Figure 5. A photochemical interpretation of the observed fluorescence deflections. (A) A representative fluorescence trace recorded upon a 50-ms depolarization to 60 mV from channels labeled with MTS-TAMRA at position 135, at the S1 extracellular flank, in the presence of the native W203 (S4), as in Figs. 2 and 4 (A–D). At rest, the fluorophore is efficiently quenched by the Trp at S4 (W203), so that the reported fluorescence is relatively dim. Upon depolarization, the quenching efficiency of W203 decreases, so that overall fluorescence emission is increased and a positive ΔF is observed. The same interpretation was previously applied for TMRM fluorophore labeling the extracellular portion of S0 (Pantazis et al., 2010b) and can also account for the ΔF signals from TMRM-labeling S2, as their amplitude is also strongly attenuated by mutation W203V (Fig. 3). (B) A representative fluorescence trace recorded upon a 50-ms depolarization to 60 mV from channels labeled with MTS-TAMRA at position 135, at the S1 extracellular flank, in the presence of the native W203 (S4) and introduced W141 (near S2; Fig. 4, E–H). At rest, W203 quenches the fluorophore (as in A), whereas W141 quenches the fluorophore relatively less, so that the overall fluorescence detected is at an intermediate level. Upon depolarization, W203 quenching is lifted, generating positive ΔF, as in A. At the same time, W141 approaches and quenches the fluorophore. It seems that the W141 is a more efficient quencher than W203, as the overall fluorescence level at the depolarized state is dimmer than when at rest. However, W203 departure appears to have faster kinetics than W141 quenching, so it is observed as an upward transient deflection. Upon membrane repolarization, the two quenching processes are reversed: W203 returns to quench the label, so that overall fluorescence becomes transiently dimmer, whereas W141 departs, reducing its quenching efficiency. (C) A representative fluorescence trace recorded upon a 50-ms depolarization to 60 mV from channels labeled with MTS-TAMRA at position 135, at the S1 extracellular flank, in the presence of the introduced W141 (near S2), with additional mutation W203V to remove the native W203 (S4; as in Fig. 4, I–L). The fluorescent label is apparently unquenched at rest, as W203 has been substituted. Upon depolarization, W141 approaches and quenches the fluorophore, a process reported as a negative ΔF. Upon membrane repolarization, W141 departs, reducing its quenching efficiency. In agreement with the ΔF interpretation in A and B, the substitution of W203 resulted in the lack of observable depolarization-induced positive ΔF.
Figure 5.

A photochemical interpretation of the observed fluorescence deflections. (A) A representative fluorescence trace recorded upon a 50-ms depolarization to 60 mV from channels labeled with MTS-TAMRA at position 135, at the S1 extracellular flank, in the presence of the native W203 (S4), as in Figs. 2 and 4 (A–D). At rest, the fluorophore is efficiently quenched by the Trp at S4 (W203), so that the reported fluorescence is relatively dim. Upon depolarization, the quenching efficiency of W203 decreases, so that overall fluorescence emission is increased and a positive ΔF is observed. The same interpretation was previously applied for TMRM fluorophore labeling the extracellular portion of S0 (Pantazis et al., 2010b) and can also account for the ΔF signals from TMRM-labeling S2, as their amplitude is also strongly attenuated by mutation W203V (Fig. 3). (B) A representative fluorescence trace recorded upon a 50-ms depolarization to 60 mV from channels labeled with MTS-TAMRA at position 135, at the S1 extracellular flank, in the presence of the native W203 (S4) and introduced W141 (near S2; Fig. 4, E–H). At rest, W203 quenches the fluorophore (as in A), whereas W141 quenches the fluorophore relatively less, so that the overall fluorescence detected is at an intermediate level. Upon depolarization, W203 quenching is lifted, generating positive ΔF, as in A. At the same time, W141 approaches and quenches the fluorophore. It seems that the W141 is a more efficient quencher than W203, as the overall fluorescence level at the depolarized state is dimmer than when at rest. However, W203 departure appears to have faster kinetics than W141 quenching, so it is observed as an upward transient deflection. Upon membrane repolarization, the two quenching processes are reversed: W203 returns to quench the label, so that overall fluorescence becomes transiently dimmer, whereas W141 departs, reducing its quenching efficiency. (C) A representative fluorescence trace recorded upon a 50-ms depolarization to 60 mV from channels labeled with MTS-TAMRA at position 135, at the S1 extracellular flank, in the presence of the introduced W141 (near S2), with additional mutation W203V to remove the native W203 (S4; as in Fig. 4, I–L). The fluorescent label is apparently unquenched at rest, as W203 has been substituted. Upon depolarization, W141 approaches and quenches the fluorophore, a process reported as a negative ΔF. Upon membrane repolarization, W141 departs, reducing its quenching efficiency. In agreement with the ΔF interpretation in A and B, the substitution of W203 resulted in the lack of observable depolarization-induced positive ΔF.

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