# Ion Channel Properties of a Cation Channelrhodopsin, \(Gt\_\)Ccr4
## 3 Results
### Ion Selectivity  
It was already reported that \(Gt\_CCR\)s are H\({}^{+}\)- and Na\({}^{+}\)-permeable cation channels [29; 30]. We here investigated the cation selectivity of \(Gt\_CCR4\) in more detail relative to \(Cr\_ChR2\). Ionic conditions of the extracellular solution were systematically exchanged with various cations including Na\({}^{+}\), K\({}^{+}\), Cs\({}^{+}\), Ca\({}^{2+}\), Mg\({}^{2+}\) and NMG. In the presence of NMG, one can assume H\({}^{+}\) as the permeated ion. Figure 2A,B show the I-V plot of \(Gt\_CCR4\) and \(Cr\_ChR2\) under several ionic conditions. Obviously, the reversal potential shift in Na\({}^{+}\) solution is larger in \(Gt\_CCR4\) than in \(Cr\_ChR2\), suggesting that \(Gt\_CCR4\) is more permeable to Na\({}^{+}\) than \(Cr\_ChR2\). Notably, I-V plots of \(Gt\_CCR4\) at pH 6.85 and 9.0 in NMG are almost identical (Figure 2A) whereas a large shift of reversal potential was observed in \(Cr\_ChR2\) under the same conditions (Figure 2B). These results indicate that \(Gt\_CCR4\) has less H\({}^{+}\) selectivity than \(Cr\_ChR2\). The photocurrent amplitude of \(Gt\_CCR4\) and \(Cr\_ChR2\) at -60 mV under each condition is summarized in Figure 2C,D. The current amplitude of \(Gt\_CCR4\) was significantly larger in the presence of Na\({}^{+}\) and K\({}^{+}\) close to -100 pA/pF and in the presence of Cs\({}^{+}\) at about -40 pA/pF. In contrast, only a negligible current was observed at low pH, or in the presence of Ca\({}^{2+}\) or Mg\({}^{2+}\). This supports the notion that \(Gt\_CCR4\) is more of a monovalent metal cation selective channel. In addition, we also tested measurements under a competitive environment in which both Na\({}^{+}\) and Ca\({}^{2+}\) were both added to bath solutions (Figure S1). Interestingly, photocurrents by \(Gt\_CCR4\) were suppressed ata higher Ca2+ concentration (40 mM), suggesting that Na+ flow is blocked by Ca2+. In contrast, such a large difference in current amplitude was not observed under various conditions in the photocurrent from _Cr_ChR2_ (Figure 2D). This is due to low ion selectivity in _Cr_ChR2_ as was reported previously. To assume the selectivity ratio, the reversal potential shift from the condition with NMG at pH 9.0 is depicted in Figure 2E. The shifts (\(\Delta\)Erev) in _Gt_CCR4_ are larger than in _Cr_ChR2_ for Na+, K+, and Cs+ indicating that _Gt_CCR4_ is selective for monovalent cations but less selective for H+. In the initial study of _Cr_ChR2_, the permeability ratio was calculated based on the current amplitude [8]. The table in Figure 2F summarizes the ratio both for _Gt_CCR4_ and _Cr_ChR2_. H+ permeability for _Cr_ChR2_ is \(0.77\times 10^{5}\), close to the reported value (\(1\times 10^{6}\)), while that for _Gt_CCR4_ is \(2.1\times 10^{4}\), indicating about 37-fold less permeability for H+ in _Gt_CCR4_.  
Figure 2: Ion selectivity of _Gt_CCR4_ and _Cr_ChR2_. Each channel-hodopsin expressed in ND7/23 cells was stimulated by green and blue LED light. I-V plot of _Gt_CCR4_ (**A**) and _Cr_ChR2_ (**B**) are depicted. Steady-state current density (pA/pF) in 20 mV steps from \(-\)100 mV to \(+\)80 mV was plotted. The present liquid junction potential was considered. The pipette solution contained 110 mM NMG-Cl at pH 9.0, and the bath solution varied: black, NMG-Cl at pH 9.0; red, NMG-Cl at pH 6.85; green, NaCl at pH 9.0; blue, KCl at pH 9.0; grey, CsCl at pH 9.0. See “Materials and Methods” for details about the solutions. (**C**,**D**) Comparison of current density of _Gt_CCR4_ (**C**) and _Cr_ChR2_ (**D**) in the presence of various cations at \(-\)60 mV. (**E**) Reversal potential shift (\(\Delta\)Erev) for each condition for _Gt_CCR4_ and _Cr_ChR2_. Erev was determined from the I-V plot shown in **A**,**B**. Each Erev value was subtracted from the Erev at NMG-Cl at pH 9.0. (**F**) Permeability ratio for H+ and Na+ in _Gt_CCR4_ and _Cr_ChR2_ as estimated from current value and ionic concentration. (n = 6–9 cells).