(1) Background: After the discovery and application of Chlamydomonas reinhardtii channelrhodopsins, the optogenetic toolbox has been greatly expanded with engineered and newly discovered natural channelrhodopsins. However, channelrhodopsins of higher Ca2+ conductance or more specific ion permeability are still in demand. (2) Methods: In this study, we mutated the conserved aspartate of the transmembrane helix 4 (TM4) within Chronos (Stigeoclonium helveticum channelrhodopsin = ShChR) and PsChR (Platymonas subcordiformis channelrhodopsin) and compared them with published ChR2 (C. reinhardtii channelrhodopsin-2) aspartate mutants. (3) Results: We found that the ChR2 D156H mutant (XXM) showed enhanced Na+ and Ca2+ conductance, which was not noticed before, while the D156C mutation (XXL) influenced the Na+ and Ca2+ conductance only slightly. Furthermore, the D173H mutant of PsChR showed a much improved photocurrent, compared to wildtype, and even higher Na+ selectivity to H+ than XXM. PsChR D173H also showed a strongly enhanced Ca2+ conductance, more than 2-fold that of the calcium translocating L132C of ChR2 (CatCh). (4) Conclusions: We found that mutating the aspartate of the TM4 to histidine influences the ion selectivity of channelrhodopsins. With the large photocurrent and enhanced Na+ selectivity and Ca2+ conductance, XXM and PsChR D139H are promising powerful optogenetic tools, especially for Ca2+ manipulation.

Second messengers, cyclic adenosine 3'-5'-monophosphate (cAMP) and cyclic guanosine 3'-5'-monophosphate (cGMP) are playing important roles in many animal cells by regulating intracellular signaling pathways and modulating cell physiology. Environmental cues like temperature, light and chemical compounds can stimulate cell surface receptors and trigger the generation of second messengers and the following regulations. Spread of cAMP and cGMP is further shaped by cyclic nucleotide phosphodiesterases (PDEs) for orchestration of intracellular microdomain signaling. However, localized intracellular cAMP and cGMP signaling requires further investigation. Optogenetic manipulation of cAMP and cGMP offers new opportunities of spatio-temporally precise study of their signaling mechanism. Light-gated nucleotide cyclases are well developed and applied for cAMP/cGMP manipulation. Recently discovered rhodopsin phosphodiesterase gene from protists established new and direct biological connection between light and PDEs. Light-regulated PDEs are under development and of demand to complete the toolkit of cAMP/cGMP manipulation. In this review, we summarize the state of the art, pros and cons of artificial and natural light-regulated PDEs and discuss potential new strategies of developing light-gated PDEs for optogenetic manipulation.

Light-gated ion channel and ion pump rhodopsins are widely used as optogenetic tools and these can control the electrically excitable cells as: (1) they are a single-component system i.e., their sensor and effector functions are encoded by the 7-transmembrane domains and (2) they show fast kinetics with small dark-thermal recovery time. In cellular signaling, a signal receptor, modulator and effector components are involved for attaining synchronous multicomponent regulation. Optical modulation of this network requires either receptor to effector encoded in a single ORF or direct modulation of the effector domain through bypassing all upstream players. Recently discovered modular rhodopsins like rhodopsin guanine cyclase (RhoGC) and rhodopsin phosphodiesterase (RhoPDE) paves the way to establish proof of concept. Light sensor coupled modular system could be expressed in a precise cell type and which holds great potential in the advancement of optogenetics 2.0. It would enable manipulating entire relevant cell signaling system. Here, we had identified 50 novels modular rhodopsins with variant rhodopsins domain and its diverse cognate signaling cascades encoded in a single ORF, which are associated with specialized functions in the cells. These novel modular algal rhodopsins have been characterized functionality based on their sequence and structural homology with previously characterized rhodopsins. Presented novel modular rhodopsins with various effector domains hold potential to expand optogenetics tool kit to regulate various cellular signaling pathways across the diverse biological model systems.

Subcellular targeting of opsins in optogenetics provides new possibilities for investigating the function of nerve cells. One of the widely used motifs for central targeting of opsins is the motif of potential-dependent potassium channel Kv2.1. We have expressed construct CHR2-Venus-Kv2.1 in the layer 2/3 pyramidal neurons of the murine cerebral cortex by means of in utero electroporation. It was found that, although the majority of neurons expressing CHR2-Venus-Kv2.1 demonstrated mainly central localization of fluorescence in the soma, proximal dendrites and axon, there was also significant population of neurons with disruption of the “correct” targeting resulting in the fluorescent protein distributed uniformly throughout the entire cell surface. We have suggested that observed mislocalization was caused by overexpression of the construct. Indeed, a decrease in the plasmid concentration during the in utero electroporation procedure resulted in almost complete absence of neurons with altered targeting. Thus, the possibility of “incorrect” targeting of CHR2 by the potassium channel motif Kv2.1 should be taken into account when using this construct in optogenetic experiments.

Brain-stimulation reward, also known as intracranial self-stimulation (ICSS), is a commonly used procedure for studying brain reward function and drug reward. In electrical ICSS, an electrode is surgically implanted into the medial forebrain bundle in the lateral hypothalamus or the ventral tegmental area in the midbrain. Operant lever responding leads to the delivery of electrical pulse stimulation. The alteration in the stimulation frequency-lever response curve is used to evaluate the impact of pharmacological agents on brain reward function. If a test drug induces a leftward or upward shift in the ICSS response curve, it implies a reward-enhancing or abuse-like effect. Conversely, if a drug causes a rightward or downward shift in the functional response curve, it suggests a reward-attenuating or aversive effect. A significant drawback of electrical ICSS is the lack of cellular selectivity in understanding the neural substrates underlying drug reward versus aversion. Excitingly, recent advancements in optical ICSS (oICSS) have facilitated the development of at least three cell type-specific oICSS models – dopamine-, glutamate-, and GABA-dependent oICSS. In these new models, a comparable frequency-rate response curve has been established and employed to study the substrate-specific mechanisms underlying brain reward function and a drug's rewarding versus aversive effects. In this review article, we summarize recent progress in this exciting research area. The findings in these new behavioral models have not only increased our understanding of the neural mechanisms underlying drug reward and addiction but have also introduced a novel experimental approach in preclinical medication development for treating substance use disorders.