A high-performance genetically encoded fluorescent indicator for in vivo cAMP imaging

The second messenger cAMP modulates a wide range of important biological processes. Here, the authors developed a fluorescent indicator termed G-Flamp1, which can accurately report levels of cAMP in living cells. cAMP is a key second messenger that regulates diverse cellular functions including neural plasticity. However, the spatiotemporal dynamics of intracellular cAMP in intact organisms are largely unknown due to low sensitivity and/or brightness of current genetically encoded fluorescent cAMP indicators. Here, we report the development of the new circularly permuted GFP (cpGFP)-based cAMP indicator G-Flamp1, which exhibits a large fluorescence increase (a maximum Delta F/F-0 of 1100% in HEK293T cells), decent brightness, appropriate affinity (a K-d of 2.17 mu M) and fast response kinetics (an association and dissociation half-time of 0.20 and 0.087 s, respectively). Furthermore, the crystal structure of the cAMP-bound G-Flamp1 reveals one linker connecting the cAMP-binding domain to cpGFP adopts a distorted beta-strand conformation that may serve as a fluorescence modulation switch. We demonstrate that G-Flamp1 enables sensitive monitoring of endogenous cAMP signals in brain regions that are implicated in learning and motor control in living organisms such as fruit flies and mice.

Automated summary

In this study, a new fluorescent indicator called G-Flamp1 was developed to accurately measure levels of cAMP in living cells. G-Flamp1 exhibited good brightness, appropriate affinity, and fast response kinetics. The results showed that G-Flamp1 enables sensitive monitoring of endogenous cAMP signals in living organisms such as fruit flies and mice.