A trafficking motif alters GEVI activity implicating persistent protein interactions at the membrane.

Efficient plasma-membrane expression is critical for genetically encoded voltage indicators (GEVIs). To improve the plasma-membrane expression, we introduced multiple combinations of plasma-membrane trafficking motifs at different positions to members of the Bongwoori family of GEVIs. An improvement from 20% to 27% in the ΔF/F/100 mV depolarization of the plasma membrane was observed when a Golgi transport motif was inserted near the N-terminus in conjunction with an endoplasmic reticulum release motif near the C-terminus of the protein.

Mechanism of ArcLight derived GEVIs involves electrostatic interactions that can affect proton wires.

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.

Improving the flexibility of genetically encoded voltage indicators via intermolecular FRET.

A new family of genetically encoded voltage indicators (GEVIs) has been developed based on intermolecular Förster 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.

Biophysical Parameters of GEVIs: Considerations for Imaging Voltage.

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.

Optical consequences of a genetically-encoded voltage indicator with a pH sensitive fluorescent protein.

Genetically-Encoded Voltage Indicators (GEVIs) are capable of converting changes in membrane potential into an optical signal. Here, we focus on recent insights into the mechanism of ArcLight-type probes and the consequences of utilizing a pH-dependent Fluorescent Protein (FP). A negative charge on the exterior of the β-can of the FP combined with a pH-sensitive FP enables voltage-dependent conformational changes to affect the fluorescence of the probe.

A dimeric fluorescent protein yields a bright, red-shifted GEVI capable of population signals in brain slice.

A bright, red-shifted Genetically Encoded Voltage Indicator (GEVI) was developed using a modified version of the fluorescent protein, tdTomato. Dimerization of the fluorescent domain for ArcLight-type GEVIs has been shown to affect the signal size of the voltage-dependent optical signal. For red-shifted GEVI development, tdTomato was split fusing a single dTomato chromophore to the voltage sensing domain.

Toward Better Genetically Encoded Sensors of Membrane Potential.

Genetically encoded optical sensors of cell activity are powerful tools that can be targeted to specific cell types. This is especially important in neuroscience because individual brain regions can include a multitude of different cell types. Optical imaging allows for simultaneous recording from numerous neurons or brain regions.

Pado, a fluorescent protein with proton channel activity can optically monitor membrane potential, intracellular pH, and map gap junctions.

An in silico search strategy was developed to identify potential voltage-sensing domains (VSD) for the development of genetically encoded voltage indicators (GEVIs). Using a conserved charge distribution in the S2 α-helix, a single in silico search yielded most voltage-sensing proteins including voltage-gated potassium channels, voltage-gated calcium channels, voltage-gated sodium channels, voltage-gated proton channels, and voltage-sensing phosphatases from organisms ranging from mammals to bacteria and plants. A GEVI utilizing the VSD from a voltage-gated proton channel identified from that search was able to optically report changes in membrane potential.

Combinatorial mutagenesis of the voltage-sensing domain enables the optical resolution of action potentials firing at 60 Hz by a genetically encoded fluorescent sensor of membrane potential.

ArcLight is a genetically encoded fluorescent voltage sensor using the voltage-sensing domain of the voltage-sensing phosphatase from Ciona intestinalis that gives a large but slow-responding optical signal in response to changes in membrane potential (Jin et al., 2012). Fluorescent voltage sensors using the voltage-sensing domain from other species give faster yet weaker optical signals (Baker et al.