RedquorinXS Mutants with Enhanced Calcium Sensitivity and Bioluminescence Output Efficiently Report Cellular and Neuronal Network Activities

Considerable efforts have been focused on shifting the wavelength of aequorin Ca-dependent blue bioluminescence through fusion with fluorescent proteins. This approach has notably yielded the widely used GFP-aequorin (GA) Ca sensor emitting green light, and tdTomato-aequorin (Redquorin), whose bioluminescence is completely shifted to red, but whose Ca sensitivity is low. In the present study, the screening of aequorin mutants generated at twenty-four amino acid positions in and around EF-hand Ca-binding domains resulted in the isolation of six aequorin single or double mutants (AequorinXS) in EF2, EF3, and C-terminal tail, which exhibited markedly higher Ca sensitivity than wild-type aequorin in vitro. The corresponding Redquorin mutants all showed higher Ca sensitivity than wild-type Redquorin, and four of them (RedquorinXS) matched the Ca sensitivity of GA in vitro. RedquorinXS mutants exhibited unaltered thermostability and peak emission wavelengths. Upon stable expression in mammalian cell line, all RedquorinXS mutants reported the activation of the P2Y2 receptor by ATP with higher sensitivity and assay robustness than wt-Redquorin, and one, RedquorinXS-Q159T, outperformed GA. Finally, wide-field bioluminescence imaging in mouse neocortical slices showed that RedquorinXS-Q159T and GA similarly reported neuronal network activities elicited by the removal of extracellular Mg. Our results indicate that RedquorinXS-Q159T is a red light-emitting Ca sensor suitable for the monitoring of intracellular signaling in a variety of applications in cells and tissues, and is a promising candidate for the transcranial monitoring of brain activities in living mice.