FRET response of Rv-Mermaid^D129E/Y235R to changes in t-tubule membrane voltage applied from 0 mV. (A) Schematic structure of Rv-Mermaid^D129E/Y235R and its confocal triadic pattern in muscle fibers. (B) Changes in mUKG (F₅₀₀, green) and mKOκ (F_>560, red) fluorescence in response to the indicated voltage protocol. Insets show the corresponding raw records before correction for voltage-independent changes in fluorescence; the fit used for correction is shown in white. (C) Changes in the FRET ratio (F_>560/F₅₀₀) elicited in response to the pulse protocols shown at top. The black trace was from the fluorescent responses shown in B. (D) Mean voltage dependence of the FRET response measured with voltage pulses from 0 mV (n = 6). FRET ratio values were normalized to the value at 0 mV (R₀). (E) Mean voltage dependence of the time constant of change in FRET ratio at the onset (τ_on) and offset (τ_off) of the pulses. Error bars represent ± SEM.