TY  - JOUR
A1  - Zhou, Yang
A1  - Ding, Meiqi
A1  - Duan, Xiaodong
A1  - Konrad, Kai R.
A1  - Nagel, Georg
A1  - Gao, Shiqiang
T1  - Extending the Anion Channelrhodopsin-Based Toolbox for Plant Optogenetics
JF  - Membranes
N2  - Optogenetics was developed in the field of neuroscience and is most commonly using light-sensitive rhodopsins to control the neural activities. Lately, we have expanded this technique into plant science by co-expression of a chloroplast-targeted β-carotene dioxygenase and an improved anion channelrhodopsin GtACR1 from the green alga Guillardia theta. The growth of Nicotiana tabacum pollen tube can then be manipulated by localized green light illumination. To extend the application of analogous optogenetic tools in the pollen tube system, we engineered another two ACRs, GtACR2, and ZipACR, which have different action spectra, light sensitivity and kinetic features, and characterized them in Xenopus laevis oocytes, Nicotiana benthamiana leaves and N. tabacum pollen tubes. We found that the similar molecular engineering method used to improve GtACR1 also enhanced GtACR2 and ZipACR performance in Xenopus laevis oocytes. The ZipACR1 performed in N. benthamiana mesophyll cells and N. tabacum pollen tubes with faster kinetics and reduced light sensitivity, allowing for optogenetic control of anion fluxes with better temporal resolution. The reduced light sensitivity would potentially facilitate future application in plants, grown under low ambient white light, combined with an optogenetic manipulation triggered by stronger green light.
KW  - optogenetics
KW  - rhodopsin
KW  - light-sensitive anion channel
KW  - surface potential recording
KW  - pollen tube
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-236617
SN  - 2077-0375
VL  - 11
IS  - 4
ER  - 
TY  - JOUR
A1  - Duan, Xiaodong
A1  - Nagel, Georg
A1  - Gao, Shiqiang
T1  - Mutated channelrhodopsins with increased sodium and calcium permeability
JF  - Applied Sciences
N2  - (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 Ca\(^{2+}\) 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 Ca\(^{2+}\) conductance, which was not noticed before, while the D156C mutation (XXL) influenced the Na\(^+\) and Ca\(^{2+}\) 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 Ca\(^{2+}\) 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 Ca\(^{2+}\) conductance, XXM and PsChR D139H are promising powerful optogenetic tools, especially for Ca\(^{2+}\) manipulation.
KW  - optogenetics
KW  - channelrhodopsins
KW  - sodium
KW  - calcium
KW  - DC gate
Y1  - 2019
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-197435
SN  - 2076-3417
VL  - 9
IS  - 4
ER  -