Conserved amino acids residing outside the voltage field can shift the voltage sensitivity and increase the signal size of Ciona based GEVIs.
- Conserved amino acids residing outside the voltage field can shift the voltage sensitivity and increase the signal size of Ciona based GEVIs.
- 브래들리 베이커; 마소드; 래리코헨
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- 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 183 voltagegated 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 pHlorin (A227D). Different mutations shifted the voltage sensitivity in a more positive or a more negative direction. Double mutants were then created by selecting constructs that shifted the optical signal to more negative potentials resulting in an improved signal in the physiological voltage range. The combination of two mutations, Y172A (intracellular loop between transmembrane segments S2 and S3) and D204K (extracellular loop between transmembrane segments S3 and S4) showed the increased the signal size from 2.5% to 13.8% for a 100 mV depolarization. Introduction of these mutations into previously developed GEVIs improved the dynamic range to 40% ？F/F/100 mV.
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