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

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. Every construct that reached the plasma membrane produced a voltage-dependent optical signal, underscoring the deep conservation of voltage sensing across the family. Yet lineage-specific substitutions generated strikingly different phenotypes. A plankton ortholog exhibited a strongly left-shifted activation range, producing robust signals at modest depolarizations. The human VSD 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 hamster 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 demonstrate that even single residue changes can reorient the coupling between VSD motion and the fused fluorescent protein, revealing the fine mechanistic resolution accessible through GEVI readouts. 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.