Hydrogen peroxide activates glibenclamide-sensitive K⁺ channels in LLC- PK₁ 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-PK₁ cell line. Exposure of cells to H₂O₂ (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 H₂O₂-induced cellular hyperpolarization. H₂O₂ (5 mM) produced a sixfold increase in the whole cell conductance. The reversal potential of the H₂O₂-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-PK₁ cells to 5 mM H₂O₂ 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 H₂O₂-induced activation of the K⁺ conductance. These findings indicate that H₂O₂ activates an ATP-sensitive, Ca²⁺-independent K⁺ conductance in LLC-PK₁ cells.