Abstract
Two major K(+) channels are expressed in T cells, (i) the voltage-dependent 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 ( approximately 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 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.
Highlights
Intracellular Ca2ϩ release triggers activation of CRAC channels resulting in longer lasting (ϳ1 h) elevated [Ca2ϩ]i mandatory for further transcription-dependent steps of T cell activation [4, 6]
TASK1 and TASK3 Channels Are Expressed on Human T Lymphocytes—In a first set of experiments purified human CD3ϩ T lymphocytes (n ϭ 6 donors) were assessed by immu
Proliferation—To correlate K2P channel expression to T cell effector functions we studied the influence of pharmacological blocking reagents for Kϩ channels on cytokine production and T cell proliferation
Summary
Materials and Reagents—Charybdotoxin, bupivacaine, spermine, and ruthenium red (diluted in H2O; Sigma), Psora-4 (DMSO; Carl Roth GmbH, Germany), and anandamide (EtOH; Tocris, Germany) were frozen as aliquots for further use. For antibody staining cells were resuspended in FACS buffer (PBS containing 0.1% bovine serum albumin and 0.1% NaN3), directly labeled antibodies were added. The MBPTCs used for this experiment were freshly restimulated for 3 days with antigen presenting cells and guinea pig MBP protein (in one Petri dish: 3 ϫ 106 MBP-TCs ϩ 150 ϫ 106 antigen presenting cells ϩ guinea pig MBP 10 g/ml in 10 ml of restimulation medium, which consists of RPMI 1640 containing 1% glutamine, 1% antibiotics, 1% rat serum, and 0.4% -mercaptoethanol). The maximum specific conductance gkbar was set to 0.0023 S/cm to reach a maximum current of about 200 pA at a membrane potential of ϩ40 mV. The Ik current amplitude was calculated according to the following equation: Ik ϭ gkbar ϫmϫhϫ (VM Ϫ Ek); m, activation state variable; h, inactivation state variable; and VM, membrane voltage. Statistical significance was set at p Ͻ 0.05, which is indicated by double asterisks
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