Visualization of synaptic inhibition with an optogenetic sensor developed by cell-free protein engineering automation
Grimley, Joshua S.
Beese, Lorena S.
Hellinga, Homme W.
Augustine, George James
Date of Issue2013
Lee Kong Chian School of Medicine
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.
Journal of neuroscience
© 2013 The Authors. This paper was published in Journal of Neuroscience and is made available as an electronic reprint (preprint) with permission of the authors. The paper can be found at the following official DOI: [http://dx.doi.org/10.1523/JNEUROSCI.4616-11.2013]. One print or electronic copy may be made for personal use only. Systematic or multiple reproduction, distribution to multiple locations via electronic or other means, duplication of any material in this paper for a fee or for commercial purposes, or modification of the content of the paper is prohibited and is subject to penalties under law.