Imaging Voltage with Microbial Rhodopsins

Abstract

Membrane potential is the critical parameter that reflects the excitability of a neuron, and it is usually measured by electrophysiological recordings with electrodes. However, this is an invasive approach that is constrained by the problems of lacking spatial resolution and genetic specificity. Recently, the development of a variety of fluorescent probes has made it possible to measure the activity of individual cells with high spatiotemporal resolution. The adaptation of this technique to image electrical activity in neurons has become an informative method to study neural circuits. Genetically encoded voltage indicators (GEVIs) can be used with superior performance to accurately target specific genetic populations and reveal neuronal dynamics on a millisecond scale. Microbial rhodopsins are commonly used as optogenetic actuators to manipulate neuronal activities and to explore the circuit mechanisms of brain function, but they also can be used as fluorescent voltage indicators. In this review, we summarize recent advances in the design and the application of rhodopsin-based GEVIs.

Rhodopsins: An Excitingly Versatile Protein Species for Research, Development and Creative Engineering

“…Meanwhile a host of additional voltage sensors have been developed. Next to optimized rhodopsins and chimeric rhodopsin fusions, fusion proteins of light-sensitive opsin cores with other fluorophores, often GFP derivatives or synthetic dyes, and of other voltage sensors with fluorescent rhodopsins have become popular ( Bando et al, 2019 ; Kannan et al, 2019 ; Lee et al, 2019 ; Berglund et al, 2020 ; Zhang X. M. et al, 2021 ).…”

Automated QM/MM Screening of Rhodopsin Variants with Enhanced Fluorescence

“…, light-gated ion channels or light-driven ion pumps), have been found to be instrumental to the development of optogenetics tools. ,,,− For this reason, they have been engineered to function as light-driven actuators, silencers, or fluorescent reporters of neuronal action potentials . Regarding the latter, specific microbial rhodopsins have been used to construct genetically encodable voltage indicators (GEVIs). − In such applications, a change in membrane voltage induces a variation in rhodopsin fluorescence intensity, which is directly used as a voltage indicator. ,, …”

“…It is evident that rhodopsins used as GEVIs must be fluorescent. A prototype fluorescent reporter is Archaerhodopsin-3 (Arch3), an archaeal rhodopsin from Halorubrum sodomense, with light-driven outward proton pumping activity. − However, since the fluorescence of Arch3 is extremely dim with a fluorescence quantum yield (ϕ f ) of ca. 1.1 × 10 –4 , extensive efforts have been made to search variants with enhanced fluorescence ( i.e.…”

“…1.1 × 10 –4 , extensive efforts have been made to search variants with enhanced fluorescence ( i.e. , via directed evolutionary approaches and random mutagenesis). − ,, Among others, variants such as QuasArs, Archons, , Archers, , Arch5, and Arch7 (see Table S1 in the Supporting Information) have been reported to feature a brighter fluorescence, enabling applications not only in the imaging of thin brain slices but also in living mammals and invertebrates. − Nevertheless, as shown in Table S2 in the Supporting Information, their ϕ f value remains in the range of 10 –3 –10 –2 and, therefore, not yet as bright as desirable.…”

“…The enhanced fluorescence exhibited by the Arch3 variants is triggered by the direct one-photon irradiation of the dark adapted (DA) state, leading to the direct formation of a fluorescent excited state (FS) upon relaxation from the Franck–Condon (FC) point of Figure B, , through a mechanism herein called “mechanism A”. This is very different and more effective than the fluorescence of Arch3 that originates from a photocycle intermediate and requires three photons to generate the spontaneously emitting state. ,,, In mechanism A (see Figure B), the spontaneous emission of FS competes with the rPSB isomerization, ultimately leading to excited-state decay through a conical intersection (CI). Such a competition is controlled by the processes capable of slowing down the progression along the isomerization coordinate.…”