A general approach to engineer positive-going eFRET voltage indicators

Abstract

We engineered electrochromic fluorescence resonance energy transfer (eFRET) genetically encoded voltage indicators (GEVIs) with "positive-going" fluorescence response to membrane depolarization through rational manipulation of the native proton transport pathway in microbial rhodopsins. We transformed the state-of-the-art eFRET GEVI Voltron into Positron, with kinetics and sensitivity equivalent to Voltron but flipped fluorescence signal polarity. We further applied this general approach to GEVIs containing different voltage sensitive rhodopsin domains and various fluorescent dye and fluorescent protein reporters.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 . Monitoring voltage signals in vivo in large populations of neurons will enable dissection of detailed mechanistic links between brain activity and animal behavior. Despite recent advances, no current GEVI has ideal properties for routine, robust in vivo imaging.

Sensing cellular biochemistry with fluorescent chemical–genetic hybrids

“…This response is due to the increase in rhodopsin absorbance in response to increased membrane voltage via changes in the protonation equilibrium of the retinal Schiff base, which equilibrates with the cytoplasm. This model was used to engineer a positive-response GEVI [27]. In order to do, the proton-donating residue was mutated to a neutral residue, effectively blocking the access of protons from the cytoplasm.…”

A positively Tuned Voltage Indicator Reveals Electrical Correlates of Calcium Activity in the Brain

“…As the inherent fluorescence of opsins is dim, intense illumination is required to detect them (8). Opsins can be fused to a brighter fluorophore, allowing the fluorophore brightness to be modulated by FRET in a voltage-dependent manner, but the degree of modulation is limited (9)(10)(11)(12). Notably, both types of opsin-based GEVIs show poor responsivity under 2-P illumination, likely because voltage sensitivity resides in a transient state of the opsin photocycle with is inefficiently interrogated with 2-P excitation (5,13,14).…”

“…However, in the shot noise-limited regime, noise is related to the square root of baseline fluorescence, and thus an indicator with low baseline fluorescence would have the benefit of lower baseline noise. A well engineered positively tuned GEVI would thus have the ability to surpass all negatively tuned GEVIs in signal-to-noise ratio (SNR) (10).…”

Large-scale voltage imaging in the brain using targeted illumination

“…Another class of fully genetically encoded indicators is single compartment and directly detects the intrinsic voltage dependent fluorescence of engineered rhodopsins, such as QuarsAR3, Archon and SomArchon 3,5,15 . To improve fluorescence signals, bright chemical fluorophores have also been explored in the design of GVEIs, yielding a class of high-performance hybrid GEVIs that requires both exogeneous chemical dyes and the corresponding voltage sensing protein counterparts 2,16,17 .…”