Gating modifier toxins are potent and selective modulators of voltage-gated ion channels that bind to their voltage-sensing domains (VSDs). However, direct measurement of these interactions has been complicated by toxin binding to the detergent micelles required to solubilise isolated VSDs, leading to a prevailing model in which membrane partitioning provides a major contribution to toxin affinity. Here, we use covalently cyclised nanodiscs to separate lipid and protein contributions to binding. At room temperature, the gating modifier toxins VSTx1 and m3-Huwentoxin-IV show little or no detectable binding to DMPG nanodiscs, yet bind their respective KvAP and NaV1.7 VSDs reconstituted into the same nanodiscs with nanomolar affinity. Binding is abolished by mutations in the VSD toxin-binding interface and is modulated by lipid composition. These affinities approach those measured functionally, demonstrating that lipid partitioning is not required for high-affinity recognition of voltage sensors.
The resulting stable, high-affinity VSD preparations also provide a platform for ligand discovery. NaV1.7 VSD nanodiscs selectively capture known and previously uncharacterised gating modifier toxins from crude venoms. In parallel, 19F NMR fragment screening identified novel small-molecule ligands of nanodisc-embedded VSDs, with binding independently validated by surface plasmon resonance. Together, these findings establish isolated VSD nanodiscs as a platform for both mechanistic studies and discovery of peptide and small-molecule modulators of voltage-gated ion channels.