# Mutated Channelrhodopsins with Increased Sodium and Calcium Permeability
## 4 Discussion  
Channelrhodopsins, originating from different organisms, show quite different properties with respect to kinetics, action spectrum, and ion selectivity. Such changes can also be engineered by point mutations. In this study we compared the properties of ChR2, Chronos [9], \(Ps\)ChR [20], and their corresponding mutants of the aspartate in TM4 (DC gate aspartate). Generally, the aspartate to histidine or cysteine mutations of the three channelrhodopsins increased the expression level (probably because the mutant became more stable against degradation [21]) and slowed the closing kinetics. Nearly all mutants showed a much-increased photocurrent, probably because of a much-prolonged open state or enhanced single channel conductance, with only Chronos D173H as an exception. The tools with slowed kinetics are unfavorable for ultra-fast multiple stimulation but preferred for experiments which require low light and longtime stimulation. The prolonged open times were accompanied by elevated light sensitivities. Among the tested constructs, \(Ps\)ChR D139C and XXL became \(\sim\) 220 times and \(\sim\) 130 times more sensitive than ChR2. If slow closing would have not been a problem nor even desired, the more light-sensitive channelrhodopsins would have been ideal for efficient deep brain stimulation with infrared light via upconversion nanoparticles (UCNPs) [24]. These tools need to be further tested in mammalian systems for a broader field application. Furthermore, we investigated the influence of mutation of the aspartate in TM4 on ion selectivity. We found that aspartate to histidine mutation of ChR2 and \(Ps\)ChR increased the Na\({}^{+}\) and Ca\({}^{2+}\) permeability dramatically. To test the Ca\({}^{2+}\) current, we used BAPTA to block the Ca\({}^{2+}\)-activated endogenous chloride channels of oocytes. The fast Ca\({}^{2+}\) chelator BAPTA may have been altering the ion currents in more ways [25]. However, as we could see from the kinetics in Figure 5a that the C\({}^{+}\) current (which shows a slower off kinetics) was well-blocked. Then we could reliably compare only the photocurrent of our channelrhodopsins. With the large photocurrent, increased Na\({}^{+}\) permeability, and bigger Ca\({}^{2+}\) current, \(Ps\)ChR D139H is a novel powerful optogenetic tool for depolarization and Ca\({}^{2+}\) manipulation. Channelrhodopsins with higher Ca\({}^{2+}\) currents have the advantage of being \"direct\" light-gated Ca\({}^{2+}\) channels, in contrast to the highly Ca\({}^{2+}\)-conductive CNG (cyclic nucleotide-gated) channels which became light-gated channels when fused with bPAC (photoactivated adenylyl cyclase) [26]. In summary, we found that mutating the conserved aspartate in TM4 influenced not only the expression level and kinetics of channel closing but also the ion selectivity; with appropriate mutations, we provided novel optogenetic tools with superior photocurrent amplitudes and high Na\({}^{+}\) and Ca\({}^{2+}\) conductance. Conceptualization, S.G. and G.N. ; methodology, X.D., S.G. and G.N. ; software, X.D. and S.G.; validation, X.D, S.G. and G.N. ; formal analysis, X.D. and S.G.; investigation, X.D. and S.G.; resources, X.D., S.G. and G.N. ; data curation, X.D. and S.G.; writing--original draft preparation, S.G.; writing--review and editing, X.D., S.G. and G.N. ; visualization, X.D. and S.G.; supervision, S.G. and G.N. ; project administration, G.N. ; funding acquisition, G.N. This research was funded by grants from the German Research Foundation to GN (TRR 166/A03 and TR 240/A04). GN acknowledges support provided by the Prix-Louis-Jeantet. We are grateful to [PERSON] for help with some of the cloning work. This publication was funded by the German Research Foundation (DFG) and the University of Wuerzburg in the funding program Open Access Publishing. [PERSON]; [PERSON]; [PERSON]; [PERSON]; [PERSON] [PERSON]; [PERSON] [PERSON] Light activation of channel-hodopsin-2 in excitable cells of _Caenorhabditis elegans_ triggers rapid behavioral responses. _Curr Biol._**2005**, _15_, 2279-2284.