# Ion Channel Properties of a Cation Channelrhodopsin, \(Gt\_\)Ccr4
## 1 Introduction  
Microbial-type rhodopsins are made up of seven or eight transmembrane helices with a covalently bound all-_trans_ retinal as the chromophore [1]. They are found in archaea, bacteria, eukaryota (such as fungi and algae) and viruses, and are physiologically responsible for energy production and the phototaxis reaction. Molecular functions of microbial rhodopsin involve ion transporters, sensors and light-regulated enzymes. As for ion-transporting rhodopsins, they are divided into ion-pumps and channels. Bacteriorhodopsin (BR) was the first identified outward directed proton-pumping rhodopsin [2]. The discovery of a Cl\({}^{-}\) pump, an Na\({}^{+}\) pump and inward-directed proton pumps has been even until recently [3; 4; 5; 6]. Structure-based and spectroscopic studies, when combined with electrophysiology and molecular dynamics studies, revealed the detailed molecular mechanism of bacteriorhodopsin and other pumps. Channelrhodopsin-1 and -2 (_Cr_ChR1 and _Cr_ChR2_) from _Chlamydomonas reinhardtii_ were the first light-gated ion channels to be discovered [7; 8]. These homologous proteins permeate cations in which the permeability ratio of H\({}^{+}\), Na\({}^{+}\), and K\({}^{+}\) is 10\({}^{6}\), 1, and 0.5, respectively. High-resolution X-ray structures revealed details of their molecular architecture and provided insight into their photoactivation and ion conduction [9; 10]. Successful expression of _Cr_ChR2_ in neurons allowed the action potential to be manipulated by light, which opened up a new field of research, optogenetics [11; 12]. A number of variant molecules have been engineered to improve the functionality of ChR, and homologous ChRs were then reported [13]. Color-tuning variants cover almost the entire visible range. _Cr_ChR2_ displays an action spectrum maximum at 470 nm [8]. ChR variants such as C1V1, which is the chimeric version of ChR1 from _Chlamydomonas reinhardtii_ and _Volvox carteri_, or C1C2 (a green receiver) absorb light at around 530-545 nm [14; 15; 16]. Another red-shifted ChR, Chrimson from _Chlamydomonas noctigama_, exhibits an absorption maximum at 590 nm which allows reliable neuronal stimulation by light exceeds 600 nm [17]. On the other hand, TsChR or _Ps_ChR absorb a shorter wavelength, making it possible to excite neurons at 440 nm [18]. The lifetime of an open channel can be extended by mutations at C128 and D156 (DC pair) which form a hydrogen bond bridge in _Cr_ChR2_. Mutations at C128 to Thr, Ala and Ser slowed the kinetics of channel closing 200, 5000 and 10,000-fold respectively [19]. _Cr_ChR2_ D156C displayed an even stronger effect, namely higher light sensitivity and prolonged lifetime of the open channel, by as much as 30 min [20]. Converting ion selectivity is challenging. The _Cr_ChR2_ L132C mutant showed improved Ca2+ permeability [21]. The permeability ratio between H\({}^{+}\) and Na\({}^{+}\) could be modified by a replacement at E143 to A in Chrimson [22]. Anion channelrhodopsins were engineered or discovered from nature and they have been applied as neuronal silencing tools [23; 24; 25]. The crystal structures of anion channelrhodopsins revealed their unique features related to the channel gating mechanism [26; 27; 28]. A novel cation channelrhodopsin family was reported in 2016 and 2017 from _Guillardia theta_, namely _Gt_CCR1-4 [29; 30]. These cation channelrhodopsins (CCRs) from cryptophyte algae are more homologous to haloarchaeal rhodopsins, such as proton pumping bacteriorhodopsin, than to chlorophyte CCRs, including _Cr_ChR2_. Actually, _Gt_CCRs conserve the characteristic amino-acid residues involved in unidirectional proton transfer, including the proton acceptor D85 and the proton donor D96 in bacteriorhodopsin (Table S1). On the other hand, a characteristic glutamic acid in TM2 (E90 in _Cr_ChR2_) which is crucial for channel gating and ion selectivity, is not conserved in _Gt_CCRs [23; 31]. _Cr_ChR2 possesses a so-called DC pair (C128 and D156 in _Cr_ChR2_), which is responsible for the channel life time [19; 32; 33].