A single historical substitution drives an increase in acetylcholine receptor complexity

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 beta-subunit can not only replace the extant beta-subunit but can also supplant the neighboring delta-subunit. By forward evolving the ancestral beta-subunit with a single amino acid substitution, we restore the requirement for a delta-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.

Automated summary

The study found that a single historical amino acid substitution can influence the assembly of muscle-type acetylcholine receptors, thereby increasing receptor complexity. Through a single amino acid substitution, the research revealed that subunit entrenchment can be driven, providing new insights into the evolution of protein subunit complexity.