Ancestral Sequence Reconstruction Approach to Acetylcholine Receptor Structure and Function

Abstract

Ligand gated ion channels (LGICs) convert the binding of small molecules into opening of an intrinsic transmembrane pore. Acetylcholine receptors (AChRs), a prototypical LGIC, is present throughout the central nervous system and at neuromuscular junctions. The muscle-type AChR is the most genetically complex, composed of two α, and one each of the β, δ, and γ (fetal) / ε (adult) - subunits, arranged in a counterclockwise α-γ/ε-α-δ-β configuration around the central ion-conducting pore. Despite being studied for decades, a complete understanding of AChRs structure - function relationships, remains elusive. Using an ancestral sequence reconstruction approach, along with single-molecule patch-clamp electrophysiology, we have uncovered mechanistic insights into AChR function, with an ancestral β-subunit. Using the βAnc subunit, we demonstrate its ability to form spontaneously opening homopentamers despite lacking an evolutionarily tailored agonist binding site. βAnc was also able to form binary mixtures with human α-subunits, consequently installing agonist regulation of channel openings. Lastly, we demonstrate that βAnc is able to participate in agonist binding with the introduction of δ-residues to restore receptor priming. Additional studies combine single-molecule patch-clamping with single-particle cryo-electron microscopy, advancing our current understanding of AChR activation, through an intermediate ‘primed’ state, supporting a sequential activation model. Together, this work provides insight into the structure and function of AChRs.