From Plankton to Primates: How VSP Sequence Diversity Shapes Voltage Sensing.

Area of Science:

• Biophysics
• Molecular Biology
• Neuroscience

Background:

• Voltage-sensing phosphatases (VSPs) are crucial for understanding voltage sensing mechanisms.
• Genetically encoded voltage indicators (GEVIs) are engineered from VSPs for optical voltage detection.
• Natural sequence diversity in VSPs offers insights into functional evolution.

Purpose of the Study:

• To compare VSP voltage-sensing domains (VSDs) from different species to understand how sequence diversity affects function.
• To evaluate the impact of lineage-specific substitutions on VSP voltage-sensing properties.
• To guide the design of improved GEVIs by analyzing VSP behavior.

Main Methods:

• Engineered VSP constructs by replacing the phosphatase domain with a fluorescent protein.
• Expressed constructs in cells to enable optical detection of VSD responses.
• Compared voltage-dependent optical signals across VSP orthologs from various species.

Main Results:

• All plasma membrane-localized constructs showed voltage-dependent optical signals, confirming conserved voltage sensing.
• Plankton and sea hare VSPs exhibited left-shifted activation ranges.
• Human VSP2 showed poor recovery, improved by a specific arginine substitution.
• Chinese hamster VSP with atypical S4 charges displayed altered fluorescence responses.

Conclusions:

• Voltage-dependent signaling is conserved across VSPs, but lineage-specific variations create distinct functional phenotypes.
• Single amino acid changes can significantly alter VSD movement and reporter output.
• VSPs serve as valuable models for studying voltage sensing and optimizing GEVI development.