title: A gene-fusion strategy for stoichiometric and co-localized expression of light-gated membrane proteins.
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.
authors: Max Planck Institute of Biophysics, Department of Biophysical Chemistry, Frankfurt am Main, Germany.
authors: Sonja
authors: S
authors: Kleinlogel
authors: Ulrich
authors: U
authors: Terpitz
authors: Barbara
authors: B
authors: Legrum
authors: Deniz
authors: D
authors: Gökbuget
authors: Edward S
authors: ES
authors: Boyden
authors: Christian
authors: C
authors: Bamann
authors: Phillip G
authors: PG
authors: Wood
authors: Ernst
authors: E
authors: Bamberg
mesh_terms: D019677:Artificial Gene Fusion; D001436:Bacteriorhodopsins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D057809:HEK293 Cells; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D008027:Light; D008969:Molecular Sequence Data; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D012243:Rhodopsin / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D001436:Bacteriorhodopsins; D012243:Rhodopsin