Hydroxyl radical activation of a Ca2+-sensitive nonselective cation channel involved in epithelial cell necrosis

In a previous work the involvement of a fenamate-sensitive Ca2+-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 Ca2+-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 (P-o) by similar to40% 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 P-o 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 P-o of both .OH and DTNB. A similar increase in P-o 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.