The adult muscle type acetylcholine receptor AChR the

The adult muscle-type ceftiofur receptor (AChR), the exemplar pLGIC, is a heteropentamer comprising two α subunits, and one each of the β, δ, and ɛ subunits (Sine, 2012). These five subunits come together in a counterclockwise α-ɛ-α-δ-β arrangement (Figure 1A). Binding of acetylcholine to the AChR's two agonist-binding sites, located at the α-ɛ and α-δ interfaces, gates open the channel's central pore by bringing about a conformational change involving all five subunits (Unwin and Fujiyoshi, 2012). Much focus has been on agonist recognition and how conformational changes initiated within the agonist-binding sites are relayed to the transmembrane gate, as well as how, once open, the channel efficiently conducts ions across a hydrophobic lipid bilayer (Sine, 2012). Structure-function studies aimed at identifying the amino acid determinants of AChR function have usually employed either a classic chimeric approach (Imoto et al., 1986), whereby contiguous stretches of primary structure are swapped between related pLGIC subunits, or site-directed mutagenesis involving the targeted substitution of relatively few amino acid residues (Grosman et al., 2000, Lee and Sine, 2005). These sequence-swap experiments neglect the importance of the evolving amino acid background, thereby limiting their ability to decipher structure-function relationships obscured by protein epistasis (Wilson et al., 2015). Several AChR properties remain enigmatic, perhaps due in part to protein epistasis. For example, it has been known for some time that AChR channel opening requires the cooperative action of all five subunits, yet little is known about the underlying amino acid origins of this cooperativity (Changeux and Edelstein, 1998, Karlin, 1967, Sine and Claudio, 1991). Global properties of proteins, such as AChR subunit cooperativity, often arise from complex interactions between an interdependent network of residues, and our knowledge gaps in these areas stem from difficulties associated with identifying such networks. Our hypothesis is that AChR subunit cooperativity has been selected for throughout AChR evolution, and that by studying the evolutionary history of AChR subunits it may be possible to uncover the amino acid origins of AChR subunit cooperativity. Here, we employ a molecular phylogenetic approach to map the evolutionary history of the AChR β subunit. We then reconstruct and resurrect a β subunit ancestor shared by humans (Homo sapiens) and cartilaginous fishes (Torpedo marmorata). Despite 132 substitutions relative to the human β subunit, as well as phylogenetic uncertainty in the placement of this ancestor, our resurrected subunit is able to form hybrid human/ancestral AChRs. Furthermore, these hybrid AChRs display altered single-channel conductance and kinetic signatures, which in the context of human AChRs are characteristic of loss-of-function phenotypes. Our findings demonstrate that the use of hybrid human/ancestral AChRs is a viable approach for studying AChR subunit evolution and functional cooperativity.
Results Our decision to initially reconstruct an ancestral muscle-type β subunit was based on several considerations. First, preliminary sequence alignments of the AChR subunits, containing a variety of taxa, revealed that the β subunit is the least conserved of the four AChR subunits. Thus, when comparing analogous ancestors for each of the AChR subunits, an ancestral β subunit will be the most divergent from its extant subunit, and therefore most likely to present a challenge to our approach. Second, previous studies have shown that co-expression of the β subunit is required for the formation of functional AChRs in a heterologous expression system (Sine and Claudio, 1991, White et al., 1985). Lastly, the β subunit is the only muscle-type AChR subunit that does not directly participate in agonist or antagonist binding. Thus, hybrid AChRs consisting of human α, ɛ, and δ subunits, as well as an ancestral β subunit, should have intact agonist and antagonist binding sites, making it possible to probe AChR cell-surface expression with radiolabeled ligands.