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.
Highlights
ArcLight [1] is a genetically encoded fluorescent voltage probe is based on the voltage sensing domain (VSD) of Ciona instestinalis voltage sensitive phosphatase (Ci-Voltage sensitive phosphatase (VSP)) [2] fused to the fluorescent protein (FP) super ecliptic pHluorin [3] carrying a single point mutation (A227D in the FP)
Replacing of the super ecliptic pHluorin A227D in ArcLight with eGFP resulted in a probe with little voltage sensitivity [1]. eGFP differs from super ecliptic pHluorin at only nine residues (R80Q, D147S, Q149N, A163V, G175S, F202S, T204Q, T206A, H231L)
D147, F202 and T204, in super ecliptic pHluorin A227D, which are required for producing ArcLight signals [1]
Summary
ArcLight [1] is a genetically encoded fluorescent voltage probe is based on the voltage sensing domain (VSD) of Ciona instestinalis voltage sensitive phosphatase (Ci-VSP) [2] fused to the fluorescent protein (FP) super ecliptic pHluorin [3] carrying a single point mutation (A227D in the FP). ArcLight allows reliable detection of single action potentials and sub-threshold electric events in cultured hippocampal neurons in single trials [1] and in vivo in Drosophila [4]. It is unclear how this mutation, A227D, produced a probe which has such a large modulation of its fluorescence output in response to changes of membrane potential it does appear to be transferable to other voltage sensors [5]. ASAP1 is based on chicken VSP and circularly permuted GFP [12] A new approach to combine the rapid kinetics of a fungal rhodopsin with the brightness of genetically engineered protein fluorophores resulted in a probe with fast kinetics and brightness [15,16]
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