# Ion Channel Properties of a Cation Channelrhodopsin, \(Gt\_\)Ccr4
## 3 Results  
### Basic Characterization of Photocurrent  
We transiently expressed \(Gt\_CCR4\) and \(Cr\_ChR2\) in ND7/23 cells by a conventional transfection method (FuGENE). Expression of these channels was visualized by tagged-GFP or Venus fluorescence. Strong membrane expression was confirmed for both channels, although cytoplasmic GFP was observed in \(Gt\_CCR4\)-expressing cells (Figure 1A microscope images). We illuminated 530 nm light to induce a photocurrent for \(Gt\_CCR4\) and 470 nm light for \(Cr\_ChR2\) at the same light intensity (6.8 mW/mm\({}^{2}\)). A large photocurrent was recorded from the \(Gt\_CCR4\)-expressing cells, reproducing previous studies (Figure 1A, left) [30]. Current amplitude reached -2 nA at -60 mV. The current showed an initial peak component (I\({}_{\text{p}}\)) which decayed slightly into a steady state level (I\({}_{\text{ss}}\)). However, amplitude of the steady state still retained about 80% of the transient peak component. The photocurrent from \(Cr\_ChR2\) showed a large peak component which reached about -2 nA at -60 mV (Figure 1A, right). The current decayed by 50% of the initial peak, suggesting that \(Cr\_ChR2\) exhibits a markedly large inactivation compared to \(Gt\_CCR4\). Figure 1C depicts the current-voltage relationship of photocurrent from \(Gt\_CCR4\) and \(Cr\_ChR2\). Both peak component (I\({}_{\text{p}}\)) and steady state current (I\({}_{\text{ss}}\)) are plotted. The shape of the I-V plot from \(Gt\_CCR4\) indicates strong inward-rectification. I\({}_{\text{ss}}\) of \(Cr\_ChR2\) displayed similarly inward-rectification, while I\({}_{\text{p}}\) was weakly rectified, in which a markedly outward-directed current was observed at positive membrane voltages. For both I\({}_{\text{p}}\) and I\({}_{\text{ss}}\), \(Gt\_CCR4\) showed a larger current density (pA/pF) than \(Cr\_ChR2\). For example, the photocurrent (I\({}_{\text{ss}}\)) of \(Gt\_CCR4\) at -100 mV exceeded -80 pA/pF, while that of \(Cr\_ChR2\) was only about -40 pA/pF.  
Kinetics in the photocurrent decay after shutting off the light is shown in Figure 1D. The time constant of \(Gt\_CCR4\) is about 25-35 ms under a membrane voltage between -100 and 80 mV, while \(Cr\_ChR2\) showed faster kinetics by about 10-20 ms. Next, we compared light sensitivity in the photocurrent of two channels (Figure 1E,F). The photocurrent amplitude from \(Cr\_ChR2\) grows as a typical sigmoidal curve for both the initial peak and the steady state components (Figure 1F). EC\({}_{50}\) was determined as 0.8 mW/mm\({}^{2}\) for I\({}_{\text{p}}\) and 0.35 mW/mm\({}^{2}\) for I\({}_{\text{ss}}\) under the conditions tested. On the other hand, the \(Gt\_CCR4\) current showed unique growth in terms of power dependency with two apparent phases in which the current first saturated at 0.1 mW/mm\({}^{2}\) at about 50% of full activation, followed by the second phase of growth from 1 to 10 mW/mm\({}^{2}\) (Figure 1E). The EC\({}_{50}\) was determined as 0.13 mW/mm\({}^{2}\) for I\({}_{\text{p}}\) and 0.18 mW/mm\({}^{2}\) for I\({}_{\text{ss}}\). These results indicate that \(Gt\_CCR4\) is more sensitive to light with respect to channel activation.