Visualization of synaptic inhibition with an optogenetic sensor developed by cell-free protein engineering automation

We describe an engineered fluorescent optogenetic sensor, SuperClomeleon, that robustly detects inhibitory synaptic activity in single, cultured mouse neurons by reporting intracellular chloride changes produced by exogenous GABA or inhibitory synaptic activity. Using a cell-free protein engineering...

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Published inThe Journal of neuroscience Vol. 33; no. 41; pp. 16297 - 16309
Main Authors Grimley, Joshua S, Li, Li, Wang, Weina, Wen, Lei, Beese, Lorena S, Hellinga, Homme W, Augustine, George J
Format Journal Article
LanguageEnglish
Published United States Society for Neuroscience 09.10.2013
Subjects
Animals
Automation, Laboratory
Cell-Free System
Mice
Neural Inhibition - physiology
Neurons - physiology
Optogenetics - methods
Protein Engineering - methods
Recombinant Fusion Proteins - chemistry
Synaptic Transmission - physiology
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Summary:We describe an engineered fluorescent optogenetic sensor, SuperClomeleon, that robustly detects inhibitory synaptic activity in single, cultured mouse neurons by reporting intracellular chloride changes produced by exogenous GABA or inhibitory synaptic activity. Using a cell-free protein engineering automation methodology that bypasses gene cloning, we iteratively constructed, produced, and assayed hundreds of mutations in binding-site residues to identify improvements in Clomeleon, a first-generation, suboptimal sensor. Structural analysis revealed that these improvements involve halide contacts and distant side chain rearrangements. The development of optogenetic sensors that respond to neural activity enables cellular tracking of neural activity using optical, rather than electrophysiological, signals. Construction of such sensors using in vitro protein engineering establishes a powerful approach for developing new probes for brain imaging.
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Author contributions: J.S.G., L.L., W.W., L.W., L.S.B., H.W.H., and G.J.A. designed research; J.S.G., L.L., W.W., and L.W. performed research; J.S.G., L.L., W.W., L.W., L.S.B., H.W.H., and G.J.A. analyzed data; J.S.G., L.L., W.W., L.W., L.S.B., H.W.H., and G.J.A. wrote the paper.
J.S. Grimley's present address: Allen Institute for Brain Science, Seattle, WA 98103.
J.S.G. and L.L. contributed equally to this work.
ISSN:0270-6474
1529-2401
1529-2401
DOI:10.1523/JNEUROSCI.4616-11.2013