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Inhibitory effects of oxidants on n-type K+ channels in T lymphocytes and Xenopus oocytes
1Physiologisches Institut der Universität Tübingen, Gmelinstrasse 5, D-72076 Tübingen, Germany.
Abstract:
Reactive oxygen species (ROS) appear to be involved in Fas-induced programmed cell death. We have previously demonstrated a tyrosine-kinase-dependent inhibition of the n-type K+ channels (Kn) by Fas stimulation. Thus, the effect of hydrogen peroxide (H2O2) on the function of Kn was examined using the patch-clamp technique. Incubation of Jurkat human T lymphocytes with 100 microM H2O2 resulted in a 46 +/- 5% inhibition of the macroscopic whole-cell current. Experiments performed at the single-channel level using the cell-attached configuration revealed that the probability of the channel being open diminished upon incubation in H2O2. The effect was not dependent on src-like kinases, since H2O2 did not trigger tyrosine phosphorylation of the Kn channel protein and herbimycin A did not prevent channel inhibition. Kv1.3 channels underly the Kn of T lymphocytes and were expressed in Xenopus oocytes and subjected to electrophysiological analysis by the two-electrode voltage-clamp technique. Application of 1 mM H2O2 and 500 microM t-BOOH (tert, butylhydroperoxide) resulted in a marked inhibition of the K+ current within 20 min. Both the membrane-permeable thiol-group oxidizing agent DTNP [2,2'-dithiobis-(5-nitropyridine)] and the membrane-impermeable DTNB [5,5'-Dithiobis-(2-nitrobenzoic acid)] (50 microM) inhibited Kv1.3 channels, suggesting that extracellular domains of Kv1.3 are affected. These results point to a direct modulation of Kn by various oxidative agents.
Insights
Oxidative stress, including hydrogen peroxide (H2O2), directly inhibits n-type K+ channels (Kn) in T lymphocytes. This modulation affects channel function and may play a role in programmed cell death pathways.
Area of Science:
- Cellular electrophysiology
- Ion channel function
- Oxidative stress research
Background:
- Reactive oxygen species (ROS) are implicated in Fas-induced programmed cell death.
- Fas stimulation inhibits n-type K+ channels (Kn) via tyrosine kinases.
- The precise role of oxidative agents on Kn function requires elucidation.
Purpose of the Study:
- To investigate the effect of hydrogen peroxide (H2O2) on the function of n-type K+ channels (Kn).
- To determine if oxidative agents directly modulate Kv1.3 channels, which underlie T lymphocyte Kn.
Main Methods:
- Patch-clamp technique on Jurkat human T lymphocytes to assess whole-cell and single-channel currents.
- Two-electrode voltage-clamp electrophysiology on Xenopus oocytes expressing Kv1.3 channels.
- Application of H2O2, tert-butylhydroperoxide (t-BOOH), DTNP, and DTNB.
Main Results:
- H2O2 (100 microM) inhibited macroscopic whole-cell current of Kn by 46% in T lymphocytes.
- Single-channel recordings showed H2O2 reduced the open probability of Kn.
- H2O2 did not induce tyrosine phosphorylation of Kn, indicating a non-src-like kinase mechanism.
- H2O2 and t-BOOH inhibited Kv1.3 channels expressed in oocytes.
- Both membrane-permeable and impermeable thiol-reactive agents inhibited Kv1.3, suggesting extracellular domain involvement.
Conclusions:
- Oxidative agents, including H2O2, directly modulate n-type K+ channels (Kn).
- Kv1.3 channels are sensitive to oxidative stress, with extracellular domains being affected.
- These findings suggest a direct link between oxidative stress and ion channel function in cellular processes like programmed cell death.