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Photoswitching of Cell Surface Receptors Using Tethered Ligands

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Photoswitching Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1148))

Abstract

Optical probing and manipulation of cellular signaling has revolutionized biological studies ranging from isolated cells to intact tissues in the live animal. A promising avenue of optical manipulation is Chemical Optogenetics (or Optogenetic Pharmacology), an approach for engineering specific proteins to be rapidly and reversibly switched on and off with light. The approach employs synthetic photoswitched ligands, which can be reversibly photo-isomerized to toggle back and forth between two conformations in response to two wavelengths of light. We focus here on the photoswitched tethered ligand (PTL) approach in which the PTL is covalently attached in a site-directed manner to a signaling protein. For this a ligand anchoring site is introduced at a location which allows the ligand to dock only in one of the light-controlled conformations, thus enabling liganding to be rapidly switched. The ligand can be an agonist, antagonist or an active site (or pore) blocker. In principle, orthogonal chemistries of attachment would make PTL anchoring completely unique. However, extremely high specificity of remote control is also obtained by cysteine attachment because of the ligand specificity and precise geometric requirements for liganding. We describe here the design of light-gated ionotropic and metabotropic glutamate receptors, the selection of a site for cysteine placement, the method for PTL attachment, and a detailed protocol of photoswitching experiments in cultured cells. These descriptions can guide applications of Chemical Optogenetics to other receptors and serve as a starting point for use in more complex preparations.

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Acknowledgments

We thank all previous and current lab members for their contributions in developing and refining the described photoswitch technologies. Moreover, we thank Richard Kramer and his lab for discussion, and Dirk Trauner and his lab for developing and providing MAG compounds. This work was supported by the National Institutes of Health Nanomedicine Development Center for the Optical Control of Biological Function (2PN2EY018241).

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Authors and Affiliations

  1. Department of Molecular and Cell Biology and Helen Wills Neuroscience Institute, University of California Berkeley, Berkeley, CA, 94720, USA

    Andreas Reiner & Ehud Y. Isacoff

  2. Physical Bioscience Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA

    Ehud Y. Isacoff

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  1. Andreas Reiner
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  2. Ehud Y. Isacoff
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Correspondence to Ehud Y. Isacoff .

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Editors and Affiliations

  1. Institute of Anatomy and Cell Biology, University of Heidelberg, Heidelberg, Germany

    Sidney Cambridge

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Reiner, A., Isacoff, E.Y. (2014). Photoswitching of Cell Surface Receptors Using Tethered Ligands. In: Cambridge, S. (eds) Photoswitching Proteins. Methods in Molecular Biology, vol 1148. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0470-9_4

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  • DOI: https://doi.org/10.1007/978-1-4939-0470-9_4

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-0469-3

  • Online ISBN: 978-1-4939-0470-9

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