Mechanisms of photoswitch conjugation and light activation of an ionotropic glutamate receptor

The analysis of cell signaling requires the rapid and selective manipulation of protein function. We have synthesized photoswitches that covalently modify target proteins and reversibly present and withdraw a ligand from its binding site due to photoisomerization of an azobenzene linker. We describe...

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Published inProceedings of the National Academy of Sciences - PNAS Vol. 104; no. 26; pp. 10865 - 10870
Main Authors Gorostiza, Pau, Volgraf, Matthew, Numano, Rika, Szobota, Stephanie, Trauner, Dirk, Isacoff, Ehud Y
Format Journal Article
LanguageEnglish
Published United States National Academy of Sciences 26.06.2007
National Acad Sciences
Subjects
Agonists
Amino acids
Binding Sites
Biochemistry
Biological Sciences
Biomedical Research - methods
Biophysics
Cell-Free System
Cells
Electric current
Glutamate receptors
HEK293 cells
Inhibitory concentration 50
Ion Channels - metabolism
Ligands
Light
Models, Biological
Neuroscience
Proteins
Proteins - physiology
Receptors
Receptors, Kainic Acid - metabolism
Receptors, Kainic Acid - radiation effects
Wavelengths
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Summary:The analysis of cell signaling requires the rapid and selective manipulation of protein function. We have synthesized photoswitches that covalently modify target proteins and reversibly present and withdraw a ligand from its binding site due to photoisomerization of an azobenzene linker. We describe here the properties of a glutamate photoswitch that controls an ion channel in cells. Affinity labeling and geometric constraints ensure that the photoswitch controls only the targeted channel, and enables spatial patterns of light to favor labeling in one location over another. Photoswitching to the activating state places a tethered glutamate at a high (millimolar) effective local concentration near the binding site. The fraction of active channels can be set in an analog manner by altering the photostationary state with different wavelengths. The bistable photoswitch can be turned on with millisecond-long pulses at one wavelength, remain on in the dark for minutes, and turned off with millisecond long pulses at the other wavelength, yielding sustained activation with minimal irradiation. The system provides rapid, reversible remote control of protein function that is selective without orthogonal chemistry.
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Present address: Laboratory Animal Research Center, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan
Edited by Charles F. Stevens, The Salk Institute for Biological Studies, La Jolla, CA, and approved May 4, 2007
Author contributions: P.G., M.V., R.N., D.T., and E.Y.I. designed research; P.G., M.V., R.N., and S.S. performed research; P.G. and M.V. contributed new reagents/analytic tools; P.G., M.V., R.N., S.S., and E.Y.I. analyzed data; and P.G., M.V., D.T., and E.Y.I. wrote the paper.
Present address: Institució Catalana de Recerca i Estudis Avancats and Institut de Bioenginyeria de Catalunya, Parc Científic de Barcelona, 08028 Barcelona, Spain.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0701274104