The ability to optically image cellular transmembrane voltages at millisecond-timescale resolutions can offer unprecedented insight into the function of living brains in behaving animals. Here, we present a point mutation that increases the sensitivity of Ace2 opsin-based voltage indicators. We use the mutation to develop Voltron2, an improved chemigeneic voltage indicator that has a 65% higher sensitivity to single APs and 3-fold higher sensitivity to subthreshold potentials than Voltron. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, although with lower baseline fluorescence. In multiple in vitro and in vivo comparisons with its predecessor across multiple species, we found Voltron2 to be more sensitive to APs and subthreshold fluctuations. Finally, we used Voltron2 to study and evaluate the possible mechanisms of interneuron synchronization in the mouse hippocampus. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.

• Klíčová slova
• biosensors, fluorescence imaging, fluorescent proteins, genetically encoded indicators, voltage imaging,
• MeSH
• akční potenciály fyziologie   MeSH
• angiotensin-konvertující enzym 2 * MeSH
• mutace genetika   MeSH
• myši MeSH
• neurony fyziologie   MeSH
• rodopsin * genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• angiotensin-konvertující enzym 2 * MeSH
• rodopsin * MeSH

Visualization of electrical activity in living cells represents an important challenge in context of basic neurophysiological studies. Here we report a new voltage sensitive fluorescent indicator which response could be detected by fluorescence monitoring in a single red channel. To the best of our knowledge, this is the first fluorescent protein-based voltage sensor which uses insertion-into-circular permutant topology to provide an efficient interaction between sensitive and reporter domains. Its fluorescent core originates from red fluorescent protein (FP) FusionRed, which has optimal spectral characteristics to be used in whole body imaging techniques. Indicators using the same domain topology could become a new perspective for the FP-based voltage sensors that are traditionally based on Förster resonance energy transfer (FRET).

• MeSH
• biosenzitivní techniky metody   MeSH
• červený fluorescenční protein MeSH
• elektrofyziologické jevy MeSH
• fluorescenční barviva metabolismus   MeSH
• HEK293 buňky MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• luminescentní proteiny chemie   MeSH
• nádorové buněčné linie MeSH
• proteinové domény MeSH
• proteinové inženýrství metody   MeSH
• rezonanční přenos fluorescenční energie metody   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fluorescenční barviva MeSH
• luminescentní proteiny MeSH

ArcLight, a genetically encoded fluorescent protein voltage probe with a large ΔF/ΔV, is a fusion between the voltage sensing domain of the Ciona instestinalis voltage sensitive phosphatase and super ecliptic pHluorin carrying a single mutation (A227D in the fluorescent protein). Without this mutation the probe produces only a very small change in fluorescence in response to voltage deflections (∼ 1%). The large signal afforded by this mutation allows optical detection of action potentials and sub-threshold electrical events in single-trials in vitro and in vivo. However, it is unclear how this single mutation produces a probe with such a large modulation of its fluorescence output with changes in membrane potential. In this study, we identified which residues in super ecliptic pHluorin (vs eGFP) are critical for the ArcLight response, as a similarly constructed probe based on eGFP also exhibits large response amplitude if it carries these critical residues. We found that D147 is responsible for determining the pH sensitivity of the fluorescent protein used in these probes but by itself does not result in a voltage probe with a large signal. We also provide evidence that the voltage dependent signal of ArcLight is not simply sensing environmental pH changes. A two-photon polarization microscopy study showed that ArcLight's response to changes in membrane potential includes a reorientation of the super ecliptic pHluorin. We also explored different changes including modification of linker length, deletion of non-essential amino acids in the super ecliptic pHluorin, adding a farnesylation site, using tandem fluorescent proteins and other pH sensitive fluorescent proteins.

• MeSH
• akční potenciály * MeSH
• aminokyseliny chemie   genetika   metabolismus   MeSH
• fluorescence MeSH
• fluorescenční barviva chemie   metabolismus   MeSH
• fluorescenční spektrometrie MeSH
• HEK293 buňky MeSH
• kinetika MeSH
• koncentrace vodíkových iontů MeSH
• konfokální mikroskopie MeSH
• krysa rodu Rattus MeSH
• kultivované buňky MeSH
• lidé MeSH
• luminescentní proteiny chemie   genetika   metabolismus   MeSH
• membránové potenciály MeSH
• metoda terčíkového zámku MeSH
• missense mutace MeSH
• neurony metabolismus   fyziologie   MeSH
• prenylace MeSH
• rekombinantní fúzní proteiny chemie   genetika   metabolismus   MeSH
• zelené fluorescenční proteiny chemie   genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, American Recovery and Reinvestment Act MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• aminokyseliny MeSH
• Arclight fusion protein MeSH Prohlížeč
• fluorescenční barviva MeSH
• luminescentní proteiny MeSH
• PHluorin MeSH Prohlížeč
• rekombinantní fúzní proteiny MeSH
• zelené fluorescenční proteiny MeSH