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A single historical substitution drives an increase in acetylcholine receptor complexity

Johnathon R. Emlaw,Christian J. G. Tessier,Gregory D. McCluskey,Melissa S. McNulty,Yusuf Sheikh,K. Burkett,Maria Musgaard,Corrie J. B. dacosta

Published 2021 in Proceedings of the National Academy of Sciences of the United States of America

ABSTRACT

Significance Human muscle-type acetylcholine receptors are heteropentameric ion channels formed from four evolutionarily related subunits, which assemble with a specific stoichiometry and arrangement. It has long been thought that each of the modern-day subunits are required for function. We dispel this notion by first showing that an ancestral β-subunit can replace both the β- and δ-subunits in human acetylcholine receptors. We then identify a single historical amino acid substitution that eliminates the ability of the ancestral β-subunit to functionally replace the human δ-subunit. Our work experimentally demonstrates how acetylcholine receptor subunit complexity could have evolved and uncovers a form of contingency that is unique to heteromeric protein complexes, in which mutations that “lock in” individual subunits determine future evolutionary paths. Human adult muscle-type acetylcholine receptors are heteropentameric ion channels formed from four different, but evolutionarily related, subunits. These subunits assemble with a precise stoichiometry and arrangement such that two chemically distinct agonist-binding sites are formed between specific subunit pairs. How this subunit complexity evolved and became entrenched is unclear. Here we show that a single historical amino acid substitution is able to constrain the subunit stoichiometry of functional acetylcholine receptors. Using a combination of ancestral sequence reconstruction, single-channel electrophysiology, and concatenated subunits, we reveal that an ancestral β-subunit can not only replace the extant β-subunit but can also supplant the neighboring δ-subunit. By forward evolving the ancestral β-subunit with a single amino acid substitution, we restore the requirement for a δ-subunit for functional channels. These findings reveal that a single historical substitution necessitates an increase in acetylcholine receptor complexity and, more generally, that simple stepwise mutations can drive subunit entrenchment in this model heteromeric protein.

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