Ca(2+)-activated K+ channel inhibition by reactive oxygen species.
We studied the effect of H(2)O(2) on the gating behavior of large-conductance Ca(2+)-sensitive voltage-dependent K(+) (K(V,Ca)) channels. We recorded potassium currents from single skeletal muscle channels incorporated into bilayers or using macropatches of Xenopus laevis oocytes membranes expressing the human Slowpoke (hSlo) alpha-subunit. Exposure of the intracellular side of K(V,Ca) channels to H(2)O(2) (4-23 mM) leads to a time-dependent decrease of the open probability (P(o)) without affecting the unitary conductance. H(2)O(2) did not affect channel activity when added to the extracellular side. These results provide evidence for an intracellular site(s) of H(2)O(2) action. Desferrioxamine (60 microM) and cysteine (1 mM) completely inhibited the effect of H(2)O(2), indicating that the decrease in P(o) was mediated by hydroxyl radicals. The reducing agent dithiothreitol (DTT) could not fully reverse the effect of H(2)O(2). 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 K(V,Ca) channel activity promoted by DTT suggests that H(2)O(2) 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 hSlo channels showed that H(2)O(2) induced a decrease in current mediated by a decrease both in the number of active channels and P(o).
Soto
Marco A
MA
Centro de Estudios Científicos, Av. Arturo Prat 514, Valdivia, Chile. marcos@cecs.cl
González
Carlos
C
Lissi
Eduardo
E
Vergara
Cecilia
C
Latorre
Ramón
R
D000818:Animals; D002118:Calcium / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D003676:Deferoxamine / Q000378:metabolism; D004229:Dithiothreitol / Q000378:metabolism; D005260:Female; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology*; D017665:Hydroxyl Radical / Q000378:metabolism; D015640:Ion Channel Gating; D007502:Iron Chelating Agents / Q000378:metabolism; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D008051:Lipid Bilayers / Q000378:metabolism; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism; D009865:Oocytes; D016877:Oxidants / Q000378:metabolism / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D024681:Potassium Channels, Calcium-Activated / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism / Q000494:pharmacology*; D014982:Xenopus laevis
D007502:Iron Chelating Agents; C494841:KCNMA1 protein, human; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D008051:Lipid Bilayers; D016877:Oxidants; D024681:Potassium Channels, Calcium-Activated; D017382:Reactive Oxygen Species; D017665:Hydroxyl Radical; D006861:Hydrogen Peroxide; D003676:Deferoxamine; D003545:Cysteine; D002118:Calcium; D004229:Dithiothreitol