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

• DOI: 10.1073/pnas.1922903117
• Publication Date: 2020-06-09T00:36:04Z
• Authors: Shuai Gao; William C. Valinsky; Nguyen Cam On; Patrick R. Houlihan; Qian Qu; Lei Liu; Xiaojing Pan; David E. Clapham; Nieng Yan
• Keywords: Models, Molecular; 0301 basic medicine; 0303 health sciences; Protein Conformation; Cryoelectron Microscopy; Lipid Bilayers; Spider Venoms; Voltage-Gated Sodium Channels; Sodium Channels; Nanostructures; 03 medical and health sciences; Bacterial Proteins; Animals; Humans

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 v channels. 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 NaChBac is nearly identical to that of the potentially inactivated Na v Ab. Determining the structure of NaChBac in nanodiscs enabled us to examine gating modifier toxins (GMTs) of Na v channels in lipid bilayers. To study GMTs in mammalian Na v channels, we generated a chimera in which the extracellular fragment of the S3 and S4 segments in the second voltage-sensing domain from Na v 1.7 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 HWTX-IV–bound human Na v 1.7, 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 v channels in a membrane environment.