Abstract
Fusion of a voltage sensitive domain (VSD) to the fluorescent protein (FP), Super Ecliptic pHluorin A227D, allows for the optical measurement of changes in membrane potential. However, the mechanism coupling fluorescence changes to changes in membrane potential is not fully understood. Primary studies of this mechanism suggest that dimerization via the cytoplasmic fluorescent protein domain is required. To further explore the dimerization effect of the genetically encoded voltage indicator (GEVI) via intermolecular Förster Resonance Energy Transfer (FRET), novel constructs fusing FRET donors or FRET acceptors to the VSD were constructed and co-transfected into HEK-293 cells. CFP-YFP FRET pairs showed a higher resting FRET than GFP-RFP FRET pairs presumably due to the dimerization of the CFP-YFP pairs. Robust voltage-dependent FRET signals were observed which suggested the movement of S4 transmembrane segment upon depolarization of the plasma membrane improved the FRET efficiency between the chromophore dipoles of VSDs tuned to different potentials. For instance, a FRET donor attached to a VSD tuned to extreme positive potentials can be paired with a FRET acceptor coupled to a VSD that responds to physiologically relevant potentials. This combination results in a GEVI that will only move one S4 transmembrane segment or both S4 transmembrane segments depending on the strength of the depolarization of the plasma membrane. Research reported in this publication was supported by the National Institute Of Neurological Disorders And Stroke of the National Institutes of Health under Award Number U01NS099691.
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