Fig. 2.
Voltage-dependence of channel kinetics and spectral properties of ChR2 mutants. (A) Photocurrents after laser flash activation (10 ns; green arrow) measured at −50 mV, normalized to the peak. ET/HR and ET/TC were significantly faster than wt ChR2, whereas HR and TC were significantly slower.***P < 0.005. (B) Photocurrents after laser flash activation at +50 mV. TC, HC, and wt ChR2 slowed down considerably at this membrane potential, whereas ET/HR and ET/TC retained their fast kinetics. (C) To quantify the voltage-dependence of channel kinetics, flash-to-peak and τoff were analyzed at different membrane potentials (n = 10 cells for each mutant). (D) Time-dependent recovery of peak currents (Ipeak) was measured under physiological conditions at −75 mV in oocytes. Recovery was defined as the ratio of ΔI2 (Ipeak2 − Istationary2) to ΔI1 (Ipeak1 − Istationary1) and plotted against the interpulse interval. Recovery time constants (τrec) of HR (green; n = 3) and ET/TC (blue; n = 3) were significantly faster than wt ChR2 (black; n = 15), whereas TC (magenta; n = 3) was slower. ***P < 0.005; *P < 0.05. (E) IV curves show that the typical inward rectification of wt ChR2 (black) is retained in all mutants. Reversal potentials (Vreversal) were close to zero under physiological conditions (Inset; n = 10, 10, 12, 8). (F) Action spectra measured in hippocampal neurons show red-shifted wavelength optimum of ET/TC (blue curve, n = 13) relative to HR (green curve, n = 10) and TC (red curve, n = 11).