Abstract
(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 in demand. (2) Methods: In this study, we mutated the conserved aspartate of the transmembrane helix 4 (TM4) within Chronos and PsChR and compared them with published ChR2 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. The aspartate to histidine and cysteine mutations of Chronos and PsChR also influenced their photocurrent, ion permeability, kinetics, and light sensitivity. Most interestingly, PsChR D139H showed a much-improved photocurrent, compared to wild type, and even higher Na+ selectivity to H+ than XXM. PsChR D139H also showed a strongly enhanced Ca2+ conductance, more than two-fold that of the CatCh. (4) Conclusions: We found that mutating the aspartate of the TM4 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.
Highlights
Channelrhodopsins were first discovered and characterized from C. reinhardtii [1,2]
PsChR D139H showed a strongly enhanced Ca2+ conductance, more than two-fold that of the CatCh. (4) Conclusions: We found that mutating the aspartate of the transmembrane helix 4 (TM4) influences the ion selectivity of channelrhodopsins
D139H, D139C, showed an increased expression compared to their type
Summary
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 Nagel 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]. 132, L132C) showed improved Ca2+ conductivity together with a larger photocurrent and higher light sensitivity [8].
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