Transmembrane Hemoprotein Optical Reporters (THORs) for Membrane Potential Sensing

Abstract

Engineering probes that can image changes in membrane potential with high spatial and temporal resolution is essential for enhancing our understanding of the mechanisms that underlie neural activity. While organic voltage sensitive dyes report voltage signaling events with high sensitivity and temporal resolution, they lack the ability to be targeted genetically to specific cell types. Genetically encoded voltage indicators (GEVIs) can overcome this targeting problem; however, GEVIs tend to be both slower and dimmer when compared to voltage sensitive dyes. Here, we present our progress on the development and characterization of a novel family of GEVIs to improve the properties of current GEVIs. Our GEVIs are called Transmembrane Hemoprotein Optical Reporters (THORs), which consist of a transmembrane 4-α-helical protein and a fluorescent protein. The core of THORs binds heme cofactors, whose oxidation states are affected by the transmembrane electric field. The speed of inter-heme electron transfer is strongly dependent on the distance between cofactors. Based on the Moser-Dutton Ruler of electron transfer, we can achieve electron transfer rates on the µs timescales between heme cofactors. A fluorescent protein reports the change in redox state of one of the hemes via FRET. Water-soluble prototypes of THORs expressed in E. coli revealed an 18% change in fluorescence upon heme reduction for mOrange2 and a 59% change for Sirius. Two different lengths of THORs were designed: one whose length barely exceeds the thickness of a membrane and a second one which extends into the cytoplasm. THORs have been expressed in HEK293t cells and rat hippocampal neurons. In rat hippocampal neurons, a depolarization of the membrane via potassium gradient decreased the fluorescence of THORs, as expected. Currently, we are using whole cell patch clamp techniques to determine the temporal response of THORs.