The first GEVI to consistently yield voltage-dependent optical signals in mammalian cells was VSFP2 which consisted of two FPs fused in tandem to the carboxy terminus of the voltage sensing domain (VSD) from the Ciona voltage sensing phosphatase ( Dimitrov et al., 2007). In recent years, several attempts have been reported to improve GEVI’s properties ( Gong et al., 2015 Piao et al., 2015 Lou et al., 2016 Storace et al., 2016 Jung et al., 2017 Lee et al., 2017 Sepehri Rad et al., 2017 Piatkevich et al., 2018 Adam et al., 2019 Lee et al., 2019). GEVIs with fast kinetics and large dynamic signals are needed to follow the voltage transients of neurons. Optimizing the properties of the GEVIs is important for improving the utility of GEVIs for imaging fast electrical activities in neural tissue and in vivo ( Storace et al., 2015 Inagaki et al., 2017). Kinetics, brightness and the signal size are some of the important properties of a GEVI ( Bando et al., 2019 Rhee et al., 2020). Genetically encoded voltage indicators (GEVIs) are potentially powerful tools for monitoring electrical activity in the brain. Thus, Plos6-v2 appears to be the GEVI of choice. The onset time constant of Plos6-v2 is also 50% faster than ArcLight. Introduction of these mutations into previously developed GEVIs resulted in Plos6-v2 which improved the dynamic range to 40% ΔF/F/100 mV, a 25% increase over the parent, ArcLight. Double mutants were then created by selecting constructs that shifted the optical signal to a more physiologically relevant voltage range. Different mutations shifted the voltage sensitivity to more positive or more negative membrane potentials. These conserved residues were mutated to nonpolar or oppositely charged amino acids in a GEVI that utilizes the voltage sensing domain of the voltage sensing phosphatase from Ciona fused to the fluorescent protein, super ecliptic pHluorin (A227D). Similar conservation of polar amino acids was found in the voltage-sensing domain of the voltage-sensing phosphatase gene family. Conserved polar residues were identified at multiple transmembrane/loop junctions in the voltage sensing domain. To identify potential regions of the voltage-sensing domain that could shift the voltage sensitivity of Ciona intestinalis based Genetically Encoded Voltage Indicators (GEVIs), we aligned the amino acid sequences of voltage-gated sodium channels from different organisms.
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