Visualization of Synaptic Inhibition with an Optogenetic Sensor Developed by Cell-Free Protein Engineering Automation

Title
Visualization of Synaptic Inhibition with an Optogenetic Sensor Developed by Cell-Free Protein Engineering Automation
Authors
Joshua S. GrimleyLi LiWeina Wang레이웬Lorena S. BeeseHommeW. Hellinga조지어거스틴
Issue Date
2013-10
Publisher
Journal of neuroscience
Citation
VOL 33, NO 41, 16297-16309
Abstract
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.
URI
http://pubs.kist.re.kr/handle/201004/46154
ISSN
02706474
Appears in Collections:
KIST Publication > Article
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