Figure 2
Kinetics, light sensitivity and control of eACRs. (A) Normalized photocurrent traces after light shutoff are displayed for fast cycling eACRs and their respective C128A step-function variants logarithmically binned to 50 data points per decade. (B) Light titration of eACRs and eACR C128A variants. Stationary photocurrents are normalized to the maximum (n = 5 Phobos, 4 PhobosCA, 7 iC++, 6 iC++CA, 5 Aurora, 5 Aurora 590 nm, 4 AuroraCA, 5 iChloC, 4 iChloCCA). 12 s light pulses were used for C128A variants to reach stationary photocurrents. (C) Light-activation ED50 values of fast cycling eACRs (WT) and their C128A variants (CA) obtained from fitted measurements shown in (B). (D) Example recording demonstrating the strategy to determine wavelength dependent inactivation for step-function eACRs. After fully opening the channel with a 10 ms light pulse at the peak activating wavelength, light with different wavelengths at identical photon irradiance was applied to accelerate channel closing. The eACR was fully closed with high-intensity red-shifted light after each trial. (E) Inactivation spectra calculated from measurements as shown in (D). Positive values show additional activation, whereas negative values denote inactivation (n = 4 PhobosCA, 6 iC++CA, 5 AuroraCA, 5 iChloCCA). Wavelengths yielding maximal activation and inactivation are displayed for each eACR C128A above or under the curves, respectively. (F) Apparent closing kinetic time constants (τoff) of fast cycling eACRs (WT), their C128A variants (CA) and the accelerated closing by application of red-shifted light. (n = 6 Phobos, 4 PhobosCA, 5 PhobosCA accelerated, 8 iC++, 7 iC++CA, 7 iC++CA accelerated, 8 Aurora, 5 AuroraCA, 5 AuroraCA accelerated, 7 iChloC, 5 iChloCCA, 8 iChloCCA accelerated). (G) ED50 vs. closing time constant of eACR variants linearly correlates during extended illumination of C128A variants (12 s) except for iChloC that shows a higher sensitivity compared to other eACRs with respect to its closing time constant. Fitting statistics are shown in the figure panel. (H) Typical photocurrent traces of the newly developed step-function eACRs PhobosCA, iChloCCA and AuroraCA compared to the established step-function eACR iC++CA (alias SwiChR++), activated by short 10 ms light pulses. Channel closing was always facilitated with red-shifted light. Mean values ± SEM are shown (B, C, E, F and G) together with single measurement data points (dots, C and F).
