Patrick Meuth

TWIK-related Acid-sensitive K+ Channel 1 (TASK1) and TASK3 Critically Influence T Lymphocyte Effector Functions

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. Here we 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. Pretreatment 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 cell effector function and identify a possible molecular target for immunomodulation in T cell-mediated autoimmune disorders.

Correlation of T-channel coding gene expression, IT, and the low threshold Ca2+ spike in the thalamus of a rat model of absence epilepsy.

T-type Ca(2+) current-dependent burst firing of thalamic neurons is thought to be involved in the hyper-synchronous activity observed during absence seizures. Here we investigate the correlation between the expression of T-channel coding genes (alpha1G, -H, -I), T-type Ca(2+) current, and the T-current-dependent low threshold Ca(2+) spike in three functionally distinct thalamic nuclei (lateral geniculate nucleus; centrolateral nucleus; reticular nucleus) in a rat model of absence epilepsy, the WAG/Rij rats, and a non-epileptic control strain, the ACI rats. The lateral geniculate nucleus and centrolateral nucleus were found to primarily express alpha1G and alpha1I, while the reticular thalamic nucleus expressed alpha1H and alpha1I. Expression was higher in WAG/Rij when compared to ACI. The T-type Ca(2+) current properties matched the predictions derived from the expression pattern analysis. Current density was larger in all nuclei of WAG/Rij rats when compared to ACI and correlated with LTS size and the minimum LTS generating slope, while T-type Ca(2+) current voltage dependency correlated with the LTS onset potential.

Postnatal Expression Pattern of HCN Channel Isoforms in Thalamic Neurons: Relationship to Maturation of Thalamocortical Oscillations

Hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels are the molecular substrate of the hyperpolarization-activated inward current (Ih). Because the developmental profile of HCN channels in the thalamus is not well understood, we combined electrophysiological, molecular, immunohistochemical, EEG recordings in vivo, and computer modeling techniques to examine HCN gene expression and Ih properties in rat thalamocortical relay (TC) neurons in the dorsal part of the lateral geniculate nucleus and the functional consequence of this maturation. Recordings of TC neurons revealed an approximate sixfold increase in Ih density between postnatal day 3 (P3) and P106, which was accompanied by significantly altered current kinetics, cAMP sensitivity, and steady-state activation properties. Quantification on tissue levels revealed a significant developmental decrease in cAMP. Consequently the block of basal adenylyl cyclase activity was accompanied by a hyperpolarizing shift of the Ih activation curve in young but not adult rats. Quantitative analyses of HCN channel isoforms revealed a steady increase of mRNA and protein expression levels of HCN1, HCN2, and HCN4 with reduced relative abundance of HCN4. Computer modeling in a simplified thalamic network indicated that the occurrence of rhythmic delta activity, which was present in the EEG at P12, differentially depended on Ih conductance and modulation by cAMP at different developmental states. These data indicate that the developmental increase in Ih density results from increased expression of three HCN channel isoforms and that isoform composition and intracellular cAMP levels interact in determining Ih properties to enable progressive maturation of rhythmic slow-wave sleep activity patterns.

Cytotoxic CD8+ T Cell–Neuron Interactions: Perforin-Dependent Electrical Silencing Precedes But Is Not Causally Linked to Neuronal Cell Death

Cytotoxic CD8+ T cells are considered important effector cells contributing to neuronal damage in inflammatory and degenerative CNS disorders. Using time-lapse video microscopy and two-photon imaging in combination with whole-cell patch-clamp recordings, we here show that major histocompatibility class I (MHC I)-restricted neuronal antigen presentation and T cell receptor specificity determine CD8+ T-cell locomotion and neuronal damage in culture and hippocampal brain slices. Two separate functional consequences result from a direct cell–cell contact between antigen-presenting neurons and antigen-specific CD8+ T cells. (1) An immediate impairment of electrical signaling in single neurons and neuronal networks occurs as a result of massive shunting of the membrane capacitance after insertion of channel-forming perforin (and probably activation of other transmembrane conductances), which is paralleled by an increase of intracellular Ca2+ levels (within <10 min). (2) Antigen-dependent neuronal apoptosis may occur independently of perforin and members of the granzyme B cluster (within ∼1 h), suggesting that extracellular effects can substitute for intracellular delivery of granzymes by perforin. Thus, electrical silencing is an immediate consequence of MHC I-restricted interaction of CD8+ T cells with neurons. This mechanism is clearly perforin-dependent and precedes, but is not causally linked, to neuronal cell death.

T-current related effects of antiepileptic drugs and a Ca2+ channel antagonist on thalamic relay and local circuit interneurons in a rat model of absence epilepsy.

Channel blocking, anti-oscillatory, and anti-epileptic effects of clinically used anti-absence substances (ethosuximide, valproate) and the T-type Ca2+ current (IT) blocker mibefradil were tested by analyzing membrane currents in acutely isolated local circuit interneurons and thalamocortical relay (TC) neurons, slow intrathalamic oscillations in brain slices, and spike and wave discharges (SWDs) occurring in vivo in Wistar Albino Glaxo rats from Rijswijk (WAG/Rij). Substance effects in vitro were compared between WAG/Rij and a non-epileptic control strain, the ACI rats. Ethosuximide (ETX) and valproate were found to block IT in acutely isolated thalamic neurons. Block of IT by therapeutically relevant ETX concentrations (0.25-0.75 mM) was stronger in WAG/Rij, although the maximal effect at saturating concentrations (>or=10 mM) was stronger in ACI. Ethosuximide delayed the onset of the low threshold Ca2+ spike (LTS) of neurons recorded in slice preparations. Mibefradil (>or=2 microM) completely blocked IT and the LTS, dampened evoked thalamic oscillations, and attenuated SWDs in vivo. Computational modeling demonstrated that the complete effect of ETX can be replicated by a sole reduction of IT. However, the necessary degree of IT reduction was not induced by therapeutically relevant ETX concentrations. A combined reduction of IT, the persistent sodium current, and the Ca2+ activated K+ current resulted in an LTS alteration resembling the experimental observations. In summary, these results support the hypothesis of IT reduction as part of the mechanism of action of anti-absence drugs and demonstrate the ability of a specific IT antagonist to attenuate rhythmic burst firing and SWDs.

Upregulation of K2P5.1 potassium channels in multiple sclerosis

Activation of T cells critically depends on potassium channels. We here characterize the impact of K2P5.1 (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).

Specific expression of low-voltage-activated calcium channel isoforms and splice variants in thalamic local circuit interneurons

It has been suggested that the specific burst firing patterns of thalamic neurons reflect differential expression of low-voltage-activated (LVA) Ca(2+) channel subtypes and their splice variants. By combining electrophysiological, molecular biological, immunological, and computational modeling techniques we here show that diverging LVA Ca(2+) currents of thalamocortical relay (TC) and GABAergic interneurons of the dLGN correlate with a differential expression of LVA Ca(2+) channel splice variations and isoforms (alpha1G-a in TC; alpha1G-bc and alpha1I in interneurons). Implementation of the observed LVA Ca(2+) current differences into a TC neuron model changed the burst firing from TC-like to interneuron-like. We conclude that alternative splicing of the alpha1G isoform in dLGN TC and interneurons, and the exclusive expression of the alpha1I isoform in interneurons play a prominent role in setting the different LVA Ca(2+) current properties of TC and interneurons, which critically contribute to the diverging burst firing behavior of these neurons.

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.

Reciprocal modulation of I (h) and I (TASK) in thalamocortical relay neurons by halothane.

By combining electrophysiological, immunohistochemical, and computer modeling techniques, we examined the effects of halothane on the standing outward current (ISO) and the hyperpolarization-activated current (Ih) in rat thalamocortical relay (TC) neurons of the dorsal lateral geniculate nucleus (dLGN). Hyperpolarizing voltage steps elicited an instantaneous current component (Ii) followed by a slower time-dependent current that represented Ih. Halothane reduced Ih by shifting the voltage dependency of activation toward more negative potentials and by reducing the maximal conductance. Moreover, halothane augmented Ii and ISO. During the blockade of Ih through Cs+, the current–voltage relationship of the halothane-sensitive current closely resembled the properties of a current through members of the TWIK-related acid-sensitive K+ (TASK) channel family (ITASK). Computer simulations in a single-compartment TC neuron model demonstrated that the modulation of Ih and ITASK is sufficient to explain the halothane-induced hyperpolarization of the membrane potential observed in current clamp recordings. Immunohistochemical staining revealed protein expression of the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel proteins HCN1, HCN2, and HCN4. Together with the dual effect of halothane on Ih properties, these results suggest that Ih in TC neurons critically depends on HCN1/HCN2 heterodimers. It is concluded that the reciprocal modulation of Ih and ITASK is an important mechanism of halothane action in the thalamus.

Differential phospholipase C-dependent modulation of TASK and TREK two-pore domain K+ channels in rat thalamocortical relay neurons

During the behavioural states of sleep and wakefulness thalamocortical relay neurons fire action potentials in high frequency bursts or tonic sequences, respectively. The modulation of specific K+ channel types, termed TASK and TREK, allows these neurons to switch between the two modes of activity. In this study we show that the signalling lipids phosphatidylinositol 4,5‐bisphosphate (PIP2) and diacylglycerol (DAG), which are components of their membrane environment, switch on and shut off TREK and TASK channels, respectively. These channel modulations contribute to a better understanding of the molecular basis of the effects of neurotransmitters such as ACh which are released by the brainstem arousal system. The present report introduces PIP2 and DAG as new elements of signal transduction in the thalamus.