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A gene-fusion strategy for stoichiometric and co-localized expression of light-gated membrane proteins

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Kleinlogel,  Sonja
Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max Planck Society;

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Terpitz,  Ulrich
Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max Planck Society;

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Legrum,  Barbara
Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max Planck Society;

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Gökbuget,  Deniz
Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max Planck Society;

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Bamann,  Christian
Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max Planck Society;

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Wood,  Phillip G.
Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max Planck Society;

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Bamberg,  Ernst
Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max Planck Society;

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Citation

Kleinlogel, S., Terpitz, U., Legrum, B., Gökbuget, D., Boyden, E. S., Bamann, C., et al. (2011). A gene-fusion strategy for stoichiometric and co-localized expression of light-gated membrane proteins. Nature Methods, 8(12), 1083-1088.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0024-D5C5-C
Abstract
The precise co-localization and stoichiometric expression of two different light-gated membrane proteins can vastly improve the physiological usefulness of optogenetics for the modulation of cell excitability with light. Here we present a gene-fusion strategy for the stable 1:1 expression of any two microbial rhodopsins in a single polypeptide chain. By joining the excitatory channelrhodopsin-2 with the inhibitory ion pumps halorhodopsin or bacteriorhodopsin, we demonstrate light-regulated quantitative bi-directional control of the membrane potential in HEK293 cells and neurons in vitro. We also present synergistic rhodopsin combinations of channelrhodopsin-2 with Volvox carteri channelrhodopsin-1 or slow channelrhodopsin-2 mutants, to achieve enhanced spectral or kinetic properties, respectively. Finally, we demonstrate the utility of our fusion strategy to determine ion-turnovers of as yet uncharacterized rhodopsins, exemplified for archaerhodopsin and CatCh, or to correct pump cycles, exemplified for halorhodopsin.