Screening Gevis with Voltage Sensing Domains from Different Species of the Voltage Sensing Phosphatase Gene Family Reveals Differences in Membrane Expression and Voltage Ranges

Leong Lee MinLaura Bilbao BrochYouna KimSIMS SHACAROL NICOLEIryna MartsishevskaMD SOFEQUL ISLAM MUKIMJu-Young Lee김지원송민주송지현주연하마리아 고들레우스카Bradley Baker
Issue Date
Published for the Biophysical Society by the Rockefeller University Press
65th Biophysical Society Annual Meeting, pp.366A
Genetically encoded voltage indicators offer an opportunity to investigate the factors determining the voltage range of the voltage sensing phosphatase (VSP) gene family. The GEVI ArcLight utilizes a mutated form of the voltage sensing domain (VSD) from the sea squirt Ciona intestinalis to optically report membrane potentials in the physiological range since the wildtype VSD from Ciona exhibits a V1/2 (the membrane potential at which half of the total fluorescence change is observed) of þ100 mV. A single mutation to the Ciona VSD changed the V1/2 to 30 mV for ArcLight. To investigate how nature determines the voltage range of a protein, we have generated several new GEVIs substituting the VSD from Ciona with other species ranging from mite to octopus to coelacanth to the zooplankton Eurytemora affinis. Most of these new GEVIs have similar V1/2 values to the Ciona wildtype VSD with some notable exceptions. The zooplankton GEVI had a V1/2 of þ35 mV exhibiting a maximal fluorescence change of 18% DF/F for a 200 mV depolarization. No GEVI utilizing mammalian versions of the VSD from the phosphatase gene family yielded a signal due to poor plasma membrane expression suggesting the presence of a regulatory element in the mammalian VSP gene. Chimeras are now being generated in order to improve membrane expression and signal size.
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