FIGURE 4.
Cytoprotective effects by SK2 channel activation in neuronal HT-22 cells. A, cell impedance was detected after incubation of HT-22 cells with CyPPA (1–50 μm) and glutamate (3 mm at 0 h; n = 8). B, xCELLigence real time impedance-based measurements of cells treated with CyPPA at different time points following glutamate application. The time point of treatment is marked as “0 h” in the graph (n = 8). C, MTT analysis of cells treated with CyPPA (25 μm) 0, 1, 3, 5, and 7 h after the application of glutamate. (*, p < 0.05; ***, p < 0.001 versus glutamate-treated neurons; ANOVA and Scheffé's test; n = 6). D and E, HT-22 cells were transfected with a 50 μm concentration of specific inhibitory peptides for SK2/KCa2.2 channels before exposure to CyPPA (5–10 μm) and glutamate (3 mm; n = 8). Cell impedance (D) and MTT assays (measured at 14 h after glutamate application) (E) show that only SK2/KCa2.2 inhibitory peptides reduce the protective effect of CyPPA (***, p < 0.001 versus CyPPA and glutamate treatment in non-transfected cells; ANOVA and Scheffé's test; n = 6). F, non-transfected and siRNA-transfected HT-22 cells targeting SK2/KCa2.2 channels were treated with CyPPA (5 μm) and challenged with glutamate (3 mm). Morphological alterations were detected by a real time impedance-based system. G, HT-22 cells were transfected with specific inhibitory peptides for SK1/KCa2.1 (G) and SK3/KCa2.3 (H) channels (50 μm). Afterward, transfected cells were treated with 5 μm CyPPA for SK1/KCa2.1 channels (G) and 10 μm CyPPA for SK3/KCa2.3 channels (H). Glutamate was applied together with CyPPA, and the cellular index was measured using the xCELLigence system (n = 8). Error bars represent S.D. inh, inhibitory; Ctrl, control.