Cell culture. The human leukemia T cell line, Jurkat E6-1, p56lck-deficient Jurkat mutant, JCAM.1, and CD45 deficient Jurkat mutant, J45.01 were obtained from ATCC. These Jurkat cell lines were grown in RPMI1640 medium containing 10% heat inactivated FBS, 10 mM Hepes, and 2 mM glutamate. Cells were cultured in culture plate at 37 °C in 5% CO2 humidified incubator.

Electrophysiological measurements. Membrane currents were measured in Jurkat cells using standard whole-cell voltage-clamping, using CEZ-2300 Amplifier (Nihon-Koden, Japan) and signals were stored on videotape using a PCM system and were later captured on an IBM computer using DT2801A as an analog–digital converter and an acquisition program for precise analysis as ahs been reported previously [14]. All experiments were carried out at 23 ± 2 °C.

Solutions. The physiological salt solution for electrophysiological experiments contained mM: NaCl 137, KCl 5.9, MgCl2 1.2, glucose 10, Hepes 10. The pH was adjusted to 7.4 with NaOH. The pipette solution for electrical recordings contained mM: KCl 30, KAspartate 110, ATPNa2 2, MgCl2 2, EGTA 0.05, Hepes 10. The pH was adjusted to 7.2 with KOH.

Chemicals. …

Effects of anti-CD3 and anti-CD28 antibodies on Kv1.3 current in Jurkat cells

In Jurkat T lymphocyte E6-1, outward currents were elicited by depolarization to +40 mV under whole-cell voltage-clamp. Kinetics of outward currents show rapid activation and slow inactivation and were typical for voltage-gated Kv1.3 channels (Fig. 1A). The current density at the peak was 31.7 ± 11.5 pA/pF (n = 9). Application of 10 nM margatoxin, a selective blocker of Kv1.3 channels [15], reduced the outward current by over 90%, confirming that Kv1.3 is the predominant component of the outward current. In this study, MgTX was added at end of each experiment to evaluate Kv1.3. The Kv1.3 currents was stably recorded for over 20 min, when depolarization was applied every 15 s. Stimulation of E6-1 with 2 or 10 μg/ml anti-human CD3 antibody slowly but significantly reduced Kv1.3 current to 85.3 ± 10.6% (relative to before administration, n = 4), and to 74.1 ± 4.6% (n = 7), respectively (Fig.1A and B). Application of anti-CD28 antibody alone did not affect Kv1.3 current (data not shown). The addition of 10 μg/ml anti-CD28 antibody in the presence of anti-CD3 antibody induced further decrease in Kv1.3 current from 76.2 ± 5.8% (n = 5) to 53.4 ± 7.2% (n = 5, p < 0.05) (Fig. 1D and E). Fig. 1. Effects of anti-CD3 antibody or both anti-CD3 and anti-CD28 antibodies on Kv1.3 current in Jurkat cells. (A) Effects of anti-CD3 antibody on Kv1.3 current in Jurkat cells. Repres…

It is clearly shown in the present study that CD3 stimulation alone reduced MgTx sensitive Kv1.3 current and that CD3/CD28 co-stimulation further reduced Kv1.3 current in Jurkat T cell. It has been well established that CD3 stimulation alone is not complete but that co-stimulation of both CD3 and CD28 is required for T lymphocyte fully activation [1], [2]. In the long-term effects following T lymphocyte activation, the Kv1.3 channel expression is highly enhanced and this promotes Ca2+ entry and T cell proliferation [1], [3]. In contrast, the inhibition of Kv1.3 current occurs as an acute response to CD3 stimulation and CD3/CD28 co-stimulation as shown in this study, and this may presumably prevent an excess Ca2+ entry from extracellular space through CRAC channels during a short time and protect cells from Ca2+ overload.

In the present study, the inhibitory effects of CD3 stimulation alone and CD3/CD28 co-stimulation on Kv1.3 current were still observed in p56lck-kinase deficient cells. These were rather unexpected results, since Kv1.3 channel inhibition and its phosphorylation by anti-Fas antibody and ceramide were abolished by the application of src kinase inhibitor herbimycin A or the deficiency of the p56lck tyrosine kinase [12], [17]. The src kinase inhibitor PP2 abolished the hypoxia-induced inhibition of Kv1.3 channels in primary human T lymphocytes, and Kv1.3 channel sensitivity to hypoxia was lost in p56lck-defic…