Figure 3.

Genetically encoded voltage indicators (GEVIs). (a) ArcLight-family GEVIs respond to depolarization by reduced green fluorescence (rightward green sinusoidal arrow) from superecliptic pHluorin (SEP) upon blue-light excitation (blue sinusoidal arrow). The mechanism is not fully known but is believed to involve voltage-dependent dimerization leading to protonation of the SEP chromophore. Kinetics are shown for ArcLightQ239 and Bongwoori as measured at 33 °C (ref. 123), with the slash (/) separating ArcLightQ239 and Bongwoori values. Among the ArcLight variants, these have the largest amplitude and fastest signaling kinetics, respectively. (b) GEVIs of the ASAP family also report depolarization by dimming of a circularly permuted GFP (cpGFP). The mechanism presumably involves coupling of VSD movement to chromophore protonation, similar to that in iGluSnFR and single-fluorophore GECIs. Kinetics were measured at 22 °C (ref. 25). (c) FlicR reports depolarization with increased red fluorescence (red sinusoidal arrow) from a circularly permuted red fluorescent protein (cpRFP) upon green excitation (leftward green sinusoidal arrow), presumably as a result of chromophore deprotonation. Kinetics shown were measured at 37 °C (ref. 59). (d) Opsins report depolarization with increased red fluorescence (red sinusoidal arrow) upon excitation by ~600-nm light (orange sinusoidal arrow), but this emission is weak (quantum yield < 0.01). Kinetics are shown for QuasAr2 measured at 34 °C (ref. 65). (e) Opsin–fluorescent protein fusions report depolarization with a dimming of fluorescence, due to absorbance shift in the opsin leading to increased FRET. In the case of Ace2N-mNeonGreen, emission is yellow-green (yellow-green sinusoidal arrow) and excitation is cyan (cyan sinusoidal arrow). Kinetics are shown for Ace2N-mNeonGreen measured at 22 °C (ref. 5).