Abstract

In a previous work the involvement of a fenamate-sensitive Ca 2+-activated nonselective cation channel (NSCC) in free radical-induced rat liver cell necrosis was demonstrated (5). Therefore, we studied the effect of radical oxygen species and oxidizing agents on the gating behavior of a NSCC in a liver-derived epithelial cell line (HTC). Single-channel currents were recorded in HTC cells by the excised inside-out configuration of the patch-clamp technique. In this cell line, we characterize a 19-pS Ca 2+-activated, ATP- and fenamate-sensitive NSCC nearly equally permeable to monovalent cations. In the presence of Fe2+, exposure of the intracellular side of NSCC to H2O2 increased their open probability (Po) by ?40% without affecting the unitary conductance. Desferrioxamine as well as the hydroxyl radical (.OH) scavenger MCI-186 inhibited the effect of H2O2, indicating that the increase in Po was mediated by .OH. Exposure of the patch membrane to the oxidizing agent 5,5?-dithio-bis-2-nitrobenzoic acid (DTNB) had a similar effect to .OH. The increase in P o induced by .OH or DTNB was not reverted by preventing formation or by DTNB washout, respectively. However, the reducing agent dithiothreitol completely reversed the effects on Po of both .OH and DTNB. A similar increase in Po was observed by applying the physiological oxidizing molecule GSSG. Moreover, GSSG-oxidized channels showed enhanced sensitivity to Ca2+. The effect of GSSG was fully reversed by GSH. These results suggest an intracellular site(s) of action of oxidizing agents on cysteine targets on the fenamate-sensitive NSCC protein implicated in epithelial cell necrosis.