## 2 In This Special Issue
The advantages of this protein as an optogenetic tool comprise the red-shifted absorption maximum (530 nm), small desensitization during continuous illumination and the relatively high Na\({}^{+}\)/H\({}^{+}\) permeability ratio, as compared to ChR2 from the chlorophyte alga _Chlamydomonas reinhardtii_ (CrChR2). H\({}^{+}\) permeability of CCRs is a serious problem in some optogenetic experiments, as it may lead to a decrease in the cytoplasmic pH [12]. [PERSON] et al. show that the D156H mutation of CrChR2, and the corresponding mutations of the fast CCR variant Chronos [13] and blue-shifted ChR from _Platformous subcordiformis_ (_Ps_ChR) [14] enhanced relative permeabilities for Na\({}^{+}\) and Ca\({}^{2+}\), as compared to that for H\({}^{+}\). Moreover, in _Ps_ChR this mutation additionally increased the current amplitude, which made it the best currently available tool for optogenetic manipulation of the intracellular Ca\({}^{2+}\) level. Despite >50 native and the innumerable number of engineered CCR variants currently known, CrChR2 and its gain-of-function H134R mutant so far have remained the most frequently used optogenetic excitatory tools [15]. Two articles in this Special Issue report mechanistic studies on _Cr_ChR2,the results of which might contribute to further improvement of this tool for optogenetic needs. [PERSON] et al. probe the role of residual hydrophobic mismatch (RHM) by a combination of computational and functional approaches. The authors identified several residues at the intracellular/lipid interface, mutations of which were predicted to significantly reduce the RHM energy penalty. They also showed, by electrophysiological analysis of these mutants, that the reduction of the RHM penalty in the closed state compromised _Cr_ChR2 conductance, selectivity and open state stability. These results show that protein-lipid interactions have to be taken into account when engineering optogenetic tools for specific cell types. [PERSON] et al. examine the functional role of Thr127 located near the retinylidene Schiff base in _Cr_ChR2. Replacement of this residue with alanine or serine did not change the position of the spectral maximum, which ruled out its contribution to the counterion complex. However, the T127A mutation, unlike the conservative T127S mutation, accelerated deprotonation of the Schiff base and strongly delayed its reprotonation. These results place Thr127 in the hydrogen-bonded network connecting the Schiff base with Asp156, which the authors identified earlier as the proton donor to the Schiff base [16]. This conclusion was further corroborated by the observation of extended lifetime of the channel open state observed in both T127A and T127S mutants, as compared to the wild type. [PERSON] et al. systematically analyzed the influence of frequency, duration and intensity of optical stimulation on performance of _Cr_ChR2 in cultured mouse hippocampal neurons. Using optimal photostimulation protocols is very important in optogenetic experiments, because e.g., insufficient illumination results in poor fidelity, whereas excessive light might lead to overheating of the tissue. The authors show that at the optimal stimulation frequency 1-5 Hz the dependence of photocurrent on the light pulse duration is described by a right-skewed bell-shaped curve, whereas the dependence on the stimulus intensity is close to linear. These results complement previously published work (e.g., [17]) and provide useful guidelines for optogenetic experimentation. Finally, the review by [PERSON] and [PERSON] summarize recent progress in the development of synthetic azobenzene switches and their optimization for two-photon excitation (2 PE). Azobenzene is the most popular chromophore used in synthetic optogenetics, owing to its high quantum yield, solubility in water and minimal photobleaching. Most importantly, under photoexcitation azobenzene undergoes a rapid, robust isomerization from the _trans_ to _cis_ conformation that can be harnessed to drive biologically relevant conformational changes in target proteins.