Neuronal Calcium Recording with an Engineered TEV Protease

Technologies for measuring the transient Ca²⁺ spikes that accompany neural signaling have revolutionized our understanding of the brain. Nevertheless, microscopic visualization of Ca²⁺ 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 Ca²⁺, instead of transient reporters, provides an avenue to investigate neural signaling in otherwise challenging systems. Here, we describe an engineered Ca²⁺-sensing enzyme consisting of a split Tobacco Etch Virus (TEV) protease with each half tethered to a calmodulin or M13 Ca²⁺ binding domain. This Split TEV, Ca²⁺ Activated Neuron Recorder (SCANR) remains separate and catalytically incompetent until a spike in cellular Ca²⁺ triggers its reconstitution and the subsequent turnover of a caged, genetically encoded reporter substrate. We report the identification of a successful Ca²⁺-sensing split TEV from a library of chimeras and deployment of the enzyme in primary rat hippocampal neurons.