G protein-assisted optimization of GPCR-activation based (GRAB) sensors

摘要

In the brain, neurotransmitters or neuromodulators play pivotal roles in chemical synaptic transmission and consequently,monitoring their dynamics, especially in vivo, is critical for understanding their physiological- or pathophysiologicalroles at molecular, cellular, and circuit levels during behaviors and/or during diseases. We recently developedgenetically-encoded GPCR-activation based (GRAB) sensors capable of reporting dynamics of acetylcholine, dopamineand norepinephrine with rapid kinetics, chemical- and cell-specificity in multiple organisms in vivo. Here, we exploredthe usage of G protein derivatives, either mini-G proteins or C-terminal peptides of Gα subunit to engineer new GRABsensors. We found that the conformational changes mediated by mini-G proteins interacting with GPCRs, or Gα Cterminalpeptides interacting with GPCRs could be harnessed to regulate fluorescence outputs of a GPCR fused circularpermuted GFP (cpGFP). In addition, inter-molecular fusion of Gα C-terminal peptides significantly suppressed ectopicactivation of G protein signaling in a GRAB acetylcholine sensor. Finally, we showed Gα C-terminal peptides fusionstrategy could be applied to generate various GRAB sensors for small molecular compounds or neuropeptides.