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Mechanistic studies of the genetically encoded fluorescent protein voltage probe ArcLight.
- Source :
-
PloS one [PLoS One] 2014 Nov 24; Vol. 9 (11), pp. e113873. Date of Electronic Publication: 2014 Nov 24 (Print Publication: 2014). - Publication Year :
- 2014
-
Abstract
- 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.
- Subjects :
- Amino Acids chemistry
Amino Acids genetics
Amino Acids metabolism
Animals
Cells, Cultured
Fluorescence
Fluorescent Dyes metabolism
Green Fluorescent Proteins genetics
Green Fluorescent Proteins metabolism
HEK293 Cells
Humans
Hydrogen-Ion Concentration
Kinetics
Luminescent Proteins genetics
Luminescent Proteins metabolism
Membrane Potentials
Microscopy, Confocal
Mutation, Missense
Neurons metabolism
Neurons physiology
Patch-Clamp Techniques
Prenylation
Rats
Recombinant Fusion Proteins genetics
Recombinant Fusion Proteins metabolism
Spectrometry, Fluorescence
Action Potentials
Fluorescent Dyes chemistry
Green Fluorescent Proteins chemistry
Luminescent Proteins chemistry
Recombinant Fusion Proteins chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 1932-6203
- Volume :
- 9
- Issue :
- 11
- Database :
- MEDLINE
- Journal :
- PloS one
- Publication Type :
- Academic Journal
- Accession number :
- 25419571
- Full Text :
- https://doi.org/10.1371/journal.pone.0113873