Recombinant expression and purification of pentameric ligand-gated ion channels for Cryo-EM structural studies

Methods Enzymol. 2021:652:81-103. doi: 10.1016/bs.mie.2021.01.022. Epub 2021 Feb 16.

Abstract

Pentameric ligand-gated ion channels (pLGICs) are central players in synaptic neurotransmission and are targets to a range of drugs used to treat neurological disorders and pain. pLGICs are intrinsically dynamic membrane proteins that upon stimulation by neurotransmitters, undergo global conformational changes across multiple domains spanning a distance of over 165Å. The inter-domain flexibility, a feature crucial for their function as signal transducers in chemical synapses, has been problematic in the efforts toward determining high-resolution structures. Earlier structural studies tackled this issue with a variety of strategies that included partial truncation of flexible domains and the use of antibodies and small-molecule inhibitors to restrict domain movement. With the recent advances in cryo-electron microscopy and single-particle analysis, many of these limitations have been overcome. Here, we describe the methods used in the recombinant expression and purification of full-length constructs of two members of the pentameric ligand-gated ion channel family and the approaches used for capturing multiple conformations in cryo-EM imaging.

Keywords: Cryo-EM; Cys-loop receptors; Gating; Nanodisc; Reconstitution; pLGIC.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cryoelectron Microscopy
  • Ligand-Gated Ion Channels* / genetics
  • Ligand-Gated Ion Channels* / metabolism
  • Synapses
  • Synaptic Transmission

Substances

  • Ligand-Gated Ion Channels