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Abstract
Imaging membrane voltage from genetically defined cells offers the unique ability to report spatial and temporal dynamics of electrical signaling at cellular and circuit levels. Here, we present a general approach to engineer 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 transform the state-of-the-art eFRET GEVI Voltron into Positron, with kinetics and sensitivity equivalent to Voltron but flipped fluorescence signal polarity. We further apply this general approach to GEVIs containing different voltage sensitive rhodopsin domains and various fluorescent dye and fluorescent protein reporters.
Highlights
Imaging membrane voltage from genetically defined cells offers the unique ability to report spatial and temporal dynamics of electrical signaling at cellular and circuit levels
We recently used the same Ace[2] rhodopsin to engineer a negative-going chemigenetic electrochromic fluorescence resonance energy transfer (eFRET) genetically encoded voltage indicators (GEVIs) 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]. In both of these GEVIs, photocurrent of Ace[2] rhodopsin (Fig. 1a) is blocked by mutating the residue that normally functions as the proton acceptor (PA)[20] (D81N) (Fig. 1a, b), analogous to the Archaerhodopsin 3 (Arch) D95N mutation described above. This mutation blocks the primary pathway for exchange of protons from the retinal Schiff base, which links retinal to the rhodopsin protein, to outside the cell[20]
Electrophysiology measurements showing transient inward photocurrents with Ace[2] D81N (Fig. 1c) and other rhodopsin-based GEVIs21, combined with previous mutagenesis and biochemical data[17,20], suggest that voltage sensitivity in Ace[2] D81N and other eFRET GEVIs results from membrane potential changes altering the equilibrium of protonation between the retinal Schiff base, the proton donor (PD) residue[20], and the cell cytoplasm (Fig. 1a, b)
Summary
Imaging membrane voltage from genetically defined cells offers the unique ability to report spatial and temporal dynamics of electrical signaling at cellular and circuit levels. We present a general approach to engineer 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 transform the state-of-the-art eFRET GEVI Voltron into Positron, with kinetics and sensitivity equivalent to Voltron but flipped fluorescence signal polarity We further apply this general approach to GEVIs containing different voltage sensitive rhodopsin domains and various fluorescent dye and fluorescent protein reporters. 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
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