RT Journal Article SR Electronic T1 Employing NaChBac for cryo-EM analysis of toxin action on voltage-gated Na+ channels in nanodisc JF bioRxiv FD Cold Spring Harbor Laboratory SP 2020.04.16.045674 DO 10.1101/2020.04.16.045674 A1 Shuai Gao A1 William C Valinsky A1 Nguyen Cam On A1 Patrick R Houlihan A1 Qian Qu A1 Lei Liu A1 Xiaojing Pan A1 David E. Clapham A1 Nieng Yan YR 2020 UL http://biorxiv.org/content/early/2020/04/18/2020.04.16.045674.abstract AB NaChBac, the first bacterial voltage-gated Na+ (Nav) channel to be characterized, has been the prokaryotic prototype for studying the structure-function relationship of Nav channels. Discovered nearly two decades ago, the structure of NaChBac has not been determined. Here we present the 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 NavAb. Determining the structure of NaChBac in nanodiscs enabled us to examine gating modifier toxins (GMTs) of Nav channels in lipid bilayers. To study GMTs in mammalian Navs, we generated a chimera in which the extracellular fragment of the S3 and S4 segments in the second voltage-sensing domain from Nav1.7 replaces 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 Å and 3.2 Å resolutions, respectively. Compared to the structure of HWTX-IV-bound human Nav1.7, which was obtained at an overall resolution of 3.2 Å, the local resolution of the toxin has been improved from ~ 6 Å 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 Nav channels in a membrane environment.Competing Interest StatementThe authors have declared no competing interest.