By combining molecular biological, electrophysiological, immunological, and computer modeling techniques, we here demonstrate a counterbalancing contribution of TASK channels, underlying hyperpolarizing K+ leak currents, and HCN channels, underlying depolarizing Ih, to the resting membrane potential of thalamocortical relay (TC) neurons. RT-PCR experiments revealed the expression of TASK1, TASK3, and HCN1-4. Quantitative determination of mRNA expression levels and immunocytochemical staining demonstrated that TASK3 and HCN2 channels represent the dominant thalamic isoforms and are coexpressed in TC neurons. Extracellular acidification, a standard procedure to inhibit TASK channels, blocked a TASK current masked by additional action on HCN channels. Only in the presence of the HCN blocker ZD7288 was the pH-sensitive component typical for a TASK current, i.e., outward rectification and current reversal at the K+ equilibrium potential. In a similar way extracellular acidification was able to shift the activity pattern of TC neurons from burst to tonic firing only during block of Ih or genetic knock out of HCN channels. A single compartmental computer model of TC neurons simulated the counterbalancing influence of TASK and HCN on the resting membrane potential. It is concluded that TASK3 and HCN2 channels stabilize the membrane potential by a mutual functional interaction, that the most efficient way to regulate the membrane potential of TC neurons is the converse modulation of TASK and HCN channels, and that TC neurons are potentially more resistant to insults accompanied by extracellular pH shifts in comparison to other CNS regions.

Two major K؉ channels are expressed in T cells, (i) the voltagedependent K V 1.3 channel and (ii) the Ca 2؉ -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 (ϳ40%) 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 K 2 P 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.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. K ϩ selective ion channels in T cells and their role in immune responses have been discussed for decades, since the discovery that non-selective K ϩ blockers could inhibit T cell proliferation in vitro (1-3). The role of K ϩ channels in the activation of T cells is pivotal, because opening the channels hyperpolarizes the membrane potential, which in turn increases the influx of Ca 2ϩ via Ca 2ϩ release-activated Ca Intracellular Ca 2ϩ release triggers activation of CRAC channels resulting in longer lasting (ϳ1 h) elevated [Ca 2ϩ ] 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 ϩ channel 1.3 (K V 1.3 channel) and the intermediate conductance Ca 2ϩ -activated channel (IKCa channel; IK Ca 1 or K Ca 3.1; which acquires its Ca 2ϩ dependence from constitutively bound calmodulin) were found to be the dominating K ϩ channel types expressed in T cells (7-10). K V 1.3 is activated by membrane depolarization, whereas K Ca 3.1 channel opening is triggered by a rise in Ca 2ϩ ions (5). Selective blockade of K V 1.3 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 K V 1.3 has been shown to mediate beneficial effects in experimental autoimmune encephalomyelitis (EAE), a rodent model for multiple sclerosis (12,13). The importance of K ϩ ch...

Cytotoxic CD8ϩ 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 ϩ 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 ϩ 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 Ca 2ϩ levels (within Ͻ10 min). (2) Antigen-dependent neuronal apoptosis may occur independently of perforin and members of the granzyme B cluster (within ϳ1 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 ϩ T cells with neurons. This mechanism is clearly perforin-dependent and precedes, but is not causally linked, to neuronal cell death.

Hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels are the molecular substrate of the hyperpolarizationactivated inward current (I h ). 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 I h 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 I h 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 I h 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 I h conductance and modulation by cAMP at different developmental states. These data indicate that the developmental increase in I h density results from increased expression of three HCN channel isoforms and that isoform composition and intracellular cAMP levels interact in determining I h properties to enable progressive maturation of rhythmic slow-wave sleep activity patterns.

A genetic knockout was used to determine the specific contribution of TWIK-related acid-sensitive K ϩ (TASK)-1 channels to the function of dorsal lateral geniculate nucleus (DLG) thalamocortical relay (TC) neurons. Disruption of TASK-1 function produced an ϳ19% decrease in amplitude of the standing outward current (I SO ) and a 3 Ϯ 1-mV depolarizing shift in resting membrane potential (V rest ) of DLG neurons. We estimated that current through TASK-1 homodimers or TASK-1/TASK-3 heterodimers contribute(s) approximately one third of the current sensitive to TASK channel modulators in DLG TC neurons. The effects of the TASK channel blocker bupivacaine (20 M), of muscarine (50 M), and of H ϩ on I SO were reduced to approximately 60%, 59%, and shifted to more acidic pH values, respectively. The blocking effect of anandamide on I SO [30 M; 23 Ϯ 3% current decrease in wild type (WT)] was absent in TASK-1 knockout (TASK-1 Ϫ/Ϫ ) mice (9 Ϯ 6% current increase). Comparable results were obtained with the more stable anandamide derivative methanandamide (20 M; 20 Ϯ 2% decrease in WT; 4 Ϯ 6% increase in TASK-1 Ϫ/Ϫ ). Current-clamp recordings revealed a muscarine-induced shift in TC neuron activity from burst to tonic firing in both mouse genotypes. Electrocorticograms and sleep/wake times were unchanged in TASK-1 Ϫ/Ϫ mice. In conclusion, our findings demonstrate a significant contribution of TASK-1 channels to I SO in DLG TC neurons, although the genetic knockout of TASK-1 did not produce severe deficits in the thalamocortical system.