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

作者：Kleinlogel Sonja; Terpitz Ulrich; Legrum Barbara; Goekbuget Deniz; Boyden Edward S; Bamann Christian; Wood Phillip G; Bamberg Ernst^*

来源：Nature Methods, 2011, 8(12): 1083-+.

DOI：10.1038/NMETH.1766

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.

• 出版日期2011-12