Improving a genetically encoded voltage indicator by modifying the cytoplasmic charge composition

Lee, Sungmoo; Geiller, Tristan; Jung, Arong; Nakajima, Riho; Song, Yoon‐Kyu; Baker, Bradley J.

doi:10.1038/s41598-017-08731-2

Cited by 41 publications

(64 citation statements)

References 33 publications

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“…ArcLight has an on tau of over 10 ms but gives a 40% ΔF/F/100 mV [ 7 17 ]. This large signal size can overcome the slow speed of the response enabling the optical resolution of action potentials firing at 35 Hz [ 12 ]. Unfortunately, none of the V220 mutants give very large signals but do provide insights into manipulating the voltage range.…”

Section: Resultsmentioning

confidence: 99%

“…A V220T mutation in the VSD was found to shift the V 1/2 by increasing the voltage range of the GEVI, CC1. CC1 is an ArcLight-type probe utilizing the VSD of the voltage-sensing phosphatase from Ciona intestinalis [ 11 ] that requires a strong depolarization of the plasma membrane to induce an optical signal [ 10 12 ]. In an attempt to modify the voltage range, saturation mutagenesis of the V220 position was performed.…”

Section: Introductionmentioning

confidence: 99%

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Modulating the Voltage-sensitivity of a Genetically Encoded Voltage Indicator

et al. 2017

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Saturation mutagenesis was performed on a single position in the voltage-sensing domain (VSD) of a genetically encoded voltage indicator (GEVI). The VSD consists of four transmembrane helixes designated S1-S4. The V220 position located near the plasma membrane/extracellular interface had previously been shown to affect the voltage range of the optical signal. Introduction of polar amino acids at this position reduced the voltage-dependent optical signal of the GEVI. Negatively charged amino acids slightly reduced the optical signal by 33 percent while positively charge amino acids at this position reduced the optical signal by 80%. Surprisingly, the range of V220D was similar to that of V220K with shifted optical responses towards negative potentials. In contrast, the V220E mutant mirrored the responses of the V220R mutation suggesting that the length of the side chain plays in role in determining the voltage range of the GEVI. Charged mutations at the 219 position all behaved similarly slightly shifting the optical response to more negative potentials. Charged mutations to the 221 position behaved erratically suggesting interactions with the plasma membrane and/or other amino acids in the VSD. Introduction of bulky amino acids at the V220 position increased the range of the optical response to include hyperpolarizing signals. Combining The V220W mutant with the R217Q mutation resulted in a probe that reduced the depolarizing signal and enhanced the hyperpolarizing signal which may lead to GEVIs that only report neuronal inhibition.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Modulating the Voltage-sensitivity of a Genetically Encoded Voltage Indicator

et al. 2017

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“…Introducing the double mutant Y172A/D204K as well as the D164N into Pos6 [8], we made a new GEVI (named Plos6-v2). Plos6-v2 has a large signal size (~40% for 100 mV depolarizations) ( Figure 6).…”

Section: S3-s4 Loop Mutantsmentioning

confidence: 99%

“…GEVIs with fast kinetics and large dynamic signals are needed to follow the voltage transients of neurons. In recent years, several attempts have been made to improve GEVI's properties [6][7][8][9][10][11][12][13][14][15].…”

Section: Introductionmentioning

confidence: 99%

“…The resulting probe, Bongwoori, had sufficient speed and sensitivity to resolve action potentials in a hippocampal neuron firing at 60 Hz. Further improvement of the Bongwoori family of GEVIs was achieved by altering the charge composition of the region linking the fluorescent protein (FP) to the VSD domain [8]. Introduction of positively charged residues in the linker region improved the signal size of Bongwoori yielding fluorescent signals as high as 20% ΔF/F during action potentials.…”

Section: Introductionmentioning

confidence: 99%

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Conserved amino acids residing outside the voltage field can shift the voltage sensitivity and increase the signal size ofCionabased GEVIs

Sepehri

Cohen

Baker

2020

Preprint

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To identify potential regions of the voltage-sensing domain that could shift the voltage sensitivity of Ciona intestinalis based Genetically Encoded Voltage Indicators (GEVIs), we aligned 183 voltage-gated sodium channels from different organisms. Conserved polar residues were identified at multiple transmembrane loop junctions in the voltage sensing domain. Similar conservation of polar amino acids was found in the voltage sensing domain of the voltage-sensing phosphatase gene family. These conserved residues were mutated to nonpolar or oppositely charged amino acids in a GEVI that utilizes the voltage sensing domain of the voltage sensing phosphatase from Ciona fused to the fluorescent protein, super ecliptic pHlorin (A227D). Different mutations shifted the voltage sensitivity in a more positive or a more negative direction. Double mutants were then created by selecting constructs that shifted the optical signal to more negative potentials resulting in an improved signal in the physiological voltage range. The combination of two mutations, Y172A (intracellular loop between transmembrane segments S2 and S3) and D204K (extracellular loop between transmembrane segments S3 and S4) showed the increased the signal size from 2.5% to 13.8% for a 100 mV depolarization. Introduction of these mutations into previously developed GEVIs improved the dynamic range to 40% ΔF/F/100 mV.

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Recent Progress of Hybrid Optical Probes for Neural Membrane Potential Imaging

Liu

Chen

et al. 2020

Biotechnology Journal

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The neural membrane potential of nerve cells is the basis of neural activity production, which controls advanced brain activities such as memory, emotion, and learning. In the past decades, optical voltage indicator has emerged as a promising tool to decode neural activities with high-fidelity and excellent spatiotemporal resolution. In particular, the hybrid optical probes can combine the advantageous photophysical properties of different components such as voltage-sensitive molecules, highly fluorescent fluorophores, membrane-targeting tags, and optogenetic materials, thus showing numerous advantages in improving the photoluminescence intensity, voltage sensitivity, photostability, and cell specificity of probes. In this review, the current state-of-the-art hybrid probes are highlighted, that are designed by using fluorescent proteins, organic dyes, and fluorescent nanoprobes as the fluorophores, respectively. Then, the design strategies, voltage-sensing mechanisms and the in vitro and in vivo neural activity imaging applications of the hybrid probes are summarized. Finally, based on the current achievements of voltage imaging studies, the challenges and prospects for design and application of hybrid optical probes in the future are presented.

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Improving a genetically encoded voltage indicator by modifying the cytoplasmic charge composition

Cited by 41 publications

References 33 publications

Modulating the Voltage-sensitivity of a Genetically Encoded Voltage Indicator

Modulating the Voltage-sensitivity of a Genetically Encoded Voltage Indicator

Conserved amino acids residing outside the voltage field can shift the voltage sensitivity and increase the signal size ofCionabased GEVIs

Recent Progress of Hybrid Optical Probes for Neural Membrane Potential Imaging

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