Figure 4.

Direct application of H₂O₂ increases the KCa3.1 current through a CaMKII-mediated pathway. (A) Hydrogen peroxide elevates intracellular Ca²⁺ but does not directly activate KCa3.1 current in MLS-9 cells. Upper panel: Representative Fura-2 recording, in which 1 mM H₂O₂ was bath applied during the period marked by the horizontal bar. The inset shows calibrated free intracellular Ca²⁺ concentration as mean ± SEM, n = 18 cells (***p < 0.001, Student’s t-test). Lower panel: Representative current in a perforated-patch recording (same solutions and voltage protocols as Figure 1) with 1 mM H₂O₂ bath applied as indicated. (B) Representative KCa3.1 current traces in perforated-patch recordings, representative currents before and after adding 300 μM riluzole, with or without 1 μM TRAM-34. From top to bottom: control cell, cell pre-treated with 1 mM H₂O₂ for 10 min at room temperature, cell pre-treated with both 1 mM H₂O₂ and the ROS scavenger, MPG (500 μM; 10 min, room temperature), cell pre-treated with 1 mM H₂O₂, and the CaMKII inhibitor, mAIP (1 μM; 10 min, room temperature). (C) Summarized data from a population study with treatments as in panel (B). The TRAM-34-sensitive KCa3.1 current is expressed as mean ± SEM for the number of cells indicated on each bar and was compared using a one-way ANOVA with Tukey’s post hoc test; ****p < 0.0001.