Technologies for measuring the transient Ca spikes that accompany neural signaling have revolutionized our understanding of the brain. Nevertheless, microscopic visualization of Ca spikes on the time scale of neural activity across large brain regions or in thick specimens remains a significant challenge. The recent development of stable integrators of Ca, instead of transient reporters, provides an avenue to investigate neural signaling in otherwise challenging systems. Here, we describe an engineered Ca-sensing enzyme consisting of a split Tobacco Etch Virus (TEV) protease with each half tethered to a calmodulin or M13 Ca binding domain. This Split TEV, Ca Activated Neuron Recorder (SCANR) remains separate and catalytically incompetent until a spike in cellular Ca triggers its reconstitution and the subsequent turnover of a caged, genetically encoded reporter substrate. We report the identification of a successful Ca-sensing split TEV from a library of chimeras and deployment of the enzyme in primary rat hippocampal neurons.
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| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6905437 | PMC |
| http://dx.doi.org/10.1021/acschembio.8b00130 | DOI Listing |
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