![showing time duration and run time in wavesurfer showing time duration and run time in wavesurfer](https://66.media.tumblr.com/6fae3a45b7ff50589db1cdd217a0d363/tumblr_nszhebyqUI1s7jx17o1_500.png)
We recently used the same Ace2 rhodopsin to engineer a negative-going chemigenetic eFRET GEVI called Voltron, which uses a HaloTag protein domain to covalently bind bright and photostable small-molecule flurophores 18, 19, extending the duration and number of neurons imaged simultaneously in vivo 14. Previous work fused the Ace2 rhodopsin from Acetabularia acetabulum 17 to the FP mNeonGreen to produce a negative-going eFRET GEVI that allowed in vivo imaging of voltage signals in several model organisms 12. Here we present a general approach to engineer eFRET GEVIs with fast, bright, and positive-going fluorescence signals in response to neuronal action potentials by modification of the natural proton transport pathway within microbial rhodopsins. Although two VSD GEVIs, FlicR 4 and Marina 16, exhibit positive-going signals in neurons, they have significantly slower response kinetics than eFRET GEVIs, making detection of action potentials difficult. All reported eFRET GEVIs therefore have negatively sloped fluorescence-voltage relationships they are brighter at resting membrane potential and become dimmer during an action potential (negative going). Although the absorbance of the retinal cofactor increases with increasing membrane potential, the emission of the eFRET-coupled fluorophore consequently decreases. These fusions enable voltage-sensitive electrochromic fluorescence resonance energy transfer (eFRET) from a bright fluorophore to the retinal cofactor within the rhodopsin, which acts as a dark quencher. Fusions of fluorescent protein (FP) domains or other bright fluorophores to rhodopsin GEVIs were therefore made to facilitate imaging 11, 13, 14. However, rhodopsin fluorescence is very dim, requiring intense illumination for imaging 8. The rhodopsin GEVI optical signal is fast and linear 8, two desirable features for a voltage indicator.
SHOWING TIME DURATION AND RUN TIME IN WAVESURFER MAC
The equivalent mutation in Ace1 12, Ace2 12, 14, and Mac 13 rhodopsins was used to generate later GEVIs. A single amino acid substitution (D95N) in Arch abolished light-driven currents and retained Arch voltage-dependent fluorescence change 8. However, Arch also generated a hyperpolarizing light-driven current upon exposure to imaging light 8, 15 by functioning as an outward proton pump 15.
![showing time duration and run time in wavesurfer showing time duration and run time in wavesurfer](https://static3.bigstockphoto.com/8/8/1/large1500/188757658.jpg)
Archaerhodopsin 3 (Arch) from Halorubrum sodomense was the first rhodopsin GEVI that accurately tracked changes in neuronal membrane potential 8. Existing GEVIs use voltage-sensitive protein domains from voltage-sensitive ion channels or phosphatases 3– 7 (VSD GEVIs), or microbial rhodopsin domains 8– 14 (rhodopsin GEVIs). Knowledge of the mechanistic function of GEVIs will lead to improved designs. Despite recent advances, no current GEVI has ideal properties for routine, robust in vivo imaging. Monitoring voltage signals in vivo in large populations of neurons will enable dissection of detailed mechanistic links between brain activity and animal behavior. Genetically encoded voltage indicators (GEVIs) allow visualization of fast action potentials and subthreshold dynamics in groups of genetically defined neurons with high spatiotemporal resolution 1, 2. Source data from experiments in this study are available from the authors upon reasonable request.