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

Nature Methods volume 8, pages 1083–1088 (2011)Cite this article

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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.

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Figure 1: Design of protein chimeras and functional evaluation of ChR2-EYFP-βbR, ChR2-EYFP-βNphR and hChR2(H134R)-mKate-hβbR.
Figure 2: Expression and functionality of bidirectional rhodopsin tandem proteins in cultured hippocampal neurons.
Figure 3: Properties of rhodopsin tandem variants measured in HEK293 cells and hippocampal neurons.

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Acknowledgements

We thank V. Busskamp and B. Roska (Friedrich Miescher Institute for Biomedical Research, Basel) for the pAAV-LTR-EGFP plasmid and help with the rAAV construction, I. Bartnik for preparation of hippocampal neuron cultures, and H. Biehl and V. Eulenburg (Max Planck Institute for Brain Research, Frankfurt) for help with the antibody screening. The transgenic mouse expressing ChR2-EYFP was a gift from H. Wässle (Max Planck Institute for Brain Research, Frankfurt). The pAAV2-Rho-EGFP vector was a gift from A. Auricchio (Telethon Institute of Genetics and Medicine, Naples). The work was supported by the Deutsche Forschungsgemeinschaft Sonderforschungsbereich 807, Centre of Excellence Frankfurt Macromolecular Complexes, the German Federal Ministry of Education and Research (01GQ0815) and by the EU FP7 OptoNeuro Project (249867) to E.B. and by the Max Planck Society.

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  1. Ulrich Terpitz & Deniz Gökbuget

    Present address: Present addresses: Department of Biotechnology and Biophysics, Julius Maximilians University Würzburg, Biocenter Am Hubland, Würzburg, Germany (U.T.) and Eidgenössische Technische Hochschule Zürich, Institute of Cell Biology, Zurich, Switzerland (D.G.).,

Authors and Affiliations

  1. Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Frankfurt am Main, Germany

    Sonja Kleinlogel, Ulrich Terpitz, Barbara Legrum, Deniz Gökbuget, Christian Bamann, Phillip G Wood & Ernst Bamberg

  2. Synthetic Neurobiology Group and Department of Biological Engineering, The Massachusetts Institute of Technology Media Laboratory, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Edward S Boyden

  3. Chemical and Pharmaceutical Sciences Department, Johann Wolfgang Goethe University Frankfurt, Frankfurt am Main, Germany

    Ernst Bamberg

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Contributions

E.B., S.K. and P.G.W. conceived the molecular and electrophysiological experiments, and S.K., B.L. and U.T. carried them out. D.G. and C.B. performed the antibody screening. S.K., U.T. and C.B. designed and carried out the data analysis. S.K. and E.B. wrote the paper. E.S.B. supplied the Arch plasmid and contributed to writing.

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Correspondence to Ernst Bamberg.

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Kleinlogel, S., Terpitz, U., Legrum, B. et al. A gene-fusion strategy for stoichiometric and co-localized expression of light-gated membrane proteins. Nat Methods 8, 1083–1088 (2011). https://doi.org/10.1038/nmeth.1766

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