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

## TL;DR

This study explores how natural sequence diversity in voltage-sensing domains affects voltage sensing across different species, using fluorescent proteins to track changes.

## Contribution

The study reveals how lineage-specific substitutions in VSPs alter voltage sensing and GEVI performance, offering insights into voltage-sensing mechanisms.

## Key findings

- VSP orthologs from different species show conserved voltage sensing but distinct activation ranges due to lineage-specific substitutions.
- Modifying specific residues in VSPs can significantly alter fluorescence responses, indicating the impact of single residue changes on GEVI behavior.
- The study demonstrates that VSPs are valuable models for understanding voltage sensing and improving GEVI design.

## Abstract

Voltage-sensing phosphatases (VSPs) provide a conserved framework for dissecting the mechanics of voltage sensing and for engineering genetically encoded voltage indicators (GEVIs). To evaluate how natural sequence diversity shapes function, we compared VSP voltage-sensing domains (VSDs) from multiple species by replacing the phosphatase domain with a fluorescent protein to enable optical detection of VSD responses. Every construct that reached the plasma membrane produced a voltage-dependent optical signal, underscoring the deep conservation of voltage sensing across VSP orthologs. Yet lineage-specific substitutions generated strikingly different phenotypes. A plankton VSP ortholog from Eurytemora carolleeae and the Sea Hare (Aplysia californica) VSP exhibited left-shifted activation ranges, producing robust fluorescence transitions during modest depolarizations of the plasma membrane. The human VSD of hVSP2 yielded weak, sluggish responses with poor recovery, but reintroduction of a conserved arginine in S1 (G95R) partially restored reversibility, implicating lipid-facing residues in conformational stability. The Chinese hamster (Cricetulus griseus) VSD, with atypical S4 sensing charges (RWIR), generated a slow fluorescence increase during depolarization, while reverting to the consensus arginine (RRIR) inverted the polarity to a decrease. These contrasting behaviors show that single residue changes can reshape how VSD movements influence the fluorescent reporter, highlighting the molecular precision revealed by GEVI measurements. Together, these results show that voltage-dependent signaling is deeply conserved across VSPs but shaped by lineage-specific sequence variation, establishing VSPs as powerful models for probing voltage sensing and guiding GEVI design.