K-CELLS DECREASE WITH ENCEPHALITOGENICITY WHILE INTERLEUKIN-2 (IL-2) RECEPTOR EXPRESSION REMAINS STABLE DURING IL-2 DEPENDENT CELL EXPANSION( CURRENTS OF ENCEPHALITOGENIC MEMORY T)

Previous studies revealed a linkage between increased K+ current and l ymphocyte activation upon non-specific stimulation with mitogenic lect ins and antibodies. So far no information is available about the behav iour of K+ currents in specifically autoantigen-stimulated lymphocytes . Therefore, we have investigated K+ currents in encephalitogenic T li ne 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 fl ow cytometry. Outward currents were observed in response to depolarizi ng voltage steps from a holding potential of -80 mV. The peak current density increased with more positive membrane potentials, where the cu rrent threshold was about -40mV and the maximum conductance was 1.22 n S/pF. This current was characterized by a fast activation and a fast i nactivation with half maximal inactivation at -67 mV. The sensitivity of the peak current to Ki channel blocking agents was as follows: 4-am inopyridine (4-AP) had a half blocking concentration of 0.4mM and a ma ximal block of 83.7% at 10 mM 4-AP, tetraethylammonium caused a block of 6% at 0.1mM, 15% at 1 mM and 40% at 10mM, charybdotoxin blocked 90% at 100 nM, whereas iberiotoxin had no effect (all values at a damped membrane potential of +30 mV). The encephalitogenic T cells used in ou r study reach their highest encephalitogenic potency on day 3 to 4 aft er the onset of restimulation. Furthermore, K+ currents were measured during the whole course of an in vitro restimulation cycle. The peak c urrents normalized to cell capacitance reached their maximum on day 2 (326+/- 52.8pA/pF, n=4) and decreased thereafter as follows: day 3: 13 9.7+/-7.87pA/pF (n = 27), day 4: 85.4 +/- 8.95 pA/pF (n = 28) and day 5. 40.9 +/- 7.45 pA/pF (n = 17). The activation and inactivation chara cteristics of the current and its responses to selective blockers were similar at all days after restimulation. In contrast to the K+ curren t, IL-2 receptor expression was maintained on >95% of cells until day 6 after restimulation.