Ca2+-activated K+ channel inhibition by reactive oxygen species

Publisher Website
Google Scholar
Abstract
We studied the effect of H2O2 on the gating behavior of large-conductance Ca2+-sensitive voltage-dependent K+ (KV,Ca) channels. We recorded potassium currents from single skeletal muscle channels incorporated into bilayers or using macropatches of Xenopus laevisoocytes membranes expressing the human Slowpoke(h Slo) α-subunit. Exposure of the intracellular side of KV,Ca channels to H2O2 (4–23 mM) leads to a time-dependent decrease of the open probability ( P o) without affecting the unitary conductance. H2O2 did not affect channel activity when added to the extracellular side. These results provide evidence for an intracellular site(s) of H2O2 action. Desferrioxamine (60 μM) and cysteine (1 mM) completely inhibited the effect of H2O2, indicating that the decrease in P o was mediated by hydroxyl radicals. The reducing agent dithiothreitol (DTT) could not fully reverse the effect of H2O2. However, DTT did completely reverse the decrease in P o induced by the oxidizing agent 5,5′-dithio-bis-(2-nitrobenzoic acid). The incomplete recovery of KV,Ca channel activity promoted by DTT suggests that H2O2 treatment must be modifying other amino acid residues, e.g., as methionine or tryptophan, besides cysteine. Noise analysis of macroscopic currents in Xenopus oocytes expressing h Slo channels showed that H2O2 induced a decrease in current mediated by a decrease both in the number of active channels and P o.