Ancestral Reconstruction Approach to Acetylcholine Receptor Structure and Function

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 (alpha, beta, delta, and epsilon). Here we reconstruct the amino acid sequence of a beta subunit ancestor shared by humans and cartilaginous fishes (i.e., Torpedo). Then, by resurrecting this ancestral beta subunit and co-expressing it with human alpha, delta, and epsilon 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.