# Mutated Channelrhodopsins with Increased Sodium and Calcium Permeability
## 1 Introduction  
Channelrhodopsins were first discovered and characterized from _C. reinhardtii_[1; 2]. After the showing of light-switched large passive cation conductance in HEK293 and BHK cells by [PERSON] et al., the ChR2 (_C. reinhardtii_ channelrhodopsin-2) was immediately applied in neuroscience by several independent groups for studies in hippocampal neurons [3; 4], _Caenorhabditis elegans_[5], inner retinal neurons [6], and PC12 cells [7]. H134R (histidine to arginine mutation at position 134) was the first ChR2 gain-of-function mutant which showed enhanced plasma membrane expression and larger stationary photocurrents in comparison to ChR2 wild type [5]. Other variants came out in rapid sequence, either of natural origin or mutated and engineered. The calcium translocating channelrhodopsin CatCh (a ChR2 leucine to cysteine mutation at position 132, L132C) showed improved Ca2+ conductivity together with a larger photocurrent and higher light sensitivity [8]. Newly discovered Chronos (_Stigecolonium helevicticum_ channelrhodopsin = _Sh_ChR) and Chrimson (_Cr_ChR1 from _Chlamydomonas nc ncotiana_) showed a faster channel closing and a red-shifted action spectrum, respectively [9]. An E90R (glutamate to arginine mutation at position 90)point mutation could extend the cation conductance of ChR2 to additional anion conductance [10]. Naturally, very specific anion conductive channelrhodopsins, _G_/ACR1 and _G_/ACR2 (_Guillardia theta_ anion channelrhodopsin 1 and 2), were discovered afterwards [11]. Mutation of ChR2 C128 (cysteine at position 128) to threonine (T), alanine (A), and serine (S) slowed the closing kinetics dramatically [12; 13; 14]. Mutation of ChR2 D156 (cysteine at position 156) to alanine also decreased the closing kinetics [14]. Spectral studies suggested that a putative hydrogen bond between C128 and D156 could be an important structural determinant of the channel's closing reaction [14] or might represent the valve of the channel [15]. Thus, hydrogen bond-linked D156 and C128 was proposed as the putative gate buried in the membrane (\"DC gate\") [14; 15]. But the first channelrhodopsin structure was a chimaera (C1C2) of truncated ChR1 and ChR2, and the distances between C167 (corresponding to C128 in ChR2) and D195 (corresponding to D156 in ChR2) are too far away to be associated by a hydrogen bond [16]. However, the recently solved structure of wild type ChR2 revealed a water molecule between C128 and D156 to bridge them, indeed, by hydrogen bonds [17]. Mutation of the DC gate has a strong effect on the open channel lifetime. The ChR2 D156C (aspartate to cysteine mutation at position 134) mutant (XXL) generated very large photocurrents and is 1000-fold more light-sensitive than wild type ChR2 in _Drosophila_ larvae [18]. The ChR2 D156H (aspartate to histidine mutation at position 134) mutant (XXM) also showed a superior photo stimulation efficiency with faster kinetics than XXL, which made it an ideal optogenetic tool for _Drosophila_ neurobiological studies [19]. The aspartate D156 in ChR2 is located close to the protonated retinal Schiff base (RSBH+). Thus, mutations of D156 logically have strong effects on the open channel lifetime by influencing the protonation state of the retinal Schiff base. However, the water-bridged C128 and D156 are not in the putative ion pore proposed by [PERSON] et al. [17]. And no attention had been paid to the potential changes of ion selectivity by DC gate mutations. In this study, we compared the ion selectivity of our previously published XXL and XXM and found that XXM showed a four-fold increased Na\({}^{+}\) selectivity over H\({}^{+}\) together with a two-fold increased K\({}^{+}\) selectivity over H\({}^{+}\), compared to wild type ChR2. Based on this finding, we made further aspartate to histidine and cysteine mutations of _Ps_ChR (_Platformons subcordiformis_ channelrhodopsin) [20] and Chronos [9].