Channelrhodopsin‐2 Oligomerization in Cell Membrane Revealed by Photo‐Activated Localization Microscopy

Author:

Bestsennaia Ekaterina1ORCID,Maslov Ivan23ORCID,Balandin Taras4ORCID,Alekseev Alexey5ORCID,Yudenko Anna6ORCID,Abu Shamseye Assalla14,Zabelskii Dmitrii47ORCID,Baumann Arnd1ORCID,Catapano Claudia8,Karathanasis Christos8,Gordeliy Valentin4ORCID,Heilemann Mike8ORCID,Gensch Thomas1ORCID,Borshchevskiy Valentin4ORCID

Affiliation:

1. Institute of Biological Information Processing 1 IBI-1 (Molecular and Cellular Physiology) Forschungszentrum Jülich 52428 Jülich Germany

2. Dynamic Bioimaging Lab Advanced Optical Microscopy Centre and the Biomedical Research Institute Hasselt University B3590 Diepenbeek Belgium

3. Laboratory for Photochemistry and Spectroscopy Division for Molecular Imaging and Photonics, Department of Chemistry KU Leuven 3001 Leuven Belgium

4. Institute of Biological Information Processing 7 IBI-7 (Structural Biochemistry) Forschungszentrum Jülich 52428 Jülich Germany

5. Institute for Auditory Neuroscience and InnerEarLab University Medical Center Göttingen 37075 Göttingen Germany

6. Department of Biomedical Sciences University Medical Center Groningen University of Groningen 9713 AV Groningen The Netherlands

7. European XFEL 22869 Schenefeld Germany

8. Institute of Physical and Theoretical Chemistry Goethe-University Frankfurt 60438 Frankfurt Germany

Abstract

AbstractMicrobial 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.

Funder

Deutsche Forschungsgemeinschaft

Deutscher Akademischer Austauschdienst

Helmholtz-Gemeinschaft

Publisher

Wiley

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