## 1 Introduction  
Channelrhodopsins (ChRs) are members of the group of microbial rhodopsins that are light-gated cation channels. They were originally found in the eyespot of the algae _Chlamydomonas reinhardtii_ (Cr),where they serve to identify optimal light conditions during phototactic movement [1; 2]. Typically for rhodopsins, ChRs consist of seven \(\alpha\)-helices spanning the membrane and a retinylidene chromophore, which is bound via a Schiff base to a lysine in the core of the protein. Within the new research field of optogenetics, the coding sequences for light-responsive proteins together with regulating promoters can be introduced into complex organisms and expressed in a tissue-specific way, where cell processes can be triggered. Channelrhodopsin-2 (\(C\)\(\tau\)ChR2) in neurons can depolarize the nerve cell when illuminated by light of the corresponding wavelength. Although this protein is already frequently used as optogenetic tool, aimed at medical approaches like restoring vision and hearing, the mechanism of action is still not completely understood. Comparison to related microbial rhodopsins where individual positions in the protein sequences are highly conserved could guide our approach towards mechanistically important amino acid residues which have demonstrated effects on photocycle kinetics, activity, and/or structural stability upon mutation. Decades ago, the [PERSON] group investigated the role of threonines in the proton pump bacteriorhodopsin (bR) from _Halobacterium salinarum_. In that study, the T89V variant exhibited a blueshift of 146 nm of the visible absorption spectrum without impairing proton pumping [3]. It was concluded from FTIR and time-resolved absorption spectroscopy in the visible range that T89 exerts steric constraints during isomerization of the chromophore and is part of a hydrogen-bonded network including a water molecule and the residues Y185 and D212 [4]. Years later, the same group demonstrated that the hydroxyl group of T89 interacts with the protonated Schiff base in light-adapted bR and influences proton transfer to D85 during the photocycle [5]. However, from X-ray structures [6; 7], it became apparent that the OH group of T89 forms a hydrogen bond to an oxygen of the side chain of D85 rather than to the Schiff base. It was concluded from polarized FTIR spectroscopy that the isomerization of the chromophore after light excitation leads to a shortening of the hydrogen bond between T89 and D85, indicating an interconnection to the Schiff base [8]. This tight complex persists from the K intermediate until the M intermediate [9]. The hydroxyl amino acid side chain of T89 of bR is conserved in many channelrhodopsins. The corresponding residue T127 of \(C\)\(\tau\)ChR2 is located in immediate vicinity to the retinal Schiff base (Figure 1), with a distance of 3.7 A of the threonine oxygen to the Schiff base nitrogen in the crystal structure. Other channelrhodopsins carry a serine [10] or a cysteine at this position. The latter appears in \(C\)\(\alpha\)ChR1 (from _Chlamydomonas augustae_) and \(C\)\(\gamma\)ChR1 (from _Chlamydomonas yellowstonensis_) [11]. Even anion-conducting channelrhodopsins, which do not conserve E123, have a threonine or a cysteine at this position [12]. Figure 1: X-ray crystallographic structure of _Chlamydomonas reinhardtii_ channelrhodopsin-2 (\(C\)\(\tau\)ChR2) (PDB: 6 EID [10]). The protonated Schiff base of retinal and the side chains of key residues are shown as grey and yellow sticks, respectively. Water molecules are shown as red spheres. Dashed lines indicate hydrogen-bonding interaction. In CrChR2, the residue T127 is connected via a hydrogen bond to E123, the homologous residue of D85 in bR [10]. The protonation state of E123 is not clear, but nearby ionized D253 serves as the acceptor of the Schiff base proton during the rise of the P\({}_{2}\)[390] intermediate [13]. D156 has been shown to interact via hydrogen bonding with C128, denoted as the DC gate [14].