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

Shuai Gao, William C. Valinsky, Nguyen Cam On, Patrick R. Houlihan, Qian Qu, Lei Liu, Xiaojing Pan, David E. Clapham, Nieng Yan

Research output: Contribution to journalArticle

Abstract

NaChBac, the first bacterial voltage-gated Na+(Nav) channel to be characterized, has been the prokaryotic prototype for studying the structure-function relationship of Navchannels. 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 NavAb. Determining the structure of NaChBac in nanodiscs enabled us to examine gating modifier toxins (GMTs) of Navchannels in lipid bilayers. To study GMTs in mammalian Navchannels, we generated a chimera in which the extracellular fragment of the S3 and S4 segments in the second voltage-sensing domain from Nav1.7 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 Nav1.7, 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 Navchannels in a membrane environment.

Original languageEnglish (US)
Pages (from-to)14187-14193
Number of pages7
JournalProceedings of the National Academy of Sciences of the United States of America
Volume117
Issue number25
DOIs
StatePublished - Jun 23 2020

All Science Journal Classification (ASJC) codes

  • General

Keywords

  • Electron cryomicroscopy (cryo-EM)
  • Gating modifier toxins
  • NaChBac
  • Nachannels
  • Nanodisc

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