Comparison of the optical response of Bongwoori-R3 to Bongwoori and Bongwoori-Pos6.
Bongwoori (봉우리)
PEAK
Bongwoori (green trace) is an Arclight derivative consisting of three mutations to the voltage-sensing domain that drastically improved the on and off kinetics of the optical response (Piao et al., 2015).
Bongwoori-Pos6 (larger signal)
Bongwoori-Pos6 (blue trace) is a derivative of Bongwoori that has positive charges in the linker region between the voltage-sensing domain and the fluorescent protein (Lee et al., 2017).
Bongwoori-R3 (voltage response shifted towards action potentials)
Bongwoori-R3 (purple trace) is a derivative of Bongwoori that has a positive charge at position 3 of the linker between the voltage-sensing domain and the fluorescent protein which shifts the optical signal to more positive potentials (Lee et al., 2017).
Pado (파도)
Wave
The only GEVI that utilizes the voltage-sensing domain from a voltage-gated proton channel. This GEVI is able to optically report multiple down states of the voltage sensor. The fluorescent protein is pH sensitive Super Ecliptic pHlorin that is also used in Arclight and Bongwoori. The voltage-gated proton channel activity is preserved (see current in Pado trace). A 200 mV depolarization of the plasma membrane activates the channel activity allowing protons to escape thereby raising the internal pH (shift in baseline). Notice that the 100 mV depolarization does not activate the channel but still yields an optical signal (Kang and Baker, 2016).
Representative fluorescent (red) and current (blue) traces of an HEK cell expressing Pado in the whole cell patch clamp configuration.
The amino acid sequence of the varying linker lengths (LK1-LK22) to optimize voltage-dependent signal. The linker segment is in green. dTomato sequence is in red. The GDP sequence between the linker and dTomato is due to a BamHI cloning site.
Optical traces in response to membrane depolarizations. The average of at least four HEK cells expressing LK1 (green), LK7 (purple), LK11 (blue), LK15 (red), and LK22 (black). The color-coordinated shaded areas are standard errors of the mean. The command voltage pulses are shown below in black. An offline, low-pass Gaussian filter was used for all cells.
Ilmol (일몰)
Sunset
Ilmol is a modified version of the fluorescent protein, tdTomato, fused to the VSD of Bongwoori. Optimization of the amino acid length and charge composition of the linker region between the voltage sensing domain and the fluorescent protein resulted in a probe that yields a 3-fold improvement in the signal-to-noise ratio compared to FlicR1. It is the brightest red-shifted GEVI to date (Yi et al., 2018).
Aahn (안)
Inside
An ArcLight-type GEVI with mutations in its VSD loops. Opposite polarity signals make it capable of optically monitoring both plasma membrane and internal membrane potentials. Responds to changes in plasma membrane potential.
The average of first 4 trials observed in the plasma membrane (blue trace) and internal membrane (red trace) without temporal filtering.
Nabi1 probes contain mKO and UKG as the FRET pair at flanking regions of the Ciona voltage sensitive domain. Nabi2 has Clover and mRuby2 as the FRET pair. Nabi1 and Nabi2 are also different in the linkers between the Ciona voltage sensing domain (CiVSD) and FP β-cans. FPs are color-coded as mKO (orange), UKG (dark green), Clover (bright green), and mRuby2 (red).
Representative donor (UKG, green) and acceptor (mKO, orange) signals of Nabi1.213, Nabi1.242, and Nabi1.244 are shown. Representative donor (Clover; green) and acceptor (mRuby2; red) signals of Nabi2.213, Nabi2.242, and Nabi2.244. All of the traces were from single trials and without temporal filtering.
Nabi (나비)
Butterfly
Developing FP voltage sensors that utilize FRET between two FPs can be useful for in vivo imaging because the ratio of the FRET signals reduces common source noise such as heartbeat and breathing artifacts. A combinatorial library of constructs, named “Nabi,” were generated with one FP at 6 different locations in the N-terminus and the other FP at 8 different locations downstream of S4 in the C-terminus. We created two groups of FRET based voltage sensors, Nabi1 with UKG and mKO as the FRET pair, and Nabi2 with Clover and mRuby2 as the pair. Each Nabi1 and Nabi2 probe displayed voltage dependent fluorescence changes with unique signal kinetics and size. Many Nabi probes showed signals with improved signal size and faster kinetics than previously described butterfly FRET based voltage sensors such as VSFP Butterfly 1.2 and VSFP-CR (Sung et al., 2015).