High-frequency repetitive stimuli amplify the GxTX-594 response. (A) Fluorescence intensity from Kv2.1-CHO cells incubated in 9 nM GxTX-594. Arrow indicates voltage-clamped cell. Fluorescence at holding potential of −80 mV (left), after 50 s of 200-Hz stimulus (middle), and 100 s after the cell is returned to holding potential of −80 mV (right). Stimulus was a 2-ms step to +40 mV. Note that in each panel, the unclamped cell (left cell in each panel) does not show a change in fluorescence. Scale bar, 10 μm. (B) Representative trace with GxTX-594 unlabeling at 200 Hz. Red line, monoexponential fit (Eq. 1): k_ΔF = 0.2327 ± 0.0099 × 10⁻¹ s⁻¹. 0% F/F_init was set by subtraction of the average intensity of a region that did not contain cells. 100% was set by the initial fluorescence intensity at −80 mV. (C) Fluorescence–stimulus frequency relation. Points indicate F/F_init from individual cells. Top: Point coloring indicates data from the same cell. Bottom: Black bars represent the average F/F_init at each voltage, and error bars represent the SEM. Green line is the prediction of the EVAP model at a concentration of 9 nM. (D)k_ΔF–stimulus frequency relation. Plotted as in C.