K+ currents of encephalitogenic memory T cells decrease with encephalitogenicity while interleukin-2 (IL-2) receptor expression remains stable during IL-2 dependent cell expansion.
Previous studies revealed a linkage between increased K+ current and lymphocyte activation upon non-specific stimulation with mitogenic lectins and antibodies. So far no information is available about the behaviour of K+ currents in specifically autoantigen-stimulated lymphocytes. Therefore, we have investigated K+ currents in encephalitogenic T line cells, specifically stimulated with myelin basic protein, using the whole-cell patch-clamp technique. In parallel, the T cell activation marker interleukin-2 (IL-2) receptor was measured quantitatively by flow cytometry. Outward currents were observed in response to depolarizing voltage steps from a holding potential of -80mV. The peak current density increased with more positive membrane potentials, where the current threshold was about -40mV and the maximum conductance was 1.22nS/pF. This current was characterized by a fast activation and a fast inactivation with half maximal inactivation at -67mV. The sensitivity of the peak current to K+ channel blocking agents was as follows: 4-aminopyridine (4-AP) had a half blocking concentration of 0.4mM and a maximal block of 83.7% at 10mM 4-AP, tetraethyl-ammonium caused a block of 6% at 0.1mM, 15% at 1mM and 40% at 10mM, charybdotoxin blocked 90% at 100nM, whereas iberiotoxin had no effect (all values at a clamped membrane potential of +30mV). The encephalitogenic T cells used in our study reach their highest encephalitogenic potency on day 3 to 4 after the onset of restimulation. Furthermore, K+ currents were measured during the whole course of an in vitro restimulation cycle. The peak currents normalized to cell capacitance reached their maximum on day 2 (326+/- 52.8pA/pF, n = 4) and decreased thereafter as follows: day 3: 139.7 +/- 7.87pA/pF (n = 27), day 4: 85.4 +/- 8.95pA/pF (n = 28) and day 5: 40.9 +/- 7.45pA/pF (n = 17). The activation and inactivation characteristics of the current and its responses to selective blockers were similar at all days after restimulation. In contrast to the K+ current, IL-2 receptor expression was maintained on > 95% of cells until day 6 after restimulation. In conclusion, the K+ currents measured in rat encephalitogenic T cells resemble n-type voltage-gated K+ currents described in mice and man. The comparison of K+ current, IL-2 receptor expression and encephalitogenic potency let us suppose that the observed K+ current represents an early event of specific T cell activation and can serve as a parameter of high functional activity of T cells corresponding to their encephalitogenicity.
Department of Neurology, University of Rostock, Germany.
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Strauss
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Schubert
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Jung
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Mix
D000818:Animals; D002460:Cell Line; D004681:Encephalomyelitis, Autoimmune, Experimental / Q000209:etiology* / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007156:Immunologic Memory; D007376:Interleukin-2 / Q000378:metabolism; D008213:Lymphocyte Activation; D008564:Membrane Potentials; D051379:Mice; D004676:Myelin Basic Protein / Q000276:immunology; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000378:metabolism*; D051381:Rats; D015375:Receptors, Interleukin-2 / Q000378:metabolism*; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology* / Q000378:metabolism*
D007376:Interleukin-2; D004676:Myelin Basic Protein; D015375:Receptors, Interleukin-2; D011188:Potassium