Ancestral Reconstruction Approach to Acetylcholine Receptor Structure and Function

Structure. 2017 Aug 1;25(8):1295-1302.e3. doi: 10.1016/j.str.2017.06.005. Epub 2017 Jul 6.


Acetylcholine receptors (AChRs) are members of a superfamily of proteins called pentameric ligand-gated ion channels, which are found in almost all forms of life and thus have a rich evolutionary history. Muscle-type AChRs are heteropentameric complexes assembled from four related subunits (α, β, δ, and ɛ). Here we reconstruct the amino acid sequence of a β subunit ancestor shared by humans and cartilaginous fishes (i.e., Torpedo). Then, by resurrecting this ancestral β subunit and co-expressing it with human α, δ, and ɛ subunits, we show that despite 132 substitutions, the ancestral subunit is capable of forming human/ancestral hybrid AChRs. Whole-cell currents demonstrate that the agonist acetylcholine has reduced potency for hybrid receptors, while single-channel recordings reveal that hybrid receptors display reduced conductance and open probability. Our results outline a promising strategy for studies of AChR evolution aimed at identifying the amino acid origins of AChR structure and function.

Keywords: acetylcholine receptor; ancestral protein reconstruction; evolutionary biochemistry; ligand-gated ion channels; patch-clamp electrophysiology.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylcholine / metabolism
  • Amino Acid Substitution
  • Binding Sites
  • Cell Line
  • Evolution, Molecular
  • Fish Proteins / chemistry*
  • Fish Proteins / genetics
  • Fish Proteins / metabolism
  • Humans
  • Protein Binding
  • Receptors, Cholinergic / chemistry*
  • Receptors, Cholinergic / genetics
  • Receptors, Cholinergic / metabolism
  • Sequence Homology, Amino Acid*


  • Fish Proteins
  • Receptors, Cholinergic
  • Acetylcholine