Journal Article

FZJ-2024-02340

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Channelrhodopsin‐2 Oligomerization in Cell Membrane Revealed by Photo‐Activated Localization Microscopy

Bestsennaia, E.FZJ* ; Maslov, I. ; Balandin, T.FZJ* ; Alekseev, A. ; Yudenko, A. ; Abu Shamseye, A.FZJ* ; Zabelskii, D. ; Baumann, A.FZJ* ; Catapano, C. ; Karathanasis, C. ; Gordeliy, V.FZJ* ; Heilemann, M. ; Gensch, T. (Corresponding author)FZJ* ; Borshchevskiy, V. (Corresponding author)

2024
Wiley-VCH Weinheim

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Please use a persistent id in citations: doi:10.1002/ange.202307555  doi:10.34734/FZJ-2024-02340

Abstract: Microbial rhodopsins are retinal membrane proteins that found a broad application in optogenetics. The oligomeric state of rhodopsins is important for their functionality and stability. Of particular interest is the oligomeric state in the cellular native membrane environment. Fluorescence microscopy provides powerful tools to determine the oligomeric state of membrane proteins directly in cells. Among these methods is quantitative photoactivated localization microscopy (qPALM) allowing the investigation of molecular organization at the level of single protein clusters. Here, we apply qPALM to investigate the oligomeric state of the first and most used optogenetic tool Channelrhodopsin-2 (ChR2) in the plasma membrane of eukaryotic cells. ChR2 appeared predominantly as a dimer in the cell membrane and did not form higher oligomers. The disulfide bonds between Cys34 and Cys36 of adjacent ChR2 monomers were not required for dimer formation and mutations disrupting these bonds resulted in only partial monomerization of ChR2. The monomeric fraction increased when the total concentration of mutant ChR2 in the membrane was low. The dissociation constant was estimated for this partially monomerized mutant ChR2 as 2.2±0.9 proteins/μm2. Our findings are important for understanding the mechanistic basis of ChR2 activity as well as for improving existing and developing future optogenetic tools.

Note: We are thankful to Fedor Tsybrov for the help with thepreparation of plasmids. V.B. acknowledges DAAD YoungTalents Programme Line A. V.G. acknowledges his HGFProfessorship. I.M. acknowledges FWO Research FoundationFlanders (G0B9922N) and BOF UHasselt(BOF21BL11). C.C., C.K. and M.H. gratefully acknowledgethe Deutsche Forschungsgemeinschaft (grants CRC1507 andCRC807) for financial support. The work was done in theframework of CEA(IBS)–HGF(FZJ) STC 5.1 specific agreement.Open Access funding enabled and organized byProjekt DEAL.

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Contributing Institute(s):

1. Molekular- und Zellphysiologie (IBI-1)
2. Strukturbiochemie (IBI-7)

Research Program(s):

1. 5241 - Molecular Information Processing in Cellular Systems (POF4-524) (POF4-524)

Appears in the scientific report 2024

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Database coverage:
; ; ; DEAL Wiley ; Ebsco Academic Search ; Nationallizenz ; SCOPUS

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