Purification of a native nicotinic receptor

Md Mahfuzur Rahman, Brady T. Worrell, Michael H.B. Stowell, Ryan E. Hibbs

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Nicotinic acetylcholine receptors are members of the Cys-loop superfamily of pentameric ligand-gated ion channels. The electric organ of the Torpedo ray is extraordinarily rich in an acetylcholine receptor that is homologous to the human nicotinic receptor found at the neuromuscular junction. Due to this abundant natural source in the fish and the relatively accessible preparation of the neuromuscular junction (compared to a central synapse), this muscle-type receptor and specifically the fish receptors have long been used as the prototype for study of nicotinic receptors. However, an absence of structural detail at high resolution has limited the chemical interpretation of this archetypal nicotinic receptor. One of the main concerns in preparing receptor for high resolution structural analysis was its documented sensitivity to particular detergents and requirements for specific lipids in order to maintain function after reconstitution in a membrane. Here, we present methods for purifying native nicotinic receptor from Torpedo electric tissue that maintains functionality after reconstitution and that is amenable to high resolution structural analysis. The specific developments we describe include detergent exchange during purification, inclusion of specific lipids during purification and for nanodisc reconstitution, and synthesis of a new affinity reagent for rapid isolation of receptors.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
DOIs
StateAccepted/In press - 2021

Publication series

NameMethods in Enzymology
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • Acetylcholine receptor
  • Cryo-EM
  • Cys-loop receptor
  • Ion channel structure
  • Ligand-gated ion channel
  • Membrane protein
  • Nicotinic receptor
  • Purification protocol
  • Torpedo

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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