Inhibitory effects of oxidants on n-type K⁺ channels in T lymphocytes and Xenopus oocytes

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 (K_n) by Fas stimulation. Thus, the effect of hydrogen peroxide (H₂O₂) on the function of K_n was examined using the patch-clamp technique. Incubation of Jurkat human T lymphocytes with 100 μM 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 K_n channel protein and herbimycin A did not prevent channel inhibition. K_v1.3 channels underly the K_n 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 μM 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 μM) inhibited K_v1.3 channels, suggesting that extracellular domains of K_v1.3 are affected. These results point to a direct modulation of K_n by various oxidative agents.