Detection of t-tubule voltage changes with Mermaid and Rv-Mermaid in muscle fibers. (A and D) Confocal pattern of the green fluorescence in muscle fibers expressing Mermaid and Rv-Mermaid, respectively. In each panel, the frame and graph on the right show a higher-magnification view together with the longitudinal profile of fluorescence within the white box. Expression of Mermaid and Rv-Mermaid yielded a transverse-banded pattern consistent with the proteins being present in the triadic region of the fibers. A schematic illustration of the structure of the proteins is shown at top. (B and E) Changes in mUKG (F₅₀₀, green) and mKOκ (F_>560, red) fluorescence and corresponding FRET ratio in response to the voltage-clamp protocol shown at top, from muscle fibers expressing Mermaid and Rv-Mermaid, respectively. In response to the voltage-clamp depolarizing steps from −80 mV, the detected changes in fluorescence (at 500 nm and >560 nm, upon 458-nm excitation) were consistent with voltage-dependent energy transfer between the two fluorophores mUKG and mKOκ, providing evidence for localization of Mermaid and Rv-Mermaid in the t-tubule membrane of the muscle fibers. Main traces in B and E have been corrected from time-dependent changes in fluorescence also observed when no voltage pulse was given. Raw fluorescence traces and superimposed fits used for the correction are shown in the insets. Details of how fits were generated and correction performed are described in Materials and methods. (C) Changes in Mermaid FRET ratio in response to depolarizing (left, black trace) and hyperpolarizing (right, blue trace) steps from −80 and 0 mV, respectively, in the same fiber. The graph at bottom shows the mean voltage dependence of the change in FRET ratio established from three fibers stimulated with a series of successive voltage-clamp depolarizing steps from −80 mV (black circles) and then with a series of successive hyperpolarizing steps from 0 mV (blue circles). FRET ratio values were normalized to the value R₀ at −80 mV. Step hyperpolarizing pulses from 0 mV generated symmetrical changes in the FRET ratio, with no detectable sign of shift in the voltage sensitivity. (F) Mean voltage dependence of the FRET response of Mermaid (n = 6) and Rv-Mermaid (n = 6) established using depolarizing steps from −80 mV. Error bars represent ± SEM.