A single historical substitution drives an increase in acetylcholine receptor complexity

Abstract

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.

Native mass spectrometry analyses of chaperonin complex TRiC/CCT reveal subunit N-terminal processing and re-association patterns

“…All CCT subunits inherited an oligomeric interface from an ancient, single-copy CCT gene that duplicated several times deep in the eukaryotic lineage 54 . In many paralogs that interface was likely only partly lost as their specific positions in the ring were entrenched, as has been demonstrated for other multimeric complexes with ring topologies 55,56 . Our results could be explained if the residual affinity between paralogs that are not adjacent in TRiC reflects incomplete loss of ancient contacts 7,17,[47][48][49] .…”

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

“…A concatameric construct confirmed that the two β Anc subunits resided next to each other, occupying positions in the heteropentameric complex usually reserved for the human β- and δ-subunits (Fig. 1) (7). Regardless of whether they bind agonist or not, all pLGIC subunits have both principal (+) and complementary (−) subunit interfaces.…”

“…We therefore wondered if the ability of β Anc to form spontaneously opening homomers was an artefact of the discordant tree used to reconstruct it. To test this, we took advantage of an alternate ancestral β-subunit, called “β AncS ”, whose reconstruction was based upon a molecular phylogeny that matched the accepted species phylogeny (7). Despite 67 substitutions and 6 indels relative to β Anc , when expressed alone, β AncS still formed homomeric channels that opened in bursts in the absence of acetylcholine (Fig.…”

“…1)(5, 6). To gain insight into the structure, function, and evolution of the AChR, we have employed an ancestral reconstruction approach (7–10). Previously we resurrected a putative ancestral AChR β-subunit.…”

“…Referred to as “β Anc ”, this subunit differed from its human counterpart by 132 amino acids (i.e. approximately 30% of the total amino acid sequence), yet was able to substitute for the human β-subunit, and also supplant the human δ-subunit, forming functional hybrid ancestral/human AChRs (7). These hybrid AChRs were ternary mixtures, containing two β Anc subunits, two human α-subunits, and one human ε-subunit.…”