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001023075 1001_ $$00009-0009-7212-978X$$aBestsennaia, Ekaterina$$b0
001023075 245__ $$aChannelrhodopsin‐2 Oligomerization in Cell Membrane Revealed by Photo‐Activated Localization Microscopy
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001023075 520__ $$aMicrobialrhodopsinsare retinalmembraneproteinsthat founda broadapplicationin optogenetics.Theoligomericstate of rhodopsinsis importantfor their functionalityand stability.Of particularinterestis the oligomericstate in the cellularnativemembraneenvironment.Fluorescencemicroscopyprovidespowerfultools to determinetheoligomericstate of membraneproteinsdirectlyin cells. Amongthese methodsis quantitativephotoactivatedlocalizationmicroscopy(qPALM)allowingthe investigationof molecularorganizationat the level of singleproteinclusters.Here,we applyqPALMto investigatethe oligomericstate of the first and most used optogenetictool Channelrhodopsin-2(ChR2)in the plasmamembraneof eukaryoticcells. ChR2appearedpredominantlyas a dimerin the cell membraneanddid not form higheroligomers.The disulfidebondsbetweenCys34and Cys36of adjacentChR2monomerswere notrequiredfor dimerformationand mutationsdisruptingthese bondsresultedin only partialmonomerizationof ChR2.The monomericfractionincreasedwhenthe total concentrationof mutantChR2in the membranewas low. Thedissociationconstantwas estimatedfor this partiallymonomerizedmutantChR2as 2.2�0.9 proteins/μm2. Our findingsare importantfor understandingthe mechanisticbasis of ChR2activityas well as for improvingexistingand developingfutureoptogenetictools.
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001023075 7001_ $$00000-0003-3371-4416$$aMaslov, Ivan$$b1
001023075 7001_ $$0P:(DE-Juel1)131949$$aBalandin, Taras$$b2
001023075 7001_ $$aAlekseev, Alexey$$b3
001023075 7001_ $$00000-0002-3115-0015$$aYudenko, Anna$$b4
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001023075 7001_ $$0P:(DE-Juel1)176570$$aZabelskii, Dmitrii$$b6
001023075 7001_ $$0P:(DE-Juel1)131911$$aBaumann, Arnd$$b7
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001023075 7001_ $$0P:(DE-HGF)0$$aKarathanasis, Christos$$b9
001023075 7001_ $$0P:(DE-Juel1)131964$$aGordeliy, Valentin$$b10
001023075 7001_ $$00000-0002-9821-3578$$aHeilemann, Mike$$b11
001023075 7001_ $$0P:(DE-Juel1)131924$$aGensch, Thomas$$b12$$eCorresponding author
001023075 7001_ $$0P:(DE-Juel1)179072$$aBorshchevskiy, Valentin$$b13
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