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