Ancestral acetylcholine receptor β-subunit forms homopentamers that prime before opening spontaneously.

Human adult muscle-type acetylcholine receptors are heteropentameric ion channels formed from two α-subunits, and one each of the β-, δ-, and ε-subunits. To form functional channels, the subunits must assemble with one another in a precise stoichiometry and arrangement. Despite being different, the four subunits share a common ancestor that is presumed to have formed homopentamers. The extent to which the properties of the modern-day receptor result from its subunit complexity is unknown. Here, we discover that a reconstructed ancestral muscle-type β-subunit can form homopentameric ion channels. These homopentamers open spontaneously and display single-channel hallmarks of muscle-type acetylcholine receptor activity. Our findings attest to the homopentameric origin of the muscle-type acetylcholine receptor, and demonstrate that signature features of its function are both independent of agonist and do not necessitate the complex heteropentameric architecture of the modern-day protein.