Hydrogen peroxide activates glibenclamide-sensitive K ⁺ channels in LLC- PK ₁ cells

Oxidant-induced damage has been implicated in the pathogenesis of several forms of cellular injury. The present study employed patch-clamp methods to determine if oxidant stress leads to activation of plasma membrane K ⁺ channels in the renal epithelial LLC-PK ₁ cell line. Exposure of cells to H ₂O ₂ (0.1 to 5 mM) induced a rapid (within 5-10 min), dose-dependent membrane hyperpolarization. Perforated patch whole cell voltage-clamp studies were performed to determine the ion selectivity of the currents underlying this H ₂O ₂-induced cellular hyperpolarization. H ₂O ₂ (5 mM) produced a sixfold increase in the whole cell conductance. The reversal potential of the H ₂O ₂-induced current was consistent with a K ⁺-selective conductance. This current was blocked almost completely by 5 mM barium and 500 μM glibenclamide but only partially by 15 mM tetraethylammonium. Exposure of LLC-PK ₁ cells to 5 mM H ₂O ₂ reduced cell ATP content by 70%. To evaluate more directly the role of ATP depletion in the activation of K ⁺ channels, conventional whole cell patch-clamp studies were performed. Inclusion of ATP in the pipette solution prevented H ₂O ₂-induced activation of the K ⁺ conductance. These findings indicate that H ₂O ₂ activates an ATP-sensitive, Ca ²⁺-independent K ⁺ conductance in LLC-PK ₁ cells.