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 - TY - THES A1 - Duan, Xiaodong T1 - Development of new channelrhodopsin versions with enhanced plasma membrane targeting and high calcium/sodium conductance T1 - Entwicklung neuer Channelrhodopsin-Versionen mit verbessertem Plasmamembrantargeting und hoher Na+- und Ca2+-Leitfähigkeit N2 - The technique to manipulate cells or living animals by illumination after gene transfer of light-sensitive proteins is called optogenetics. Successful optogenetics started with the use of the light-gated cation channel channelrhodopsin-2 (ChR2). After early demonstrations of the power of ChR2, further light-sensitive ion channels and ion pumps were recruited to the optogenetic toolbox. Furthermore, mutations and chimera of ChR2 improved its versatility. However, there is still a need for improved optogenetic tools, e.g. with higher permeability for calcium or better expression in the plasma membrane. In this thesis, my work focuses on the design of highly functional channelrhodopsins with enhanced Na+ and Ca2+ conductance. First, I tested different N-terminal signal peptides to improve the plasma membrane targeting of Channelrhodopsins. We found that a N-terminal peptide, named LR, could improve the plasma membrane targeting of many rhodopsins. Modification with LR contributed to three to ten-fold larger photocurrents (than that of the original version) of multiple channelrhodopsins, like ChR2 from C. reinhardtii (CrChR2), PsChR, Chrimson, CheRiff, CeChR, ACRs, and the light-activated pump rhodopsins KR2, Jaw, HR. Second, by introducing point mutation, I could further improve the light sensitivity and photocurrent of different channelrhodopsins. For instance, ChR2-XXM 2.0, ChR2-XXL 2.0 and PsChR D139H 2.0 exhibited hundred times larger photocurrents than wild type ChR2 and they show high light sensitivity. Also, the Ca2+ permeable channelrhodopsins PsCatCh 2.0f and PsCatCh 2.0e show very large photocurrents and fast kinetics. In addition, I also characterized a novel bi-stable CeChR (from the acidophilic green alga Chlamydomonas eustigma) with a much longer closing time. Third, I analysed the ion selectivity of different ChRs, which provides a basis for rational selection of channelrhodopsins for different experimental purposes. I demonstrate that ChR2, Chronos, Chrimson, CheRiff and CeChR are highly proton conductive, compared with wild type PsChR. Interestingly, Chronos has the lowest potassium conductance among these channelrhodopsins. Furthermore, I found that mutation of an aspartate in TM4 of ChR2 (D156) and PsChR (D139) to histidine obviously increased both the sodium and calcium permeability while proton conductance was reduced. PsChR D139H 2.0 has the largest sodium conductance of any published channelrhodopsin variants. Additionally, I generated PsCatCh 2.0e which exhibits a ten-fold larger calcium current than the previously reported Ca2+ transporting CrChR2 mutant CatCh. In summary, my research work 1.) described strategies for improving plasma membrane trafficking efficiency of opsins; 2.) yielded channelrhodopsins with fast kinetics or high light sensitivity; 3.) provided optogenetic tools with improved calcium and sodium conductance. We could also improve the performance of channelrhodopsins with distinct action spectra, which will facilitate two-color neural excitation, both in-vitro and in-vivo. N2 - Die Technik, Zellen oder lebende Tiere nach dem Gentransfer lichtempfindlicher Proteine durch Belichtung zu manipulieren, wird als Optogenetik bezeichnet. Erfolgreiche Optogenetik begann mit der Verwendung des lichtgesteuerten Kationenkanals Channelrhodopsin-2 (ChR2). Nach frühen erfolgreichen Versuchen mit ChR2 wurden weitere lichtempfindliche Ionenkanäle und Ionenpumpen als optogenetische Werkzeuge etabliert. Darüber hinaus verbesserten Mutationen und Chimären von ChR2 seine Vielseitigkeit. Es besteht jedoch immer noch ein Bedarf an verbesserten optogenetischen Werkzeugen, z. mit höherer Permeabilität für Calcium oder besserer Expression in der Plasmamembran. In dieser Arbeit beschäftige ich mich mit dem Design hochfunktioneller Channelrhodopsine mit verbesserter Na+- und Ca2+-Leitfähigkeit. Zuerst habe ich verschiedene N-terminale Signalpeptide getestet, um die Anreicherung von Channelrhodopsinen in der Plasmamembran (“Plasmamembran-Targeting”) zu verbessern. Wir fanden heraus, dass ein N-terminales Peptid namens LR das Plasmamembran-Targeting vieler Rhodopsine verbessern kann. Die Modifikation mit LR trug zu drei- bis zehnfach größeren Photoströmen (als die der Originalversion) von mehreren Channelrhodopsinen bei, wie ChR2 von C. reinhardtii (CrChR2), PsChR, Chrimson, CheRiff, CeChR, ACRs und der lichtaktivierten Pump-Rhodopsine KR2, Jaw, HR. Zweitens konnte ich durch Mutagenese die Lichtempfindlichkeit und/oder den Photostrom verschiedener Channelrhodopsine weiter verbessern. Beispielsweise zeigten ChR2-XXM 2.0, ChR2-XXL 2.0 und PsChR D139H 2.0 hundertmal größere Photoströme als Wildtyp-ChR2 und sie zeigen eine hohe Lichtempfindlichkeit. Auch die Ca2+-permeablen Kanalrhodopsine PsCatCh 2.0f und PsCatCh 2.0e zeigen sehr große Photoströme und eine schnelle Kinetik. Außerdem habe ich ein neues bistabiles CeChR (aus der azidophilen Grünalge Chlamydomonas eustigma) mit einer viel längeren Schließzeit charakterisiert. Drittens analysierte ich die Ionenselektivität verschiedener ChRs, die eine Grundlage für die rationale Selektion von Channelrhodopsinen für verschiedene experimentelle Zwecke bietet. Ich zeige, dass ChR2, Chronos, Chrimson, CheRiff und CeChR im Vergleich zu Wildtyp-PsChR eine hohe Protonenleitfähigkeit aufweisen. Interessanterweise weist Chronos die niedrigste Kaliumleitfähigkeit unter diesen Channelrhodopsinen auf. Außerdem fand ich, dass die Mutation eines Aspartats in TM4 von ChR2 (D156) und PsChR (D139) zu Histidin offensichtlich sowohl die Natrium- als auch die Calciumpermeabilität erhöht, während die Protonenleitfähigkeit verringert ist. PsChR D139H 2.0 weist die größte Natriumleitfähigkeit aller veröffentlichten Channelrhodopsin-Varianten auf. Zusätzlich erzeugte ich PsCatCh 2.0e, das einen zehnmal größeren Calciumstrom als die zuvor berichtete Ca2+-transportierende CrChR2-Mutante CatCh aufweist. Zusammenfassend ergab meine Dissertationsarbeit: 1.) Strategien zur Verbesserung der Expression von Opsinen in der Plasmamembran; 2.) Gut exprimierende Channelrhodopsine mit schneller Kinetik oder hoher Lichtempfindlichkeit; 3.) Neue optogenetische Werkzeuge mit verbesserter Calcium- und Natriumleitfähigkeit. Auch konnte ich die Leistung von Channelrhodopsinen mit unterschiedlichen Aktionsspektren verbessern, was die zweifarbige neuronale Anregung sowohl in vitro als auch in vivo erleichtern sollte. KW - Optogenetik KW - Channelrhodopsinen KW - optogenetic KW - channelrhodopsin KW - molecular engineering KW - voltage clamp Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-188397 ER -