Heteropentameric architecture predisposes functional inequivalence of acetylcholine receptor agonist sites

Muscle-type acetylcholine receptors are heteropen-tameric ion channels composed of four different but evolutionarily related subunits. These subunits assemble with a precise stoichiometry and arrangement, such that two distinct agonist-binding sites are formed at interfaces between a principal α-subunit and a complementary δ or ε/γ-subunit. Chemical differences between the two complementary subunits are presumed to confer functional inequivalence to the two agonist sites. This interpretation, however, overlooks the asymmetric architecture of the acetylcholine receptor, which places each subunit, and therefore each agonist site, at unique positions within the heteropentamer. The extent to which functional inequivalence of the agonist sites stems from this structural asymmetry, as opposed to chemical differences, remains unexplored. Here, we reconstruct an ancestral subunit capable of substituting for both complementary subunits, thereby engineering hybrid ancestral/human acetylcholine receptors with chemically identical agonist sites. Despite being chemically identical, these agonist sites remain functionally inequivalent. We show that this functional inequivalence stems from distinct intersubunit interactions dictated by the receptor’s heteropentameric architecture, which also underlie the subunit-dependent effects of a human disease-causing mutation. Thus, structural asymmetry emerges as a fundamental determinant of receptor function, capable of imposing functional inequivalence even in chemically identical sites, with implications for both the evolution of heteromeric protein complexes and the molecular basis of disease.