Sepehri Rad, Masoud Cohen, Lawrence B. Baker, Bradley J. 2024-01-19T12:00:25Z 2024-01-19T12:00:25Z 2022-07-14 2022-06 2296-634X https://pubs.kist.re.kr/handle/201004/115105 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. Conserved polar residues were identified at multiple transmembrane/loop junctions in the voltage sensing domain. Similar conservation of polar amino acids was found in the voltage-sensing domain of the voltage-sensing phosphatase gene family. 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). Different mutations shifted the voltage sensitivity to more positive or more negative membrane potentials. Double mutants were then created by selecting constructs that shifted the optical signal to a more physiologically relevant voltage range. Introduction of these mutations into previously developed GEVIs resulted in Plos6-v2 which improved the dynamic range to 40% Delta F/F/100 mV, a 25% increase over the parent, ArcLight. The onset time constant of Plos6-v2 is also 50% faster than ArcLight. Thus, Plos6-v2 appears to be the GEVI of choice. English Frontiers Media S.A. Conserved Amino Acids Residing Outside the Voltage Field Can Shift the Voltage Sensitivity and Increase the Signal Speed and Size of Ciona Based GEVIs Article 10.3389/fcell.2022.868143 1 Frontiers in Cell and Developmental Biology, v.10 Frontiers in Cell and Developmental Biology 10 Y scie scopus 000819140300001 Cell Biology Developmental Biology Cell Biology Developmental Biology Article ACTION-POTENTIALS TRANSMISSION MECHANISM NEURONS voltage voltage imaging GEVI fluorescence voltage sensing domain