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Articles in PresS, published online ahead of print September 21, 2001
Am J Physiol Cell Physiol, 10.1152/ajpcell.00167.2001
Submitted on April 2, 2001
Accepted on September 13, 2001
1 Biophysic, Centro de Estudios Cientificos, Valdivia, Valdivia, Chile; Chemistry and Biology, University of Santiago, Santiago, Santiago, Chile; Science, University of Chile, Santiago, Santiago, Chile
2 Biophysic, Centro de Estudios Cientificos, Valdivia, Valdivia, Chile; Science, University of Chile, Santiago, Santiago, Chile
3 Chemistry and Biology, University of Santiago, Santiago, Santiago, Chile
4 Science, University of Chile, Santiago, Santiago, Chile; Biophysic, Centro de Estudios Cientificos, Valdivia, Valdivia, Chile
* To whom correspondence should be addressed. E-mail: marcos{at}cecs.cl.
We studied the effect of hydrogen peroxide (H2O2) upon the gating behavior of a Ca2+-sensitive voltage-dependent K+ (KV,Ca) channels of large conductance. Potassium currents were recorded from single skeletal muscle channels incorporated into bilayers or using macropatches of Xenopus leavis oocytes membranes expressing the human Slowpoke (hSlo)
subunit. Exposure of the intracellular side of KV,Ca channels to H2O2 (8-23 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 effect of H2O2 could not be fully reverted by the reducing agent dithiothreitol (DTT). However, the decrease in Po induced by the oxidizing agent 5,5,-dithio-bis-(2-nitrobenzoic acid) (DTNB) was completely reverted by DTT. The incomplete recovery of KV,Ca channel activity promoted by DTT, suggests that H2O2 treatment must be modifying other amino acid residues such as methionine or tryptophan besides cysteine. Noise analisys 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.
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