SENSIT, a Modular Single-Chain Fluorescent Integrator Platform for GPCR Ligands.

G-protein-coupled receptors (GPCRs) transduce the effects of many neurotransmitters such as epinephrine, norepinephrine, and acetylcholine. However, understanding neurotransmitter release via GPCRs at high resolution across large brain volumes remains a significant challenge. Here, we report a modular platform called single-chain expressing neurotransmitter sensing integrator tool (SENSIT) featuring the design of bifunctional chimeric nanobodies. We applied the SENSIT platform by designing reporters for epinephrine and norepinephrine that exhibit 20-fold and 5-fold selectivity over another catecholamine, respectively. To further demonstrate the modularity of the platform, we also developed a muscarinic acetylcholine receptor 2 (CHRM2)-based sensor and chimeric miniG proteins. The SENSIT platform reports the first single-chain integrator sensor capable of distinguishing epinephrine and norepinephrine as well as enabling the generalized design of integrator sensors for diverse neurotransmitters with the potential for whole-brain mapping.