similar to KV1.3 channels). However, based on the abundant expression of K2P channel in a number of different cell types (20,34,43-46) it might be speculated that these channels are expressed throughout different T cell subtypes and that influence of TASK on K+ conductances represents a “baseline” feature common to all T cells. Our findings imply the possibility that earlier results concerning the net outward current in human T lymphocytes could be contaminated by a TASK channel component, not known or extrapolated at that time. However, the exact role of each component contributing to the outward current in T cell can only be estimated: based on our pharmacological and modelling results we suggest a nearly equal contribution of

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currents through TASK channels (~40%) and KV1.3 channels (~40%) to the outward current in human CD3+ T cells while other potassium conductances (e.g. IKCa1) contribute to the remaining ~20%. Taken together, our data support the following hypothetical model for K2P channels in the cascade of TCR-mediated T cell activation, as summarized in a cartoon indicated in Fig. 8.

Selective blockade of T lymphocyte TASK channels ameliorates experimental autoimmune encephalomyelitis, a model of multiple sclerosis. In order to prove the pathophysiological relevance of T lymphocyte TASK channels we investigated its modulation in experimental autoimmune encephalomyelitis (27). Selective blockade of T lymphocyte K+ channels was achieved by pre-incubation of encephalitogenic MBP-lines prior to transfer with various blockers. This approach was used in order to dissect the relevance on T cells, which would have been contaminated when applying the channel blockers systemically. In accordance with the literature, Kv1.3 blockade by Psora-4 ameliorated EAE: animals showed a delayed disease onset (non significant), less severe disease course (significant) and a tendency towards faster recovery (Fig. 7). Importantly, TASK channel modulation via anandamide resulted in very comparable results indicating that TASK mediated effects on T lymphocytes are of pathophysiological relevance in a model of T cell mediated autoimmunity. It is

therefore tempting to speculate that attenuation of T cell function by selective pharmacological interference with TASK channels may translate into clinical benefits in T cell mediated (auto)immune disorders. However, based on the broad expression pattern of TASK channels pharmacological treatment of EAE animals can only partially be linked to TASK channels expressed on immune cells because the effects might be contaminated through effects of TASK channels expressed e.g. on neurons (13,23). As a note of caution, long term effects of ion channel blockers inhibiting TASK channels are not known. Therefore further work, including the detailed characterization of TASK1- and TASK3-knock out mice is clearly warranted.

In summary our study introduces TASK channels on T lymphocytes as critical components for the maintenance of the resting membrane potential and T cell function. Accordingly, channel modulation results in a marked reduction of the outward current in human T cells accompanied by significantly altered effector functions. T cell selective pharmacological blockade in an animal model of human MS serves as a proof of concept concerning the (patho)physiological relevance of these channels. Further studies are warranted to delineate the potential of TASK channels as a target und TASK channel blockers as potential drug candidates in T cell mediated autoimmune diseases such as multiple sclerosis.

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FOOTNOTES Thanks are due to Ms. Astrid Schmitt and Mrs. Sabrina Braunschweig for excellent technical assistance. This work was supported by IZKF N39-1 to S.G.M. and H.W. This work was done in partitial fulfilment of the doctoral thesis of SB.

FIGURE LEGENDS

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Fig. 1. Expression of the K+ channels KV1.3, TASK1 and TASK3 on human T lymphocytes.

(A) Immunocytochemical staining of human CD3+ T lymphocytes with DAPI (left panel), anti-KV1.3 (middle panel) and overlay (right panel). (B, C) Antibodies raised against TASK1 (B, middle panel) and TASK3 (C, middle panel) and corresponding DAPI counterstainings (B, C, left panels) indicate a channel expression on human T lymphocytes (B, C, right panels). Scale bars represents 50 µm (upper left) and counts for all images (enlarged details scale bar: 10 µm). (D) Western blot analysis using a TASK1 specific antibody revealed two distinct signals at 50 and 65 kDa (→).Left lane indicates marker bands. 1 - negative control. 2 –albumin blotting (no band). 3 – Whole mouse brain. 4, 5, 6 - Lysates of CD3-positive isolated T cells. (E) Western blot of TASK3 protein indicates a clear signal at 60 kDa (→) using CD3-positive isolated T cells of healthy humans (lanes 3-5). Marker bands are indicated on the left lane. 1 - negative control. 2 – Whole mouse brain. 3, 4, 5 – Three lysates of CD3-positive isolated T cells from 3 independent healthy donors. (F) Flow cytometry for TASK1 (upper panel) and TASK3 (lower panel). Fluorescence intensity is plotted against event counts. Dotted lines: unstained controls; thin black lines: staining with secondary antibodies alone; bold black lines: staining with TASK1/3 antibodies. Arrows (↓) indicate overlapping signals for thin and bold black lines. One out of three representative experiment is shown.

Fig. 2. Production of interferon γγγγ and IL2 by human T lymphocytes is reduced in the presence of ion channel modulators against the K+ channels KV1.3, TASK1 and TASK3. (A) Cell viability assessment in the presence and absence of K+ channel modulators was performed by flow cytometry. Gating procedures (side and forward scatter), results under control conditions and after treatment with H2O2 (100 µM) are depicted. (B) Upper panel: superimposed images exemplify flow cytometry analysis for apoptosis and necrosis under control conditions (con) and in the presence of the different channel modulators charybdotoxin (100 nM, C) and Psora-4 (100 nM, P). Lower panel: Interferon γ production of isolated human T lymphocytes after stimulation with CD3/CD28 beads under control conditions (positive control; black column, +) and in the presence of the KV1.3 channel inhibitors charybdotoxin (C1 - 10 nM; C2 - 100 nM) and Psora-4 (P1 - 1 nM; P2 - 10 nM; P3 - 100 nM). (C) IFNγ production after application of bupivacaine (B1 - 30 µM; B2 - 100 µM; B3 - 250 µM) and anandamide (A1 - 3 µM; A2 - 30 µM; A3 - 100 µM). (D) TASK3 specific inhibition by spermine (S1 - 50 µM, S2 - 500 µM, S3 - 1 mM) and ruthenium red (R1 - 100 nM, R2 - 1 µM, R3 - 10 µM). (E) Blockade of KV1.3 and TASK channels using selected concentrations leads to a similar effect on interleukin-2 (IL-2) production (abbreviations see B-D).

Fig. 3. Modulation of TASK inhibits proliferation of human T lymphocytes. Human CD3 T lymphocytes were labelled with CFSE and cultured in the presence of CD3/CD28 beads (A) and in the presence of various K+ channel modulators (B-D). Application of the potassium channel modulators Psora-4 (P, 10 nM, n = 5), anandamide (A, 30 µM, n = 5) and ruthenium red (R, 100 nM, n = 5) indicated a marked reduction of T cell proliferation. Expemplary histograms are shown. See corresponding Table 2.

Fig. 4. Whole-cell patch-clamp recordings of potassium outward currents in human T cells reveal a pharmacological profile indicative of KV1.3 and TASK channels. (A) Voltage step protocols (see inset) from -80 mV to +40 mV (500 ms, repeated every 30 sec) were used to evoke stable potassium outward currents on human T lymphocytes (left panel). Application of Psora-4 (100 nM, inhibitor of KV1.3 channels) induced a clear current reduction (right panel). (B) The endogenous cannabinoid anandamide (30 µM and 100 µM) significantly reduced the current. (C) Bar graph representation of run down effects under control conditions (con) compared to TASK-specific inhibition by anandamide (30 µM, left panel). Amplitude of the net outward current plotted against time under control conditions (black squares, middle panel). Period of anandamide treatment (ana, grey circles) is indicated by a horizontal line (middle panel). Bar graph representation of time constants under control conditions and after application of anandamide (right panel) as indicated by

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mathematical single-exponential decay fits (right panel, inset). (D) Blocker sensitive current components obtained by subtraction of currents after application of an inhibitor from control values. ** represents p < 0.05

Fig. 5. Effects of charybdotoxin and ruthenium red on the potassium outward currents in human T cells. (A) Effects of charybdotoxin (50 nM) on the net outward current are shown in the left panel. Mean bar graph representation (inset) and amplitude versus time plot of charybdotoxin (right panel). (B) Ruthenium red (RR) applied on T cell outward current induced a significant current reduction at 1 and 10 µM (left panel). Amplitude of the net outward current plotted against time in the presence of 1 µM ruthenium red as indicated by a horizontal line (middle panel). Bar graph representation of ruthenium red effects on the net outward current of human T lymphocytes compared to control values (right panel).

Fig. 6. Computational modeling of K+ channel contributions to the net outward current in human T cells. A single compartment cell model calculating the effects of KV1.3, TASK1 and 3 channels. We modeled a T cell outward current consisting of IKv1.3 (A), ITASK (B) and IC (C). (A-C) Left column indicates the currents evoked by depolarizing voltage steps (inset) if only a single current is included in the model. Right panel demonstrates the activation and inactivation curve of KV1.3 (A, right column), the current-voltage relationship of TASK channels (B, right column) and the activation curve of IC (C, right column). Analysis of the net outward current (40% IKv1.3, 40% ITASK, 20% IC) demonstrated characteristic features comparable to the recorded outward current of human T lymphocytes (D, left panel). Different K+ channel contributions to the net outward current were modeled and revealed results comparable to the elctrophysiological recordings under Psora-4 (D, middle panel) and anandamide (D, right panel).

Fig. 7. T cell selective modulation of Kv1.3 and K2P attenuates disease in an animal model of multiple sclerosis. (A) Expression of KV1.3 (upper row) and TASK1 (lower row) protein in rat MBP-specific T lymphocytes. Left column shows DAPI stainings, right columns the overlay. Representative stainings are shown, scale bar represents 10 µm. (B) Activated MBP cells pretreated with channel modulators were incubated another 24 hours and supernatants were assessed for IFNγ protein levels (un: control cells, C: charybdotoxin 100 nM, P: Psora-4 10 nM, A: anandamide 30 µM). (C-E) Disease score after transfer of unmanipulated MBP-specific encephalitogenic T cells into Lewis rats is blotted as a solid line (solid line). Mean score of all animals including standard errors are shown (n=8-9 each experiment). Pretreatment of encephalitogenic T cells MBP-TC with Psora-4 (C), charybdotoxin (D) and anandamide (E) to MBP-specific T cells resulted in an altered disease course.

Fig. 8. Hypothetical model for K2P channels in the cascade of TCR-mediated T cell activation. T cell receptor (TCR) activation is accompanied by an increased intracellular ([Ca2+

i]) Ca2+ concentration. This up-regulation of Ca2+ is transferred to a sustained intracellular Ca2+ elevation by activation of Ca2+ release-activated Ca2+ channels (CRAC), resulting in a membrane depolarization. Furthermore the increased Ca2+ concentration leads to long lasting changes in transcription processes and T cell proliferation. To maintain the driving force for Ca2+ entry through the plasma membrane the Ca2+-induced depolarization is counterbalanced by potassium channels. Our data suggests that ∼40% of the K+ outward current in human CD3+ T lymphocytes is mediated by KV1.3, ∼20% by IKCa1 and ∼40% by K2P channels, a third and novel K+ conductance in human T cells. Based on their pharmacological profile (e.g. current inhibition by O2 depletion or extracellular acidification) we hypothesize that TASK channels influence T cell actions under (patho-) physiological conditions (adapted from (42)).

TABLES

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substance abbreviation concentration Result n p-value Psora-4 P1 1 nM 64 ± 8 % 5 0.01 P2 10 nM 61 ± 8 % 5 0.007 P3 100 nM 54 ± 12 % 5 0.02 Bupivacaine B1 30 µM 96 ± 4 % 5 0.44 B2 100 µM 74 ± 6 % 5 0.01 B3 250 µM 59 ± 12 % 5 0.00001 Anandamide A1 3 µM 81 ± 4 % 5 0.01 A2 30 µM 68 ± 8 % 5 0.02 A3 100 µM 26 ± 2 % 5 0.00001 Spermine S1 50 µM 82 ± 8 % 5 0.1 S2 500 µM 62 ± 4 % 5 0.001 S3 1 mM 55 ± 5 % 5 0.0007 Ruthenium red

R1 100 nM 47 ± 6 % 5 0.0007

R2 1 µM 40 ± 8 % 5 0.002 R3 10 µM 33 ± 6 % 5 0.0005 Tab. 1 IFNγ production in the absence and presence of K+ channel modulators.

responder frequency (%)

standard error

p-value

unstimulated 6 1 0.00001 CD3/CD28 stimulated 100 0 0 Charybdotoxin, 100 nM 61 6 0.002 Psora-4, 10 nM 27 7 0.004 Bupivacaine, 250 µM 52 8 0.004 Anandamide, 30 µM 70 9 0.02 Spermine, 500 µM 67 12 0.046 Spermine, 1 mM 60 11 0.02 Ruthenium red, 1 nM 98 4 0.46 Ruthenium red,10 nM 76 4 0.003 Ruthenium red, 0.1 µM 28 8 0.0007 Tab.2 The table summarizes all investigated compounds and concentrations of the inhibitors used in this study concerning human T-cell proliferation.

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Figure 1

A

F

B

DAPI KV1.3 overlay

TASK1

TASK1

overlayDAPI

C

DAPI

TASK1

TASK3

TASK3overlayDAPI

overlay

D

E

TASK1

E

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Figure 2

A

PI

con H2O2

SS

C PI

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Annexin

B pos C P

Annexin

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Annexin

PI

0.40

0.60

0.80

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0.40

0.60

0.80

1.00

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+ C1 C2 P1 P2 P3

D pos S RPI

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+ B1 B2 B3 A1 A2 A3

E

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0.60

0.80

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1.00

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+ S1 S2 S3 R1 R2 R3

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Figure 3

A Bpos P

C D RA

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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Figure 8

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WiendlSven G. Meuth, Stefan Bittner, Patrick Meuth, Ole J. Simon, Thomas Budde and Heinz

influence T lymphocyte effector functionsTWIK-related acid sensitive K+ channel 1 (TASK1) and TASK3 critically

published online March 28, 2008J. Biol. Chem.

10.1074/jbc.M800637200Access the most updated version of this article at doi:

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