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Related Experiment Video

Updated: Feb 19, 2026

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors
09:57

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Modulating the Voltage-sensitivity of a Genetically Encoded Voltage Indicator.

Arong Jung1,2, Dhanarajan Rajakumar1, Bong-June Yoon2

  • 1The Center for Functional Connectomics, Korea Institute of Science and Technology, Seoul, Korea.

Experimental Neurobiology
|November 3, 2017
PubMed
Summary

Saturation mutagenesis of voltage-sensing domains in genetically encoded voltage indicators (GEVIs) revealed how specific amino acid changes alter optical signals. Bulky mutations at V220 expanded the optical response range, potentially enabling new GEVI probes.

Keywords:
FluorescenceGEVIVoltage rangeVoltage sensing domain

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Area of Science:

  • Molecular Biology
  • Biophysics
  • Neuroscience

Background:

  • Genetically encoded voltage indicators (GEVIs) are crucial tools for monitoring cellular electrical activity.
  • The voltage-sensing domain (VSD) of GEVIs, composed of transmembrane helices, governs their voltage sensitivity.
  • Previous studies identified the V220 position in the VSD as critical for the optical signal's voltage range.

Purpose of the Study:

  • To investigate the impact of saturation mutagenesis at the V220 position of a GEVI's VSD on its optical voltage sensing properties.
  • To explore how different amino acid properties (charge, size, polarity) at V220 influence GEVI performance.
  • To assess the potential for engineering novel GEVIs with tailored voltage response characteristics.

Main Methods:

  • Saturation mutagenesis was employed at the V220 position within the VSD of a GEVI.
  • Various amino acid substitutions (polar, charged, bulky) were introduced at V220.
  • Optical signals were measured to evaluate changes in voltage-dependent responses.
  • Mutations at adjacent positions (219, 221) and combined mutations were also analyzed.

Main Results:

  • Introduction of polar amino acids at V220 reduced the voltage-dependent optical signal.
  • Negatively charged amino acids caused a 33% reduction, while positively charged amino acids caused an 80% reduction.
  • Mutations V220D and V220K showed similar optical responses shifted towards negative potentials.
  • The V220E mutant's response mirrored V220R, suggesting side chain length is a factor.
  • Bulky amino acid substitutions at V220 expanded the optical response range to include hyperpolarizing potentials.
  • A combined V220W/R217Q mutation reduced depolarizing signals and enhanced hyperpolarizing signals.

Conclusions:

  • Amino acid identity at the V220 position significantly modulates the voltage-sensing properties of GEVIs.
  • Side chain length and charge play critical roles in determining the voltage range and response characteristics.
  • Engineering GEVIs with expanded voltage sensitivity, particularly for hyperpolarizing potentials, is achievable.
  • The V220W/R217Q double mutant shows promise for developing GEVIs that specifically report neuronal inhibition.