# Mutated Channelrhodopsins with Increased Sodium and Calcium Permeability
## 3 Results  
Mutating the Conserved Aspartate of TM Helix 4 Influences the Expression, Photocurrent, and Kinetics  
Similar to previously published results [18; 19], fluorescence measurements of whole oocyte membranes with YFP-tagged XXM and XXL showed a \(\sim\) three-fold increased expression level, compared to ChR2 (Figure 1). The steady-state photocurrents were increased \(\sim\)30- and \(\sim\)48-fold for XXM (D156H) and XXL (D156C), respectively, compared to ChR2 (Figure 1 and Table 1). The enhanced photocurrent might have been a comprehensive outcome of the higher plasma membrane expression level, higher light-sensitivity, or increased single channel conductance. Both XXM and XXL showed much-prolonged closing kinetics, leading to higher light-sensitivity (Figure 1a and Table 1). Figure 1: Comparison of ChR2, _Ps_ChR, and Chronos variants. (**a**) Representative photocurrent traces of ChR2, _Ps_ChR, Chronos, and their mutants. (**b**) Comparison of stationary photocurrents of above channelrhodopsin variants. All data points were plotted in the figure and mean \(\pm\) standard error mean (SEM) (\(n=7\)–\(8\)) was indicated. Figure 2: Comparing the light sensitivities of ChR2, _Ps_ChR, and Chronos variants. (**a**) 5 s, 30 s, and 100 s continuous 473 nm blue light illumination were applied to ChR2, XXM and XXL, respectively. Photocurrent at maximum light power density of each oocyte was normalized as 1. (**b**) 5 s continuous 532 nm blue light illumination were applied to Chronos, Chronos D173H, and Chronos D173C. Photocurrent at maximum light power density of each oocyte was normalized as 1. (**c**) 5 s, 30 s, and 100 s continuous 445 nm blue light illumination were applied to _Ps_ChR, _Ps_ChR D139H, and _Ps_ChR D139C. Photocurrent at maximum light power density of each oocyte was normalized as 1. Photocurrents of XXM, XXL, _Ps_ChR D139H, and _Ps_ChR D139C were measured at \(-60\) mV because of the larger current and slower kinetics; other constructs were measured at \(-100\) mV. Data points were presented as mean \(\pm\) SD, \(n=3\)–\(4\). We further synthesized Chronos [9] and _Ps_ChR [20] and characterized the corresponding aspartate to cysteine, and the histidine mutations as the aspartate in transmembrane helix 4 (TM4) were conserved in all three channel-hodopsins (Appendix B). Chronos D173C, D173H, and _Ps_ChR D139H, D139C, all showed an increased expression level, compared to their wild type (Figure 1a). All mutants also showed increased light-sensitivities along with prolonged off kinetics (Figure 2 and Table 1). Chronos D173C, _Ps_ChR D139H and _Ps_ChR D139C showed dramatically increased photocurrents while the Chronos D173H was similar to the wild type Chronos (Figure 1 and Table 1). Among these variants, _Ps_ChR D139C was the most light-sensitive with an effective light power density (LPD) for 50% photocurrent (EPD\({}_{50}\)) of \(\sim\) 3.2 \(\mu\)W/mm\({}^{2}\), which was \(\sim\) 250 times more sensitive than _Ps_ChR (Figure 2 and Table 1). The EPD\({}_{50}\) for XXL was \(\sim\) 5.4 \(\mu\)W/mm\({}^{2}\), which was \(\sim\) 130 times more sensitive than ChR2 (Figure 2 and Table 1). Figure 4: Comparison of K\({}^{+}\) and H\({}^{+}\) permeabilities of ChR2, Chronos, _Ps_ChR, and their mutants. Reversal potentials were determined after photocurrent measurements from \(-\)90 mV to +10 mV. Reversal potential shift for K\({}^{+}\) was calculated by the reversal potential differences in two outside buffers containing 120 mM KCl pH 7.6 and 1 mM KCl pH 7.6. Reversal potential shift for H\({}^{+}\) was determined by the reversal potential differences in two outside buffers of pH 7.6 and 9.6 containing 120 mM KCl. Data points were presented as mean \(\pm\) SD, \(n\) = 4–6. Figure 3: Comparison of Na\({}^{+}\) and H\({}^{+}\) permeabilities of ChR2, Chronos, _Ps_ChR, and their mutants. Reversal potentials (V\({}_{r}\)) were determined after photocurrent measurements from \(-\)90 mV to + 10 mV.