Voltage sensing by voltage-gated ion channels (VGICs) underlies neuronal electrical signalling. The voltage-sensing domains (VSDs) of VGICs play an important role in regulating ion transport across the cell membrane. Some biological toxins selectively interact with VSDs, which unsurprisingly also involves the lipid bilayer. A bacterial VSD, KvAP-VSD, is a significant prototype for studying voltage sensing. Functional studies in reconstituted lipid bilayers suggest that KvAP undergoes lipid-dependent gating in liposomes, although the details of this process remain unknown. Therefore, it is possible to manipulate the state of the channel through changes in the lipid composition. There are currently few binding studies of the isolated VSDs and biological toxins in a lipid bilayer, which would allow access to additional states of the channel to further elucidate the structural basis of channel gating and enable the characterisation of ligands that bind to the resting state.

In this study, we reconstituted the KvAP-VSD in nanodiscs encircled by cyclized MSPs with different diameters to provide a stable membrane mimic for the VSD. The 2D (TROSY) NMR spectra of ¹⁵N KvAP-VSD in cyclized nanodiscs (encircled by cNW7) were recorded, revealing a well-resolved spectrum consistent with a folded monomer. VSTx1, a gating modifier toxin (GMT) and potent inhibitor of KvAP was titrated into the KvAP-VSD nanodiscs to investigate the toxin-lipid-channel interaction. Our isothermal titration calorimetry (ITC) results and NMR experiments show that VSTx1 strongly binds (at the nanomolar range) to KvAP-VSD in nanodiscs containing anionic lipids. Based on the lipid-dependent gating, we are now able to interrogate the structure and function of the KvAP-VSD in its elusive resting state. The thermodynamic parameters of the toxin-lipid-channel interaction were first determined using ITC, which may provide access to the interaction among ligands, lipids, and human VSDs in the future.