Mechanism of inhibitory actions of oxidizing agents on calcium-activated potassium current in cultured pigment epithelial cells of the human retina

PURPOSE. To identify the mechanisms by which oxidative stress with oxidizing agents alters the activity of ion channels in human retinal pigment epithelial (RPE) cells. METHODS. The effects of oxidizing agents on ion currents were investigated in human RPE R-50 cells with the aid of the whole-cell, cell-attached, and inside-out configurations of the patch-clamp technique. RESULTS. In the whole-cell configuration, t-butyl hydroperoxide (t-BHP; 1 mM), thimerosal (30 μM), and 4,4′-dithiodipyridine (DTDP; 30 μM) suppressed voltage-dependent K⁺ current (I_K) that was sensitive to inhibition by iberiotoxin or paxillin, yet not by apamin or 5-hydroxydecanoate sodium. Meclofenamic acid or Evans blue, but not diazoxide, reversed the decrease in I_K caused by t-BHP. In cells dialyzed with ceramide (30 μM), neither t-BHP (1 mM) nor thimerosal (30 μM) had any effect on IK, whereas DTDP (30 μM) slightly suppressed it. In cell-attached recordings, t-BHP (1 mM), thimerosal (30 μM), and DTDP (30 μM) suppressed the activity of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels. Dithiothreitol (10 μM) reversed DTDP-induced decrease in channel activity. Under current-clamp conditions, cell exposure to oxidizing reagents caused membrane depolarization. In cells dialyzed with ceramide (30 μM), membrane potential remained unaltered in the presence of t-BHP. CONCLUSIONS. The results demonstrate that hydrophilic oxidants (e.g., t-BHP and thimerosal) suppress I_K and suggest that the underlying mechanism of this inhibitory action may involve the generation of intracellular ceramide. However, the inhibition of BK_Ca, channels by DTDP, a membrane-permeable oxidant, in human RPE cells may result from the direct inhibition of BK_Ca channels and indirectly from an increase in the intracellular production of ceramide.