# Ion Channel Properties of a Cation Channelrhodopsin, \(Gt\_\)Ccr4
## 4 Discussion  
In this study, we aimed to elucidate the ion-channel properties of a recently discovered light-gated cation channel \(Gt\_CCR4\) from a cryptophyte and compare it to well-known \(Cr\_ChR2\) from _Chlamydomonas reinhardtii_ by using an electrophysiological method. In ND7/23 cells, \(Gt\_CCR4\) showed a large current density (Figures 1C and 2A,B). Inactivation of the photocurrent obtained from \(Gt\_CCR4\) was smaller than in \(Cr\_ChR2\). In other words, a large current was observed under constant light (Figure 1A,B). This was also obvious in the current trace after illumination at a high frequency (Figure 4). These characteristics promise stable and reproducible stimulation of neuronal excitability by \(Gt\_CCR4\). The light sensitivity of \(Gt\_CCR4\) is higher than that of \(Cr\_ChR2\) with a particular steady state component (I\({}_{\text{ss}}\)) (Figure 1E,F). ChR variants with high light sensitivity have already been developed [19; 20], but those have a long channel life time with at least two orders of magnitude or even much longer. Therefore, these are inappropriate for high-frequency light stimulation. In contrast, \(Gt\_CCR4\) has a short open life time of 25-30 ms, which is about the same range as \(Cr\_ChR2\), i.e., 10-15 ms (Figure 1D). Together, \(Gt\_CCR4\) is light sensitive and useful as an optogenetics tool with high time resolution. Optical irradiation causes heat and elevates temperature by 0.2-2 \({}^{\circ}\)C, especially in cranial nerve experiments [37]. Moreover, it has been demonstrated that the rise in temperature suppressed neuronal spiking in multiple brain regions, serving as a warning of the use of strong light for neuronal stimulation. Such an undesirable artefact has to be avoided by lowering light intensity, while effective depolarization has to be stably maintained. \(Gt\_CCR4\) has the potential for overcoming this problem. H\({}^{+}\) permeability is high for \(Cr\_ChR2\)[8]. Permeability for Ca\({}^{2+}\) has been reported, and not only for monovalent cations such as Na\({}^{+}\) and K\({}^{+}\)[8; 21]. On the other hand, \(Gt\_CCR4\) showed high selectivity in monovalent metal cations and low H\({}^{+}\) permeability. The permeability of a divalent cation such as Ca\({}^{2+}\) seems to be very low or negligible. The position that is important to ion selectivity has been studied in \(Cr\_ChR2\). E90 in the central gate is crucial for cation/anion selection [23]. L132 in TM3 influences on Ca\({}^{2+}\) permeability [21]. [PERSON] and coworkers recently demonstrated that D156H and D156C mutation increase permeability for Na\({}^{+}\) and K\({}^{+}\)[38]. The outer gate in Chrimson (E139) on the extracellular side is important for Na\({}^{+}\) extrusion [22]. These key residues are not conserved in \(Gt\_CCR4\), implying that a different ion selection property resides in DTD channels. It would be necessary to study selectivity based on variant analysis and structural information in the future. Considering its application in optogenetics, \(Gt\_CCR4\) would not cause a significant change to pH in the cell membrane because of its very low H\({}^{+}\) permeability, which could be advantageous when an unknown effect by pH needs to be prevented. To enable optical stimulation without improper calcium signaling, \(Gt\_CCR4\) might work better than \(Cr\_ChR2\). A single turnover photocurrent of \(Gt\_CCR4\) by laser irradiation provided a time constant (\(\tau_{\text{off}}\)) of 15-20 ms, which is smaller than that obtained by constant light (25-30 ms). This suggests two processes for channel opening and shutting. A dual photocycle model was indeed proposed for \(Cr\_ChR2\)[39]. It is expected that a similar reaction is caused in \(Gt\_CCR4\), but more experiments are needed to prove this. We found a characteristic inactivation of channel activity by long wavelength absorption in \(Gt\_CCR4\) (Figure 5). Since inactivation lasted at least a few minutes, formation of a stable intermediate with long wavelength absorption is anticipated.