Genetically encoded voltage indicators (GEVIs) continue to evolve, resulting in many different probes with varying strengths and weaknesses. Developers of new GEVIs tend to highlight their positive features. A recent article from an independent laboratory has compared the signal/noise ratios of a number of GEVIs. Such a comparison can be helpful to investigators eager to try to image the voltage of excitable cells. In this perspective, we will present examples of how the biophysical features of GEVIs affect the imaging of excitable cells in an effort to assist researchers when considering probes for their specific needs.
The genetically encoded voltage indicators ArcLight and its derivatives mediate voltage-dependent optical signals by intermolecular, electrostatic interactions between neighboring fluorescent proteins (FPs). A random mutagenesis event placed a negative charge on the exterior of the FP, resulting in a greater than 10-fold improvement of the voltage-dependent optical signal. Repositioning this negative charge on the exterior of the FP reversed the polarity of voltage-dependent optical signals, suggesting the presence of ''hot spots'' capable of interacting with the negative charge on a neighboring FP, thereby changing the fluorescent output. To explore the potential effect on the chromophore state, voltage-clamp fluorometry was performed with alternating excitation at 390 nm followed by excitation at 470 nm, resulting in several mutants exhibiting voltage-dependent, ratiometric optical signals of opposing polarities. However, the kinetics, voltage ranges, and optimal FP fusion sites were different depending on the wavelength of excitation. These results suggest that the FP has external, electrostatic pathways capable of quenching fluorescence that are wavelength specific. One mutation to the FP (E222H) showed a voltage-dependent increase in fluorescence when excited at 390 nm, indicating the ability to affect the proton wire from the protonated chromophore to the H222 position. ArcLight-derived sensors may therefore offer a novel way to map how conditions external to the b-can structure can affect the fluorescence of the chromophore and transiently affect those pathways via conformational changes mediated by manipulating membrane potential.
A new family of genetically encoded voltage indicators (GEVIs) has been developed based on intermolecular Fö rster resonance energy transfer (FRET). To test the hypothesis that the GEVI ArcLight functions via interactions between the fluorescent protein (FP) domains of neighboring probes, the FP of ArcLight was replaced with either a FRET donor or acceptor FP. We discovered relatively large FRET signals only when cells were cotransfected with both the FRET donor and acceptor GEVIs. Using a cyan fluorescent protein donor and an RFP acceptor, we were able to observe a voltage-dependent signal with an emission peak separated by over 200 nm from the excitation wavelength. The intermolecular FRET strategy also works for rhodopsin-based probes, potentially improving their flexibility as well. Separating the FRET pair into two distinct proteins has important advantages over intramolecular FRET constructs. The signals are larger because the voltage-induced conformational change moves two FPs independently. The expression of the FRET donor and acceptor can also be restricted independently, enabling greater cell type specificity as well as refined subcellular voltage reporting.
A new family of Genetically Encoded Voltage Indicators (GEVIs) has been developed based on inter-molecular Förster Resonance Energy Transfer (FRET). To test the hypothesis that the GEVI, ArcLight, functions via interactions between the fluorescent protein (FP) domain of neighboring probes, the FP of ArcLight was replaced with either a FRET donor or acceptor FP. We discovered relatively large FRET signals only when cells were co-transfected with both the FRET donor and acceptor GEVIs. Using a CFP donor and an RFP acceptor, we were able to observe a voltage dependent signal with a Stokes shift of over 200 nm. The intermolecular FRET strategy also works for rhodopsin-based probes potentially improving their flexibility as well. Separating the FRET pair into two distinct proteins has important advantages over intramolecular FRET constructs. First, the signals are larger. Apparently the voltage-induced conformational change moves the two FPs independently thereby increasing the dynamic range. Second, the expression of the FRET donor and acceptor can be restricted independently enabling greater cell type specificity as well as refined subcellular voltage reporting.
Electrodynamic pathways through the fluorescent protein domain enable the genetically encoded voltage indicator (GEVI) ArcLight and its derivatives to optically report conformational changes induced by membrane potential transients. These electrical pathways through the fluorescent protein domain of Super Ecliptic pHluorin exhibit remarkable precision as demonstrated by the divergent optical behavior of Gln versus Asn mutations. At position D147 on the external surface of the fluorescent protein domain, Gln exhibits a voltage-dependent behavior like other negatively charged acidic amino acids at that location while the Asn mutant exhibits a response similar to the charged positive amino acids at that position. Replacement of Thr with Gln at the 203 position with the amino acid side chain in close proximity to the internal chromophore does not affect the speed of the voltage-dependent optical signal, but replacement with Asn slows the voltage-dependent optical signal implicating a twisting of the chromophore in response to transient charge interactions on the exterior of the β-can being altered during the voltage-induced conformational change. In addition to polar amino acids affecting these electrodynamic pathways, hydrophobic residues play a noticeable role in the migration of charge from the exterior of the fluorescent protein to the chromophore potentially by shielding/creating inducible dipoles on the surface of the fluorescent protein. Using these novel insights, a new GEVI that gets significantly brighter upon depolarization of the plasma membrane was developed enabling voltage imaging of population signals with low light levels at a frame rate of 5 kHz in acute brain slice as well as the reporting of neuronal activity in vivo.
The genetically encoded voltage indicators, ArcLight and its derivatives, mediate voltage dependent optical signals by intermolecular, electrostatic interactions between neighboring fluorescent proteins (FPs) via proton wires. A random mutagenesis event placed a negative charge on the exterior of the FP resulting in a greater than 10-fold improvement of the voltage-dependent optical signal. Repositioning this negative charge on the exterior of the FP reversed the polarity of voltage-dependent optical signals suggesting the presence of ‘hot spots’ capable of interacting with the negative charge on a neighboring FP thereby changing the fluorescent output. To explore the potential effect on the chromophore state, voltage-clamp fluorometry was performed with alternating excitation at 390 nm followed by excitation at 470 nm resulting in several mutants exhibiting voltage-dependent, ratiometric optical signals of opposing polarities. However, the kinetics, voltage ranges, and optimal FP fusion sites were different depending on the wavelength of excitation. These results suggest that the FP has external, electrostatic pathways capable of quenching fluorescence that are wavelength specific. ArcLight-derived GEVIs may therefore offer a novel way to map how conditions external to the β-can structure can affect the fluorescence of the chromophore and transiently manipulate those pathways via conformational changes mediated by whole cell voltage clamp.Statement of SignificanceArcLight-type GEVIs utilize proton pathways that send charge information outside of the FP to the internal chromophore enabling voltage induced conformational changes to affect fluorescence. These pathways are excitation wavelength specific suggesting that different external positions affect the protonated and deprotonated states of the chromophore.
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