Abstract
Electrophysiological approaches to the study of the activity of retinal-containing protein bacteriorhodopsin (bR) or other proteins of this family are based usually on measurements of electrical current through a planar bilayer lipid membrane (BLM) with proteoliposomes attached to the BLM surface at one side of the membrane. Here, we describe the measurements of the pumping activity of bR and channelrhodopsin 2 (ChR2) with special attention to the study of voltage dependence of the light-induced currents. Strong voltage dependence of ChR2 suggests light-triggered ion channel activity of ChR2. We also describe electrophysiological measurements with the help of collodion film instead of BLM for the measurements of fast stages of a rhodopsin photocycle as well as the estimation of the activity of proteoliposomes without a macro membrane using fluorescent probes such as oxonol VI or 9-aminoacridine.
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Acknowledgments
We are grateful to Oleksandr Volkov for producing channelrhodopsin-2 and Christian Baeken for providing us with purple membranes.
The research was supported by the Ministry of Science and Higher Education of the Russian Federation (agreement # 075-00337-20-03, project FSMG -2020-0003). Measurements of electrogenic properties were supported by RFBR grants 19-04-00238 (to TIR) and 18-04-00503a (to SAS).
This work was supported by Russian Science Foundation (RSF) Project 21-64-00018.
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Rokitskaya, T.I., Maliar, N.L., Siletsky, S.A., Gordeliy, V., Antonenko, Y.N. (2022). Electrophysiological Characterization of Microbial Rhodopsin Transport Properties: Electrometric and ΔpH Measurements Using Planar Lipid Bilayer, Collodion Film, and Fluorescent Probe Approaches. In: Gordeliy, V. (eds) Rhodopsin. Methods in Molecular Biology, vol 2501. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2329-9_12
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DOI: https://doi.org/10.1007/978-1-0716-2329-9_12
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