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1 Centro de Estudios Científicos, Valdivia; 2 Facultad de Ciencias, Universidad de Chile, Santiago; and 3 Facultad de Química y Biología, Universidad de Santiago, Santiago, Chile
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 laevis
oocytes membranes expressing the human Slowpoke (hSlo) 
-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
(Po) 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 Po 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 Po 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 hSlo channels showed that H2O2 induced a decrease in current mediated by
a decrease both in the number of active channels and
Po.
KV,Ca channels; H2O2
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