Figure 13.

Voltage-dependent excitation shift observed near the S2 segment. (A) This diagram illustrates how a theoretical shift in excitation spectrum can lead to a change in the direction of the fluorescence signal as a function of excitation wavelengths. At −90 mV, a theoretical excitation spectrum (dark trace, top panel) yields increased fluorescence emission when excited at wavelengths Δλ₂ compared with Δλ₁. At 0 mV, when a shift of the excitation spectrum to shorter wavelengths (to the left) occurs, as seen by the new excitation spectrum (dark trace, bottom panel), the relative fluorescence intensities at wavelengths Δλ₁ and Δλ₂ reverse. Thus, when exciting at Δλ₁ during a pulse from −90 to 0 mV, the fluorescence intensity would increase; when exciting at Δλ₂ during the same pulse, the fluorescence intensity would decrease. (B) Changes in fluorescence for construct D270C T449Y when exciting at two different wavelengths: 510–560 nm (top, average of 10 sweeps), and 453–487 nm (bottom, average of 25 sweeps). These changes were evoked in response to 40-ms pulses from −90 mV to potentials from −60 to 50 mV. Upward deflection of the trace indicates a decrease in fluorescence intensity (−ΔF). (C) Fluorescence change (−ΔF) at the end of a 40-ms pulse from −90 mV for the two different excitation wavelengths. Negative values indicate an increase in fluorescence. The curves correspond to excitation wavelengths 453–487 nm (•) and 510–560 nm (▵).