Employing NaChBac for cryo-EM analysis of toxin action on voltage-gated Na+ channels in nanodisc

NaChBac, the first bacterial voltage-gated Na⁺(Na_v) channel to be characterized, has been the prokaryotic prototype for studying the structure-function relationship of Na_vchannels. Discovered nearly two decades ago, the structure of NaChBac has not been determined. Here we present the single particle electron cryomicroscopy (cryo-EM) analysis of NaChBac in both detergent micelles and nanodiscs. Under both conditions, the conformation of NaCh- Bac is nearly identical to that of the potentially inactivated Na_vAb. Determining the structure of NaChBac in nanodiscs enabled us to examine gating modifier toxins (GMTs) of Na_vchannels in lipid bilayers. To study GMTs in mammalian Na_vchannels, we generated a chimera in which the extracellular fragment of the S3 and S4 segments in the second voltage-sensing domain from Na_v1.7 replaced the corresponding sequence in NaChBac. Cryo-EM structures of the nanodisc-embedded chimera alone and in complex with HuwenToxin IV (HWTX-IV) were determined to 3.5 and 3.2 Å resolutions, respectively. Compared to the structure of HWTXIV- bound human Na_v1.7, which was obtained at an overall resolution of 3.2 Å, the local resolution of the toxin has been improved from ~6 to ~4 Å. This resolution enabled visualization of toxin docking. NaChBac can thus serve as a convenient surrogate for structural studies of the interactions between GMTs and Na_vchannels in a membrane environment.