Silent but not dumb: how cellular trafficking and pore gating modulate expression of TWIK1 and THIK2.

Among K2P channels, a few of them turned out to be difficult to express in heterologous systems and were coined "silent subunits". Recent studies have shed light on the mechanisms behind this apparent lack of channel activity at the plasma membrane. For TWIK1 and THIK2 channels, silence is related to a combination of intracellular retention and low intrinsic activity. TWIK1 is constitutively endocytosed from the plasma membrane before being transported to recycling endosomes, whereas THIK2 is restricted to endoplasmic reticulum. These intracellular localizations are related to trafficking signals located in the cytoplasmic parts of the channels. When these motifs are mutated or masked, channels are redistributed at the plasma membrane and produce measurable currents. However, these currents are of modest amplitude. This weak basal activity is due to a hydrophobic barrier in the deep pore that limits water and ions in the conduction pathway. Other silent channels KCNK7, TWIK2, and TASK5 are still under study. Expression and characterization of these K2P channels pave the way for a better understanding of the mechanisms controlling intracellular trafficking of membrane proteins, ion conduction, and channel gating.

The two-pore domain potassium channel KCNK5 deteriorates outcome in ischemic neurodegeneration.

Potassium channels can fulfill both beneficial and detrimental roles in neuronal damage during ischemic stroke. Earlier studies have characterized a neuroprotective role of the two-pore domain potassium channels KCNK2 (TREK1) and KCNK3 (TASK1). Protective neuronal hyperpolarization and prevention of intracellular Ca(2+) overload and glutamate excitotoxicity were suggested to be the underlying mechanisms. We here identify an unexpected role for the related KCNK5 channel in a mouse model of transient middle cerebral artery occlusion (tMCAO). KCNK5 is strongly upregulated on neurons upon cerebral ischemia, where it is most likely involved in the induction of neuronal apoptosis. Hypoxic conditions elevated neuronal expression levels of KCNK5 in acute brain slices and primary isolated neuronal cell cultures. In agreement, KCNK5 knockout mice had significantly reduced infarct volumes and improved neurologic function 24 h after 60 min of tMCAO and this protective effect was preserved at later stages of infarct development. KCNK5 deficiency resulted in a significantly reduced number of apoptotic neurons, a downregulation of pro-apoptotic and upregulation of anti-apoptotic factors. Results of adoptive transfer experiments of wild-type and Kcnk5 (-/-) immune cells into Rag1 (-/-) mice prior to tMCAO exclude a major role of KCNK5 in poststroke inflammatory reactions. In summary, KCNK5 expression is induced on neurons under ischemic conditions where it most likely exerts pro-apoptotic effects. Hence, pharmacological blockade of KCNK5 might have therapeutic potential in preventing ischemic neurodegeneration.

Murine K2P5.1 Deficiency Has No Impact on Autoimmune Neuroinflammation due to Compensatory K2P3.1- and KV1.3-Dependent Mechanisms.

Lymphocytes express potassium channels that regulate physiological cell functions, such as activation, proliferation and migration. Expression levels of K2P5.1 (TASK2; KCNK5) channels belonging to the family of two-pore domain potassium channels have previously been correlated to the activity of autoreactive T lymphocytes in patients with multiple sclerosis and rheumatoid arthritis. In humans, K2P5.1 channels are upregulated upon T cell stimulation and influence T cell effector functions. However, a further clinical translation of targeting K2P5.1 is currently hampered by a lack of highly selective inhibitors, making it necessary to evaluate the impact of KCNK5 in established preclinical animal disease models. We here demonstrate that K2P5.1 knockout (K2P5.1-/-) mice display no significant alterations concerning T cell cytokine production, proliferation rates, surface marker molecules or signaling pathways. In an experimental model of autoimmune neuroinflammation, K2P5.1-/- mice show a comparable disease course to wild-type animals and no major changes in the peripheral immune system or CNS compartment. A compensatory upregulation of the potassium channels K2P3.1 and KV1.3 seems to counterbalance the deletion of K2P5.1. As an alternative model mimicking autoimmune neuroinflammation, experimental autoimmune encephalomyelitis in the common marmoset has been proposed, especially for testing the efficacy of new potential drugs. Initial experiments show that K2P5.1 is functionally expressed on marmoset T lymphocytes, opening up the possibility for assessing future K2P5.1-targeting drugs.

Identification of two-pore domain potassium channels as potent modulators of osmotic volume regulation in human T lymphocytes.

Many functions of T lymphocytes are closely related to cell volume homeostasis and regulation, which utilize a complex network of membrane channels for anions and cations. Among the various potassium channels, the voltage-gated K(V)1.3 is well known to contribute greatly to the osmoregulation and particularly to the potassium release during the regulatory volume decrease (RVD) of T cells faced with hypotonic environment. Here we address a putative role of the newly identified two-pore domain (K(2P)) channels in the RVD of human CD4(+) T lymphocytes, using a series of potent well known channel blockers. In the present study, the pharmacological profiles of RVD inhibition revealed K(2P)5.1 and K(2P)18.1 as the most important K(2P) channels involved in the RVD of both naïve and stimulated T cells. The impact of chemical inhibition of K(2P)5.1 and K(2P)18.1 on the RVD was comparable to that of K(V)1.3. K(2P)9.1 also notably contributed to the RVD of T cells but the extent of this contribution and its dependence on the activation status could not be unambiguously resolved. In summary, our data provide first evidence that the RVD-related potassium efflux from human T lymphocytes relies on K(2P) channels.

Excitotoxic neuronal cell death during an oligodendrocyte-directed CD8+ T cell attack in the CNS gray matter.

BACKGROUND: Neural-antigen reactive cytotoxic CD8+ T cells contribute to neuronal dysfunction and degeneration in a variety of inflammatory CNS disorders. Facing excess numbers of target cells, CNS-invading CD8+ T cells cause neuronal cell death either via confined release of cytotoxic effector molecules towards neurons, or via spillover of cytotoxic effector molecules from 'leaky' immunological synapses and non-confined release by CD8+ T cells themselves during serial and simultaneous killing of oligodendrocytes or astrocytes. METHODS: Wild-type and T cell receptor transgenic CD8+ T cells were stimulated in vitro, their activation status was assessed by flow cytometry, and supernatant glutamate levels were determined using an enzymatic assay. Expression regulation of molecules involved in vesicular glutamate release was examined by quantitative real-time PCR, and mechanisms of non-vesicular glutamate release were studied by pharmacological blocking experiments. The impact of CD8+ T cell-mediated glutamate liberation on neuronal viability was studied in acute brain slice preparations. RESULTS: Following T cell receptor stimulation, CD8+ T cells acquire the molecular repertoire for vesicular glutamate release: (i) they upregulate expression of glutaminase required to generate glutamate via deamination of glutamine and (ii) they upregulate expression of vesicular proton-ATPase and vesicular glutamate transporters required for filling of vesicles with glutamate. Subsequently, CD8+ T cells release glutamate in a strictly stimulus-dependent manner. Upon repetitive T cell receptor stimulation, CD25high CD8+ T effector cells exhibit higher estimated single cell glutamate release rates than CD25low CD8+ T memory cells. Moreover, glutamate liberation by oligodendrocyte-reactive CD25high CD8+ T effector cells is capable of eliciting collateral excitotoxic cell death of neurons (despite glutamate re-uptake by glia cells and neurons) in intact CNS gray matter. CONCLUSION: Glutamate release may represent a crucial effector pathway of neural-antigen reactive CD8+ T cells, contributing to excitotoxicity in CNS inflammation.

Volume regulation of murine T lymphocytes relies on voltage-dependent and two-pore domain potassium channels.

A variety of ion channels are supposed to orchestrate the homoeostatic volume regulation in T lymphocytes. However, the relative contribution of different potassium channels to the osmotic volume regulation and in particular to the regulatory volume decrease (RVD) in T cells is far from clear. This study explores a putative role of the newly identified K(2P) channels (TASK1, TASK2, TASK3 and TRESK) along with the voltage-gated potassium channel K(V)1.3 and the calcium-activated potassium channel K(Ca)3.1 in the RVD of murine T lymphocytes, using genetic and pharmacological approaches. K(2P) channel knockouts exerted profound effects on the osmotic properties of murine T lymphocytes, as revealed by reduced water and RVD-related solute permeabilities. Moreover, both genetic and pharmacological data proved a key role of K(V)1.3 and TASK2 channels in the RVD of murine T cells exposed to hypotonic saline. Our experiments demonstrate a leading role of potassium channels in the osmoregulation of T lymphocytes under different conditions. In summary, the present study sheds new light on the complex and partially redundant network of potassium channels involved in the basic physiological process of the cellular volume homeostasis and extends the repertoire of potassium channels by the family of K(2P) channels.

Upregulation of K2P5.1 potassium channels in multiple sclerosis.

OBJECTIVE: Activation of T cells critically depends on potassium channels. We here characterize the impact of K(2P)5.1 (KCNK5; TASK2), a member of the 2-pore domain family of potassium channels, on T-cell function and demonstrate its putative relevance in a T-cell-mediated autoimmune disorder, multiple sclerosis (MS). METHODS: Expression of K(2P)5.1 was investigated on RNA and protein level in different immune cells and in MS patients' biospecimens (peripheral blood mononuclear cells, cerebrospinal fluid cells, brain tissue specimen). Functional consequences of K(2P)5.1 expression were analyzed using pharmacological modulation, small interfering RNA (siRNA), overexpression, electrophysiological recordings, and computer modeling. RESULTS: Human T cells constitutively express K(2P)5.1. After T-cell activation, a significant and time-dependent upregulation of K(2P)5.1 channel expression was observed. Pharmacological blockade of K(2P)5.1 or knockdown with siRNA resulted in reduced T-cell functions, whereas overexpression of K(2P)5.1 had the opposite effect. Electrophysiological recordings of T cells clearly dissected K(2P)5.1-mediated effects from other potassium channels. The pathophysiological relevance of these findings was demonstrated by a significant K(2P)5.1 upregulation in CD4(+) and CD8(+) T cells in relapsing/remitting MS (RRMS) patients during acute relapses as well as higher levels on CD8(+) T cells of clinically isolated syndrome, RRMS, and secondary progressive multiple sclerosis patients during clinically stable disease. T cells in the cerebrospinal fluid from MS patients exhibit significantly elevated K(2P)5.1 levels. Furthermore, K(2P)5.1-positive T cells can be found in inflammatory lesions in MS tissue specimens. INTERPRETATION: Selective targeting of K(2P)5.1 may hold therapeutic promise for MS and putatively other T-cell-mediated disorders.

Recombinant tandem of pore-domains in a Weakly Inward rectifying K+ channel 2 (TWIK2) forms active lysosomal channels.

Recombinant TWIK2 channels produce weak basal background K+ currents. Current amplitudes depend on the animal species the channels have been isolated from and on the heterologous system used for their re-expression. Here we show that this variability is due to a unique cellular trafficking. We identified three different sequence signals responsible for the preferential expression of TWIK2 in the Lamp1-positive lysosomal compartment. Sequential inactivation of tyrosine-based (Y308ASIP) and di-leucine-like (E266LILL and D282EDDQVDIL) trafficking motifs progressively abolishes the targeting of TWIK2 to lysosomes, and promotes its functional relocation at the plasma membrane. In addition, TWIK2 contains two N-glycosylation sites (N79AS and N85AS) on its luminal side, and glycosylation is necessary for expression in lysosomes. As shown by electrophysiology and electron microscopy, TWIK2 produces functional background K+ currents in the endolysosomes, and its expression affects the number and mean size of the lysosomes. These results show that TWIK2 is expressed in lysosomes, further expanding the registry of ion channels expressed in these organelles.

Inhibition of Noninfectious Uveitis Using Intravenous Administration of Collagen II-Specific Type 1 Regulatory T Cells.

PURPOSE: To evaluate the therapeutic potential of Col-Treg, a collagen II-specific type 1 regulatory T-cell immunotherapy for the treatment of noninfectious uveitis (NIU). METHODS: Col-Treg cells were produced from collagen II-specific T cell receptor (TCR) transgenic mice or peripheral blood of healthy donors. Phenotypic characterization was performed by flow cytometry, and cytokine secretion was evaluated with Flowcytomix or ELISA. In vitro functional characterization included ATP hydrolysis, cytotoxicity, and contact-independent T-cell suppression and plasticity assays. Col-Treg migration was assessed by quantitative PCR specific to Col-Treg TCR. Col-Treg cells were administered intravenously in mice displaying experimental autoimmune uveitis (EAU) induced by interphotoreceptor retinoid-binding protein (IRBP) immunizations. Efficacy of Col-Treg was assessed by ophthalmology, histology, and immunohistochemistry. RESULTS: Mice Col-Treg cells displayed identity features of type 1 Treg cells with expression of CD25, FoxP3, low surface expression of CD127, and cytokine secretion profile (IL-10(high), IL-4(low), IFN-γ(int)). In vitro functional assays demonstrated Col-Treg suppressive capacity via soluble factor-dependent immunosuppression, cytotoxicity, and ATP hydrolysis. Col-Treg cells expressed granzyme B, CD39, and glucocorticoid-induced TNF-related protein (GITR). Administration of Col-Treg in EAU mice inhibited clinical and morphologic signs of uveitis and decreased ocular leukocyte infiltration. Col-Treg cells homed in the ocular tissues 24 hours after intravenous injection. Human Col-Treg cells were comparable to mice Col-Treg cells in identity and function and did not show the capacity to differentiate into Th17 cells in vitro. CONCLUSIONS: These results demonstrate the therapeutic potential of Col-Treg cells as a targeted approach for the treatment of NIU and the feasibility of translating this approach to the human clinical setting.

Endothelial TWIK-related potassium channel-1 (TREK1) regulates immune-cell trafficking into the CNS.

The blood-brain barrier (BBB) is an integral part of the neurovascular unit (NVU). The NVU is comprised of endothelial cells that are interconnected by tight junctions resting on a parenchymal basement membrane ensheathed by pericytes, smooth muscle cells and a layer of astrocyte end feet. Circulating blood cells, such as leukocytes, complete the NVU. BBB disruption is common in several neurological diseases, but the molecular mechanisms involved remain largely unknown. We analyzed the role of TWIK-related potassium channel-1 (TREK1, encoded by KCNK2) in human and mouse endothelial cells and the BBB. TREK1 was downregulated in endothelial cells by treatment with interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Blocking TREK1 increased leukocyte transmigration, whereas TREK1 activation had the opposite effect. We identified altered mitogen-activated protein (MAP) kinase signaling, actin remodeling and upregulation of cellular adhesion molecules as potential mechanisms of increased migration in TREK1-deficient (Kcnk2(-/-)) cells. In Kcnk2(-/-) mice, brain endothelial cells showed an upregulation of the cellular adhesion molecules ICAM1, VCAM1 and PECAM1 and facilitated leukocyte trafficking into the CNS. Following the induction of experimental autoimmune encephalomyelitis (EAE) by immunization with a myelin oligodendrocyte protein (MOG)35-55 peptide, Kcnk2(-/-) mice showed higher EAE severity scores that were accompanied by increased cellular infiltrates in the central nervous system (CNS). The severity of EAE was attenuated in mice given the amyotrophic lateral sclerosis drug riluzole or fed a diet enriched with linseed oil (which contains the TREK-1 activating omega-3 fatty acid α-linolenic acid). These beneficial effects were reduced in Kcnk2(-/-) mice, suggesting TREK-1 activating compounds may be used therapeutically to treat diseases related to BBB dysfunction.

The TASK1 channel inhibitor A293 shows efficacy in a mouse model of multiple sclerosis.

The two-pore domain potassium channel TASK1 (KCNK3) has recently emerged as an important modulator in autoimmune CNS inflammation. Previously, it was shown that T lymphocytes obtained from TASK1(-/-) mice display impaired T cell effector functions and that TASK1(-/-) mice show a significantly reduced disease severity in myelin oligodendrocyte glycoprotein (MOG(35-55)) peptide induced experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. We here evaluate a potent and specific TASK1 channel inhibitor, A293, which caused a dose-dependent reduction of T cell effector functions (cytokine production and proliferation). This effect was abolished in CD4(+) T cells from TASK1(-/-) mice but not in cells from TASK3(-/-) mice. In electrophysiological measurements, A293 application induced a significant reduction of the outward current of wildtype T lymphocytes, while there was no effect in TASK1(-/-) cells. Preventive and therapeutic application of A293 significantly ameliorated the EAE disease course in wildtype mice while it had no significant effect in TASK1(-/-) mice and was still partly effective in TASK3(-/-) mice. In summary, our findings support the concept of TASK1 as an attractive drug target for autoimmune disorders.

Expression of K2P5.1 potassium channels on CD4+ T lymphocytes correlates with disease activity in rheumatoid arthritis patients.

INTRODUCTION: CD4+ T cells express K(2P)5.1 (TWIK-related acid-sensitive potassium channel 2 (TASK2); KCNK5), a member of the two-pore domain potassium channel family, which has been shown to influence T cell effector functions. Recently, it was shown that K(2P)5.1 is upregulated upon (autoimmune) T cell stimulation. The aim of this study was to correlate expression levels of K(2P)5.1 on T cells from patients with rheumatoid arthritis (RA) to disease activity in these patients. METHODS: Expression levels of K(2P)5.1 were measured by RT-PCR in the peripheral blood of 58 patients with RA and correlated with disease activity parameters (C-reactive protein levels, erythrocyte sedimentation rates, disease activity score (DAS28) scores). Twenty patients undergoing therapy change were followed-up for six months. Additionally, synovial fluid and synovial biopsies were investigated for T lymphocytes expressing K(2P)5.1. RESULTS: K(2P)5.1 expression levels in CD4+ T cells show a strong correlation to DAS28 scores in RA patients. Similar correlations were found for serological inflammatory parameters (erythrocyte sedimentation rate, C-reactive protein). In addition, K(2P)5.1 expression levels of synovial fluid-derived T cells are higher compared to peripheral blood T cells. Prospective data in individual patients show a parallel behaviour of K(2P)5.1 expression to disease activity parameters during a longitudinal follow-up for six months. CONCLUSIONS: Disease activity in RA patients correlates strongly with K(2P)5.1 expression levels in CD4+ T lymphocytes in the peripheral blood in cross-sectional as well as in longitudinal observations. Further studies are needed to investigate the exact pathophysiological mechanisms and to evaluate the possible use of K(2P)5.1 as a potential biomarker for disease activity and differential diagnosis.

Two pore domain potassium channels in cerebral ischemia: a focus on K2P9.1 (TASK3, KCNK9).

BACKGROUND: Recently, members of the two-pore domain potassium channel family (K2P channels) could be shown to be involved in mechanisms contributing to neuronal damage after cerebral ischemia. K2P3.1-/- animals showed larger infarct volumes and a worse functional outcome following experimentally induced ischemic stroke. Here, we question the role of the closely related K2P channel K2P9.1. METHODS: We combine electrophysiological recordings in brain-slice preparations of wildtype and K2P9.1-/- mice with an in vivo model of cerebral ischemia (transient middle cerebral artery occlusion (tMCAO)) to depict a functional impact of K2P9.1 in stroke formation. RESULTS: Patch-clamp recordings reveal that currents mediated through K2P9.1 can be obtained in slice preparations of the dorsal lateral geniculate nucleus (dLGN) as a model of central nervous relay neurons. Current characteristics are indicative of K2P9.1 as they display an increase upon removal of extracellular divalent cations, an outward rectification and a reversal potential close to the potassium equilibrium potential. Lowering extracellular pH values from 7.35 to 6.0 showed comparable current reductions in neurons from wildtype and K2P9.1-/- mice (68.31 +/- 9.80% and 69.92 +/- 11.65%, respectively). These results could be translated in an in vivo model of cerebral ischemia where infarct volumes and functional outcomes showed a none significant tendency towards smaller infarct volumes in K2P9.1-/- animals compared to wildtype mice 24 hours after 60 min of tMCAO induction (60.50 +/- 17.31 mm3 and 47.10 +/- 19.26 mm3, respectively). CONCLUSIONS: Together with findings from earlier studies on K2P2.1-/- and K2P3.1-/- mice, the results of the present study on K2P9.1-/- mice indicate a differential contribution of K2P channel subtypes to the diverse and complex in vivo effects in rodent models of cerebral ischemia.

Evidence against involvement of Bmp receptor 1b signaling in fate specification of the chick mesencephalic alar plate at HH16.

Studies in the developing spinal cord have established that morphogenes secreted from the roof- and floor plate influence pattern formation along the dorsal-ventral axis of the neural tube. Bone morphogenetic proteins (Bmps), secreted from the roof plate, act on the more laterally located alar plates to induce position dependent gene expression and cell fate changes. The dorsalizing activity of Bmps is counteracted by Sonic hedgehog (Shh), which is secreted from the floor plate and underlying notochord. Bmps are also expressed in the roof plate of the mesencephalic vesicle, yet it is unclear at present if they also provide patterning information to the mesencephalic alar plates. We have experimentally tested the hypothesis that Bmp signaling is required for fate specification of the mesencephalic alar plate by manipulating Bmp receptor signaling in the early chick embryo through ectopic expression of mutated forms of Bmp receptor 1b (BmpR1b), which render the receptor constitutively active or dominant negative, respectively. In contrast to published data on the embryonic spinal cord, neither activation nor blockage of BmpR1b signaling in stage 16 embryos altered expression of markers of the mesencephalic alar plates including Pax3, Pax7, Meis2 and efnb1. Moreover, simultaneous activation of BmpR1b signaling and blockage of Shh signaling was not sufficient to induce Meis2 expression in the ventral mesencephalon. Therefore, whereas the importance of Bmp signaling for dorsal differentiation in the spinal cord is well established, it appears to play a less prominent role in the dorsal specification of the developing mesencephalon during the same developmental stages.