tram-34 and margatoxin

Functional imbalance between T helper subsets plays important role in the pathogenesis of autoimmune disorders. Transient increase of cytoplasmic calcium level, and sustention of negative membrane potential by voltage sensitive Kv1.3 and calcium-dependent IKCa1 potassium channels are essential for short-term lymphocyte activation, thus present possible target for selective immunomodulation. We aimed to investigate calcium influx sensitivity to the inhibition of potassium channels in the main T helper subsets. Peripheral blood from 11 healthy individuals was drawn and calcium influx kinetics following activation with phytohemagglutinin in Th1, Th2, Th17, and Treg cells were evaluated. Alteration of calcium influx induced by specific inhibitors of Kv1.3 and IKCa1 potassium channels, and the expression of Kv1.3 channels were also assessed. Highest cytoplasmic calcium concentration was observed in stimulated Th1 cells, while the lowest level was measured in Treg cells. In Th1 and Th17 cells, inhibition of both investigated potassium channels decreased calcium influx. In Th2 cells only the inhibitor of Kv1.3 channels, while in Treg cells none of the inhibitors had significant effect. Upon the inhibition of IKCa1 channels, short-term activation of proinflammatory cells was specifically decreased without affecting anti-inflammatory subsets, indicating that selective immunomodulation is possible in healthy individuals.

Topics: Adult; Antibodies, Monoclonal; Autoimmune Diseases; Calcium; Calcium Signaling; Female; Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Kv1.3 Potassium Channel; Leukocytes, Mononuclear; Lymphocyte Activation; Male; Phytohemagglutinins; Potassium Channel Blockers; Potassium Channels; Pyrazoles; Scorpion Venoms; T-Lymphocytes, Regulatory; Th1 Cells; Th17 Cells; Th2 Cells; Young Adult

Cytokine production in activated T lymphocytes of the term neonate is reduced compared with adults. We aimed to characterize the calcium influx kinetics of activated T lymphocytes in the neonate and to test the functionality and expression of Kv1.3 and IKCa1 lymphocyte potassium channels, important regulators of calcium influx. We isolated lymphocytes from the peripheral blood of nine adults and cord blood of nine term neonates. We measured the calcium influx kinetics with flow cytometry in the T(h)1, T(h)2, CD4 and CD8 T-lymphocyte subsets activated with PHA. We determined the sensitivity of calcium influx to specific inhibitors of the Kv1.3 and IKCa1 channels. We also measured Kv1.3 channel expression using specific antibody. With the exception of the CD4 subset, calcium influx kinetics was decreased upon activation in neonatal T lymphocytes compared with adults. Neonatal T lymphocytes were found to be less sensitive to the specific inhibition of Kv1.3 and IKCa1 channels. The expression of Kv1.3 channels was higher on major T-lymphocyte subsets of newborns except for T(h)1 lymphocytes. Our findings suggest that the characteristics of short-term activation of major neonatal T-lymphocyte subsets are altered compared with adults. The altered function of neonatal lymphocyte potassium channels may contribute to this phenomenon.

Topics: Adult; Calcium Signaling; CD4 Antigens; CD8 Antigens; Cells, Cultured; Female; Humans; Immunomodulation; Infant, Newborn; Intermediate-Conductance Calcium-Activated Potassium Channels; Kv1.3 Potassium Channel; Lymphocyte Activation; Male; Middle Aged; Pyrazoles; Scorpion Venoms; Th1 Cells; Th2 Cells

Kv1.3 and IKCa1 potassium channels participate in the maintenance of calcium-influx during lymphocyte activation. Kv1.3 channels have a prominent role in specific T cell subsets, presenting a possible target for selective immunomodulation. We investigated the impact of Kv1.3 and IKCa1 channel inhibitors on calcium-influx characteristics in human T cells in type 1 diabetes mellitus. We isolated lymphocytes from 9 healthy and 9 type 1 diabetic individuals and measured the alteration of calcium-influx with flow cytometry in the Th1, Th2, CD4 and CD8 subsets after treatment of samples with specific channel inhibitors. Our results indicate an increased reactivity of type 1 diabetes lymphocytes, which is correlated to their increased sensitivity to Kv1.3 channel inhibition. However, the contribution of Kv1.3 channels to calcium flux is not exclusive for a specific lymphocyte subset as previous reports suggest, but is characteristic for each subset investigated. Therefore, the proposed inhibition of Kv1.3 channels as a novel therapeutic approach for the treatment of type 1 diabetes mellitus may have a major effect on overall lymphocyte function in this disease.

Topics: Adult; Calcium Signaling; CD4 Antigens; CD8 Antigens; Cells, Cultured; Diabetes Mellitus, Type 1; Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Kv1.3 Potassium Channel; Lymphocyte Activation; Male; Middle Aged; Pyrazoles; Scorpion Venoms; T-Lymphocyte Subsets; Th1 Cells; Th2 Cells

Microglia are the intrinsic immune cells of the brain. As such, they are crucially involved in neuro-protection as well as neuro-degeneration. Their activation leads to the induction of cytokine and chemokine release, the production of reactive oxygen species and nitric oxide and an increased outward potassium conductance. In this study, we focus our interest on potassium currents and channels in the C8-B4 murine microglial cell line and compare them with those of primary cultured microglia from neo-natal mice. Using the whole cell patch-clamp technique, we have recorded prominent inward and outward rectifying voltage-dependent potassium currents but no calcium-activated potassium currents. Using pharmacological, biophysical and molecular approaches, we demonstrate that Kv1.3 and Kir2.1 channels underlie outward and inward rectifying potassium currents, respectively. In contrast to primary cultured microglia, we observe that an outward rectifying potassium current is already present in unstimulated C8-B4 cells. However, as seen in primary microglia, this current increases after treatment with LPS, IFN-gamma, TGF-beta and GM-CSF and is suppressed by treatment with protein kinase inhibitors. Our study indicates that the C8-B4 cell line shows similar though not identical potassium channel properties compared to primary cultured microglia. We demonstrate that despite some differences, they are a useful tool to study potassium currents in microglial activation mechanisms by means of electrophysiological methods without the need for preparation of cells as primary culture.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 4-Aminopyridine; Animals; Apamin; Barium; Calcium; Cations; Cell Line; Charybdotoxin; Dose-Response Relationship, Drug; Drug Combinations; Elapid Venoms; Electrodes; Electrophysiology; Fluorescent Antibody Technique, Direct; Genistein; Granulocyte-Macrophage Colony-Stimulating Factor; Interferon-gamma; Lipopolysaccharides; Membrane Potentials; Mice; Mice, Inbred C57BL; Microglia; Neurotoxins; Patch-Clamp Techniques; Potassium Channel Blockers; Potassium Channels; Potassium Channels, Inwardly Rectifying; Pyrazoles; Scorpion Venoms; Transforming Growth Factor beta