Conserved Amino Acids Residing Outside the Voltage Field Can Shift the Voltage Sensitivity and Increase the Signal Speed and Size of Ciona Based GEVIs

Abstract

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 the amino acid sequences of 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 pHluorin (A227D). Different mutations shifted the voltage sensitivity to more positive or more negative membrane potentials. Double mutants were then created by selecting constructs that shifted the optical signal to a more physiologically relevant voltage range. Introduction of these mutations into previously developed GEVIs resulted in Plos6-v2 which improved the dynamic range to 40% ΔF/F/100 mV, a 25% increase over the parent, ArcLight. The onset time constant of Plos6-v2 is also 50% faster than ArcLight. Thus, Plos6-v2 appears to be the GEVI of choice.

An Ultrasensitive Genetically Encoded Voltage Indicator Uncovers the Electrical Activity of Non‐Excitable Cells

“…Aiming to maintain the improvements obtained with the Q396R mutation and shift V half into the target V m range, we generated a structural model of ASAP3:Q396R that most closely resembled the up state of the VSD [ 28 , 40 , 41 ] and performed site‐saturation mutagenesis based on mKate2‐ASAP3:Q396R at three residues (T134, N138, and Y141) in transmembrane segment 3 (Figure 1E ). These residues are in close proximity to two of the voltage‐sensing charges in S4 and are therefore likely to influence the voltage‐sensor movement.…”

“…The existence of states below the down state and above the up state is supported by a recent study by Shen et al, which reports the presence of additional states (up-plus and down-minus) in the VSD of the Ciona intestinalis (Ci) VSP. [28] Aiming to maintain the improvements obtained with the Q396R mutation and shift V half into the target V m range, we generated a structural model of ASAP3:Q396R that most closely resembled the up state of the VSD [28,40,41] and performed sitesaturation mutagenesis based on mKate2-ASAP3:Q396R at three residues (T134, N138, and Y141) in transmembrane segment 3 (Figure 1E). These residues are in close proximity to two of the voltage-sensing charges in S4 and are therefore likely to influence the voltage-sensor movement.…”

An ultrasensitive genetically encoded voltage indicator uncovers the electrical activity of non-excitable cells

“…We aimed to maintain the improvements obtained with the Q396R mutation and shift V half into the target V m range. Therefore, we generated a structural model of ASAP3:Q396R that most closely resembled the up state of the VSD (28,40,41) and performed site-saturation mutagenesis based on mKate2-ASAP3:Q396R at three residues (T134, N138, and Y141) in transmembrane segment 3 (Fig. 1E).…”

Imaging different cell populations in the mouse olfactory bulb using the genetically encoded voltage indicator ArcLight

“… 19 , 72 Several biophysical properties of a GEVI are important in evaluating its ability to optically report voltage signals in neural tissue and in the interpretation of its optical signals. 73 – 75 The voltage-sensitivity range of a GEVI determines the absolute voltage changes that will be translated into optical changes. 75 – 77 The sensitivity of a GEVI is defined as the dynamic range of the fluorescence change across its voltage-sensitivity range, which is a key determinant of the signal-to-noise ratio.…”

“… 73 – 75 The voltage-sensitivity range of a GEVI determines the absolute voltage changes that will be translated into optical changes. 75 – 77 The sensitivity of a GEVI is defined as the dynamic range of the fluorescence change across its voltage-sensitivity range, which is a key determinant of the signal-to-noise ratio. GEVI brightness is important because relatively few protein molecules can be introduced into the membrane and rapid voltage changes require fast frame rates that limit photon integration time.…”