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

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.