FRET response of T306-Rv-Mermaid^D129E/Y235R during Ca²⁺ release. (A) Patchy expression (regions pointed by green arrows) and triadic confocal pattern (bottom image and associated graph showing the longitudinal fluorescence profile along the white box) of T306-Rv-Mermaid^D129E/Y235R in muscle fibers. Schematic structure of the protein is shown at top. (B) Changes in T306-Rv-Mermaid^D129E/Y235R FRET ratio in response to the indicated voltage-clamp depolarizing pulses. Inset shows the fluorescent traces in response to the pulse from −80 to −10 mV. (C) Mean voltage dependence of the peak amplitude of the drop in FRET ratio measured in response to single 0.5-s-long pulses from −80 mV. For this, successive single depolarizing pulses of increasing amplitude were applied, separated by a time interval of 30 s. Data points are mean values from several fibers ranging from a minimum of seven and a maximum of 13. (D) Mean voltage dependence of the time constant of change in FRET ratio at the onset (τ_on) of the depolarizing pulses and upon return (τ_off) to the holding value of −80 mV. (E) Acceptor photobleaching of the resting fluorescence from a fiber expressing Rv-Mermaid^D129E/Y235R: x,y fluorescence frames after photobleaching of a square region in the middle of the fiber with excitation light at 543 nm. Bar, 20 µm. (F) Effect of acceptor photobleaching (postbleach) on the changes in fluorescence detected in response to a depolarizing pulse from −80 to 0 mV in FRET configuration (458-nm excitation) from a fiber expressing T306-Rv-Mermaid^D129E/Y235R. (G) Mean values for the resting fluorescence ratio (left) and the relative changes in F₅₀₀ and F_>560 fluorescence (right) triggered by a strong depolarizing pulse, from five fibers expressing T306-Rv-Mermaid^D129E/Y235R. Error bars represent ± SEM.