Ligand-gated ion channels convert chemical signals into electrical impulses by coupling the binding of small molecules to the opening of an ion-conducting transmembrane pore (Hille, 2001). Of the many types of ligand-gated channels, the superfamily of pentameric ligand-gated ion channels (pLGICs) is the largest and most structurally and functionally diverse (Jaiteh et al., 2016). Formed from five identical or homologous subunits arranged around a central ion-conducting pore, pLGICs are found in almost all forms of life (Corringer et al., 2012). In prokaryotes, pLGICs appear to be exclusively homopentameric, while in eukaryotes both homo- and heteropentameric channels are common. Humans express more than 40 different pLGIC subunits, which are subdivided based on whether they form cation-selective channels activated by acetylcholine or serotonin (5-hydroxytryptamine [5-HT₃]), or anion-selective channels activated by γ-aminobutyric acid or glycine. This repertoire of pLGIC subunits, combined with their ability to form homo- and heteropentamers, leads to a wealth of structural and functional diversity, presumably to meet a variety of synaptic needs (Sine and Engel, 2006).

The archetypal pLGIC is the heteropentameric muscle-type nicotinic acetylcholine receptor (AChR), with the human adult form composed of two α-subunits, and one each of the β-, δ-, and ε-subunits (Figure 1; Changeux, 2020a; Changeux, 2020b). To gain insight into the structure, function, and evolution of the AChR, we have employed an ancestral reconstruction approach (Emlaw et al., 2021; Emlaw et al., 2020; Prinston et al., 2017; Tessier et al., 2017). 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 (Emlaw et al., 2021). These hybrid AChRs were ternary mixtures, containing two β_Anc subunits, two human α-subunits, and one human ε-subunit. 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 (Figure 1; Emlaw et al., 2021). Regardless of whether they bind agonist or not, all pLGIC subunits have both principal (+) and complementary (−) subunit interfaces. To sit next to each other, the (+) and (−) interfaces of any two neighbouring subunits must be structurally compatible. Thus, for β_Anc to replace both the human β- and δ-subunits, the (+) and (−) interfaces of β_Anc must be compatible with each other, raising the possibility that multiple β_Anc subunits could coassemble to form β_Anc homomers (Figure 1).

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Here, using single-channel measurements, we demonstrate that β_Anc readily forms homopentameric channels that open spontaneously in the absence of acetylcholine. This spontaneous activity displays hallmarks of the muscle-type AChR, including steady-state single-channel burst behaviour. These findings demonstrate how fundamental characteristics of AChR activation are independent of agonist, and not a result of the complex heteropentameric architecture of the muscle-type AChR. Finally, an alternate ancestral β-subunit, reconstructed using a phylogenetic tree that matched the accepted species relationships (Emlaw et al., 2021), and which only shared ~85% identity with β_Anc, revealed that these unexpected characteristics of β_Anc are robust to phylogenetic uncertainty, and thus deeply embedded within AChR β-subunit structure and evolutionary history.

Our first inkling that β_Anc may be able to form homomeric channels came from heterogeneity in single-channel recordings acquired after alterations to our original heterologous expression/transfection protocol. Typical cotransfection of cDNAs encoding human α-, δ-, and ε-subunits with a cDNA encoding β_Anc leads to robust cell surface expression of β_Anc-containing hybrid AChRs (Prinston et al., 2017). In an attempt to lower overall AChR expression with the purpose of facilitating single-channel analysis, we reduced the total amount of subunit cDNA in our transfections (~sixfold), while maintaining the same 2:1:1:1 subunit cDNA ratio (by weight; α:β_Anc:δ:ε). Reducing the amount of cDNA lowered overall AChR expression as expected, but also led to heterogeneity in our patches (Figure 2A), which was not present in original single-channel recordings of β_Anc-containing AChRs (Prinston et al., 2017). Instead of a single population of channels with a uniform amplitude of ~10 pA, and a burst behaviour indicative of β_Anc-containing AChRs (Figure 2A; left inset), we also observed a second class of channels with a different kinetic signature, and an increased amplitude (Figure 2A; right inset). A similar trend was not observed when cDNA encoding the wild-type human β-subunit was cotransfected instead of β_Anc (Figure 2—figure supplement 1), indicating that β_Anc was the source of the heterogeneity. Consistent with this, lowering the proportion of β_Anc cDNA in the transfection mixture reduced the fraction of high amplitude channels (Figure 2B and D), while transfecting exclusively with β_Anc cDNA resulted in patches where all channel openings had a uniformly high amplitude (Figure 2C and D). This demonstrated that when transfected alone, β_Anc forms functional ion channels. The present work stems from this unexpected observation and describes characterisation of these previously unobserved channels.

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The traces in Figure 2 were recorded in the presence of agonist (30 μM acetylcholine). In the human adult muscle-type AChR, the agonist-binding sites are located at the α–δ and α–ε interfaces, and the β-subunit is the only subunit that does not participate directly in agonist binding (Figure 1; Rahman et al., 2022; Rahman et al., 2020; Zarkadas et al., 2022). We were therefore surprised to see single-channel activity in patches from cells transfected exclusively with β_Anc, as channels formed from a muscle-type β-subunit alone would not be expected to have intact agonist-binding sites. To determine if the activity of β_Anc-alone channels was dependent upon acetylcholine, we recorded single-channel activity in the absence of acetylcholine (Figure 3). When no acetylcholine was present, patches from cells that were transfected exclusively with β_Anc cDNA still displayed single-channel activity, indicating that β_Anc-alone channels open spontaneously under these conditions (Figure 3A). Furthermore, spontaneous activity occurred as bursts of closely spaced openings, separated by brief closings (Figure 3B; Colquhoun and Hawkes, 1982). The briefest of these intervening closings were reminiscent of classic ‘nachschlag shuttings’ (Figure 3B, ‘i’ in inset), observed in early patch clamp recordings from frog end-plate nicotinic receptors, and originally thought to relate to agonist efficacy (Colquhoun and Sakmann, 1981). Thus, despite being homomeric and lacking agonist-binding sites, β_Anc-alone channels display single-channel hallmarks of the muscle-type AChR.

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Detected single-channel event durations of spontaneously opening β_Anc homomers. Compressed file includes three TAC 4.3.3 event files (*.evt format) of the single-channel detections for the three recordings used in the presented kinetic analysis, as well as the associated R scripts (*.txt format) for defining and sorting bursts.

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To gain insight into the spontaneous activity of β_Anc-alone channels, we performed kinetic analysis of our single-channel data. First, we determined a critical closed duration (τ_crit) to define bursts arising from a single ion channel. Then we determined the minimum number of components in our apparent open and closed dwell duration histograms by fitting each with a sum of exponentials. Open duration histograms were fit well by a single exponential component, while closed duration histograms required at least two components (Figure 3C). This suggested that a minimal scheme with a single open state and two closed states is necessary and sufficient to describe the spontaneous activity of β_Anc-alone channels. Based on this, we then fit the sequence of single-channel dwells using the three possible kinetic schemes, two linear and one cyclic, comprising a single open state and two closed states (Figure 3D; Figure 3—figure supplement 1). As a control, we also fit a simplified two-state scheme, where a single open state was connected to a single closed state, which, based on the relatively poor fit of the closed durations, confirmed that inclusion of a second closed state was justified (Figure 3—figure supplement 1). Overlaying the resulting fits on top of duration histograms revealed that each of the possible three-state schemes fits the observed dwells equally well, thus discriminating between the possible kinetic schemes is not trivial (Figure 3—figure supplement 1). We settled upon the simple linear scheme, where β_Anc-alone channels transition from a closed state, C, to an intermediate closed state, C′, before opening to O′. The form of this scheme, with an intermediate closed state that precedes channel opening, is guided by models of AChR activation that include a single ‘flipping’ or multiple ‘priming’ steps (Lape et al., 2008; Mukhtasimova et al., 2016; Mukhtasimova et al., 2009). Given that for β_Anc-alone channels there is a single intermediate closed state that precedes channel opening, we refer to this state in our scheme as ‘flipped’ or ‘primed’.

In the presence of acetylcholine, the single-channel current traces appeared different (Figure 4A). As the concentration of acetylcholine increased from 10 to 100 μM, there was a progressive decrease in the duration of openings, as well as a reciprocal increase in the number of short-lived closings within each burst (Figure 4A). This can be observed as a leftward shift in the open duration histograms as the concentration of acetylcholine is increased. This single-channel behaviour is a hallmark of open-channel block, a ubiquitous property of AChR agonists, including acetylcholine (Lape et al., 2009; Mukhtasimova et al., 2016; Ogden and Colquhoun, 1985; Sine and Steinbach, 1984). Consistent with this blockage profile, the same trend, albeit with longer-lived blocking events, was observed with the well-characterized AChR open-channel blocker, 2-[(2,6-dimethylphenyl)amino]-N,N,N-trimethyl-2-oxoethaniminium chloride (QX-222) (Figure 4B; Charnet et al., 1990; Leonard et al., 1988; Pascual and Karlin, 1998).

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Detected single-channel event durations of spontaneously opening β_Anc homomers in the absence (agonist-free) and presence of increasing concentrations of acetylcholine (ACh; 10 µM, 30 µM, 60 µM, and 100 µM) or 2-[(2,6-dimethylphenyl)amino]-N,N,N-trimethyl-2-oxoethaniminium chloride (QX-222; QX; 10 µM, 30 µM, 60 µM, and 100µM). Compressed file includes 27 TAC 4.3.3 event files (*.evt format) of the single-channel detections for the three recordings for each condition (fileA, fileB, and fileC in each case) in the presented kinetic analysis, as well as the associated R scripts (*.txt format) for defining and sorting bursts.

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The kinetics of open-channel block are determined by interactions between the blocking molecule and residues that line the channel pore in the open state, and therefore provide indirect structural insight into the open state. To compare the open state structures of β_Anc-alone homomers with wild-type AChRs, we determined the kinetics of acetylcholine and QX-222 block for both types of channels (Figure 4; Figure 4—figure supplements 1 and 2). To fit our β_Anc-alone single-channel data recorded in the presence of a blocker, we introduced an additional open, but blocked (i.e. non-conducting) state connected to our open state, where the forward rate of block was dependent upon the concentration of the blocking molecule (Figure 4A and B; Neher and Steinbach, 1978). We then globally fit each of our β_Anc-alone datasets encompassing between 0 and 100 μM acetylcholine or QX-222. Initially, we restricted the rates of the core (C-C′-O′) scheme to those inferred in the absence of blocker; however, allowing all parameters to be estimated, led to negligible changes in the inferred rates of block. For β_Anc-alone and wild-type channels, the rates of acetylcholine and QX-222 block were comparable (see Table 1 and Table 2). Of note, while the forward rate of QX-222 block (k_+B) was almost the same for both types of channels, the reverse rate of QX-222 unblocking (k_–B) was nearly twofold faster for β_Anc-alone channels, indicating that the open pore of β_Anc-alone channels has a slightly reduced affinity for QX-222. Regardless of this nuance, the similarity in the profiles of acetylcholine and QX-222 block suggests that the structure of the open pore in the two types of channels is similar.

Given that β_Anc-alone channels are expressed in the absence of other AChR subunits, a reasonable hypothesis is that they are homopentamers. To determine the subunit stoichiometry of β_Anc-alone channels, we employed a single-channel electrical fingerprinting strategy, where mutations altering unitary conductance were used to count the number of individual β_Anc subunits in β_Anc-alone channels. A similar strategy has been employed with tetrameric potassium channels (Niu and Magleby, 2002), and other pLGICs (Andersen et al., 2011), including both the homopentameric α7 AChR (Andersen et al., 2013; daCosta et al., 2015; daCosta and Sine, 2013) and the heteropentameric muscle-type AChR (Emlaw et al., 2021). The approach relies on identifying high-conductance (HC) and low-conductance (LC) variants of the β_Anc subunit, and then co-expressing them to reveal a number of amplitude classes. Openings in each amplitude class originate from channels incorporating the same ratio of HC to LC subunits, and based on the total number of amplitude classes, the number of β_Anc subunits within β_Anc-alone channels can be inferred.

When β_Anc is expressed alone, the resulting channels exhibit a single, uniform amplitude, distributed around a mean of ~16 pA (Figure 5A and D), making the wild-type β_Anc subunit an ideal HC subunit for electrical fingerprinting. To identify a LC variant of β_Anc, we took advantage of a structural feature inherent to eukaryotic pLGICs: as conducting ions exit the channel’s transmembrane pore, they are obliged to pass through one of five portals in the cytoplasmic domain (Rahman et al., 2020; Unwin, 2005). Framed by charged or polar residues from each subunit, these portals influence single-channel conductance. Mimicking the homologous 5-HT_3A receptor, which has an unusually low single-channel conductance (Hales et al., 2006; Kelley et al., 2003; Peters et al., 2004), we substituted three arginine residues (E420R, D424R, and E428R) into this region of β_Anc. When β_Anc harbouring three arginines in this region was transfected by itself, the resulting channels exhibited a reduced single-channel amplitude centred around~1–2pA (Figure 5B and D). With its markedly reduced amplitude, β_Anc harbouring three arginine residues is a suitable LC subunit for electrical fingerprinting.

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When cDNAs encoding HC and LC variants of β_Anc were transfected together, a variety of single-channel amplitudes were observed in each patch (Figure 5C). The relative proportion of channels with high versus low amplitude could, to some degree, be tuned by the ratio of HC to LC β_Anc cDNA used for transfection (Figure 5—figure supplement 1). Constructing event-based amplitude histograms, and pooling amplitudes from more than one recording, revealed that the amplitudes segregated into as many as six amplitude classes, with the highest and lowest amplitude classes matching that of the HC and LC forms of β_Anc-alone channels. The difference in amplitude between successive classes was somewhat regular, demonstrating five approximately equal contributions to single-channel conductance (Figure 5D), consistent with the hypothesis that β_Anc-alone channels are homopentamers.

As noted previously, reconstruction of β_Anc was based upon a molecular phylogeny that diverged from the accepted species phylogeny (Emlaw et al., 2021; Emlaw et al., 2020; Prinston et al., 2017; Tessier et al., 2017). Reconstruction of ancestral protein sequences is based upon a best-fit model of amino acid evolution, a multiple sequence alignment, as well as a phylogenetic tree relating the sequences within the alignment (Thornton, 2004). 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 (Emlaw et al., 2021). 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 (Figure 6). This demonstrated that the ability of β_Anc to form homomers that spontaneously open is not an artefact of the phylogeny used to reconstruct it. Instead, this surprising ability of β_Anc is robust to the phylogenetic uncertainties inherent in ancestral sequence reconstruction, as well as substantial variation in the amino acid sequence of the reconstructed ancestral β-subunits.

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Detected single-channel event durations of spontaneously opening β_AncS homomers. Compressed file includes three TAC 4.3.3 event files (*.evt format) of the single-channel detections for the three recordings used in the presented kinetic analysis, as well as the associated R scripts (*.txt format) for defining and sorting bursts.

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While β_AncS forms homomers that open spontaneously, inspection of the β_AncS single-channel activity revealed additional complexity not seen with β_Anc. For β_AncS, single-channel bursts were heterogeneous, displaying at least three distinct kinetic behaviours (Figure 6B). In some cases, the kinetic behaviour changed within a burst, demonstrating that the different kinetics were possible within the same channel (Figure 6B; boxed). This heterogeneity was also reflected in apparent open and closed duration histograms, with each displaying a minimum of three exponential components (Figure 6C). The increased number of exponential components indicated additional open and closed states relative to β_Anc, and thus that the three-state scheme used to fit β_Anc was insufficient to describe the spontaneous activity of β_AncS. To account for the additional states, we expanded our original scheme to include two additional ‘priming’ steps, where openings could occur from one of three primed states (Figure 6D). This scheme, with multiple priming steps, builds directly upon the one used to fit muscle-type AChRs that had been engineered to open spontaneously (Mukhtasimova et al., 2009). Given the heterogeneity of the β_AncS single-channel activity, and the complexity of this scheme, we caution against over interpretation of the inferred rates. Nevertheless, we note that in accord with the muscle-type AChR, the equilibrium gating constants appear to increase (Table 3; compare Θ₁, Θ₂, and Θ₃), and thus the open states become more and more favoured, for each successive priming step. In any case, the fits suggest that a scheme of this form, with multiple stages of priming, is adequate to describe the complex spontaneous single-channel activity of β_AncS homomers.

The goal of this work was to identify the source of heterogeneity in single-channel recordings from cells transfected with our previously described, reconstructed ancestral AChR β-subunit (Prinston et al., 2017). The heterogeneity in single-channel activity was only evident upon modification of our original transfection protocol. Specifically, when the total amount of transfected AChR subunit cDNA was reduced ~sixfold, a second class of single-channel openings with increased amplitude and a different kinetic signature was observed. Evidently, β_Anc can both incorporate into heteropentameric AChRs (Emlaw et al., 2021), and self-associate to form homopentameric β_Anc channels. Reducing the overall expression of AChR subunits was sufficient to tip the balance between the incorporation of β_Anc into hetero- versus homopentamers, and thus unmask this previously unobserved ability of β_Anc to form homomeric channels.

At first glance, the ability of β_Anc to form homopentamers is unexpected. Modern-day muscle-type β-subunits do not appear to form homopentamers, and instead appear fully entrenched within heteropentameric muscle-type AChRs. Since reconstruction of β_Anc was informed almost exclusively by modern muscle-type β-subunits, there was no reason to expect that β_Anc would behave differently (Prinston et al., 2017). However, as mentioned previously, β_Anc can replace both the human muscle-type β- and δ-subunits in hybrid ancestral/human AChRs (Emlaw et al., 2021). For this to be possible the principal (+) and complementary (−) subunit interfaces of β_Anc must be compatible with each other (Figure 1), leading to the logical hypothesis, confirmed here, that β_Anc can form homopentamers. From an evolutionary perspective, this ability to revert the muscle-type β-subunit, which is entrenched in a heteropentamer, back to a subunit capable of forming homopentamers, attests to the presumed homopentameric origins of pentameric ligand-gated ion channel subunits (Ortells and Lunt, 1995).

We have also shown that β_Anc homopentamers open spontaneously. This is surprising. What is even more surprising is that the spontaneous single-channel activity of β_Anc homopentamers resembles that of the agonist-activated muscle-type AChR. Some of the first single-channel recordings of frog AChRs activated by agonists revealed that agonist-activated openings occur in quick succession, as bursts of activity originating from the same channel (Sakmann et al., 1980). The main effect of increasing the concentration of agonist was to increase the open probability within bursts, with saturating concentrations of full agonists, such as acetylcholine, leading to bursts where the open probability exceeded 0.90 (i.e. the channel was open for more than 90% of the duration of the burst). Spontaneous openings of β_Anc homopentamers also occur in bursts, and despite the absence of agonist, activation appears efficient, with the probability of being open within a burst also exceeding 0.90. The kinetic structure of bursts of spontaneous β_Anc openings also resembles that of the agonist-activated AChR, with bursts containing several types of closings, the briefest of which are reminiscent of classic ‘nachschlag shuttings’. This is significant because it was originally proposed that the duration of nachschlag shuttings was related to agonist efficacy (Colquhoun and Sakmann, 1981). However, subsequent work showed that nachschlag shuttings were independent of agonist, which necessitated the introduction of an additional closed state appended to the agonist-activated open state in early schemes of AChR activation (Sine and Steinbach, 1986). This latter finding was some of the impetus for refined schemes, where the additional closed states preceded channel opening and were referred to as ‘flipped’ or ‘primed’ (Lape et al., 2008; Mukhtasimova et al., 2009). Our kinetic analysis has shown that the spontaneous activity of β_Anc fits an analogous scheme, containing an intermediate closed state that also precedes channel opening, but where the agonist binding steps have been omitted due to the absence of agonist. Evidently, these functional hallmarks of AChR activation do not arise from the complex heteropentameric architecture of the muscle-type receptor, nor do they depend upon the presence of agonist. Instead, they are fundamental properties preserved and encoded in the reconstructed ancestral amino acid sequence of β_Anc (and β_AncS).

Relative to the human β-subunit, which does not form spontaneously opening homopentamers, β_Anc contains 125 individual substitutions and 7 deletions (Prinston et al., 2017). These 132 amino acid differences are scattered throughout the entire β-subunit, and presumably not all of them are required for β_Anc to form spontaneously opening homopentamers. Thus, although the differences in amino acid sequence between β_Anc and the human β-subunit are sufficient to convert the human β-subunit into a subunit capable of (1) forming homopentamers and (2) spontaneously opening, the large number of residues involved, as well as their delocalised nature, obscures the subset of residues strictly necessary for either (or both) functions. Nevertheless, several of the residues substituted between the human β-subunit and β_Anc align with known determinants of homomeric assembly and spontaneous activity in various pLGICs (Appendix 1—figure 1).

Compared to the human wild-type and β_Anc-containing AChRs, β_Anc homopentamers exhibit increased single-channel amplitude and thus conductance (Figure 2D; Figure 5; Figure 2—figure supplement 1). This increased conductance can be explained by amino acid differences in the second transmembrane segment (i.e. M2) of each subunit, which is one of the more conserved regions across all pLGICs. M2 lines the channel pore at its narrowest constriction and is a well-known determinant of single-channel conductance (Imoto et al., 1986). With exception of the 2′ and 6′ positions, M2 is conserved between β_Anc and the human β-subunit (Emlaw et al., 2020). However, to reconcile the differences in conductance, it is important to consider the M2 segments of not just the β-subunits, but all the subunits that make up the various channels. Owing to the symmetric (homopentamers) or pseudosymmetric (heteropentamers) architecture of pLGICs, analogous M2 residues from each subunit occupy similar positions in the pore, forming concentric rings of amino acids. Several of these rings influence conductance, with the –4′, –1′, 2′, and 20′ positions being particularly important, and referred to as the ‘cytoplasmic’, ‘intermediate’, ‘central’, and ‘extracellular’ rings, respectively (Imoto et al., 1991; Imoto et al., 1988). In β_Anc homopentamers, with five identical β_Anc subunits, the amino acids contributed from individual subunits are identical. This is significant because β_Anc contains acidic residues at the –4′ (Asp), –1′ (Glu), and 20′ (Asp) positions, meaning that all 15 residues forming these three decisive rings are acidic, effectively maximising their negative potential (Appendix 1—figure 2). In heteropentamers, such as human wild-type and β_Anc-containing AChRs, the residues contributed by individual subunits are in some cases different, with polar/neutral (Gln) and basic (Lys) residues contributed by the ε- and δ-subunits, respectively (Appendix 1—figure 2). Substitution of neutral or basic residues into any of these three rings has been shown to reduce conductance (Imoto et al., 1988). Thus, unlike for β_Anc homopentamers that have higher conductance, the negative potential of these rings is not maximised in human wild-type and β_Anc-containing heteropentamers.

Based upon the rates of open-channel block by acetylcholine and the well-known open-channel blocker, QX-222, the structure of the open pore in β_Anc homopentamers and the wild-type AChR appear to be similar. Nevertheless, kinetic fitting revealed that compared to wild-type, β_Anc homopentamers exhibit an almost twofold reduced affinity for QX-222. While we have employed the simplest model for open-channel block (Neher and Steinbach, 1978), which neglects potential increased complexity at higher QX-222 concentrations (Neher, 1983), the simplest interpretation is that, like conductance, this reduced apparent affinity can be explained by amino acid differences in M2. Specifically, the 6′ and 10′ sites are known determinants of QX-222 block (Charnet et al., 1990; Leonard et al., 1988; Pascual and Karlin, 1998), with a more polar environment at 6′, and a less polar environment at 10′, favouring interaction with QX-222 (Charnet et al., 1990). In the wild-type AChR, the polarity of the 6′ ring is limited by a hydrophobic phenylalanine residue contributed by the human β-subunit (Appendix 1—figure 2). In β_Anc homopentamers, the 6′ ring does not contain this phenylalanine residue and is instead composed of five polar serine residues, predicting an increased affinity for QX-222. Conversely, the 10′ ring in β_Anc homopentamers contains five threonine residues making it more polar, and thus a less favourable environment for QX-222, than in the wild-type AChR, which contains two nonpolar alanine residues (from the δ- and ε-subunits) (Appendix 1—figure 2). Evidently, the energetic contributions of these two rings to QX-222 blockage balance out such that β_Anc homopentamers exhibit only a slightly reduced apparent affinity for QX-222 compared to the wild-type AChR.

Phylogenetic analyses of pLGICs suggests that present-day heteropentameric subunits, such as those entrenched in the muscle-type AChR, evolved from ancestral subunits capable of self-associating to form homopentamers (Dent, 2010; Jaiteh et al., 2016; Le Novère and Changeux, 1995; Ortells, 2016; Ortells and Lunt, 1995; Pedersen et al., 2019; Tsunoyama and Gojobori, 1998). Within this framework, strictly entrenched subunits have lost compatibility between their (+) and (–) interfaces, and thus the ability to self-associate. This is significant because the pentameric architecture of pLGICs places a constraint upon when the ability to self-associate became dispensable, and thus when strict subunit entrenchment could have evolved. For the simplest heteropentamers, made up of two types of subunits, at least one of the subunit types will have to occupy a minimum of three positions within the pentamer. In these cases, two copies of the same subunit must sit next to each other, and thus retain the ability to self-associate. Indeed, in neuronal α4β2 heteropentameric AChRs, both the α4- and β2-subunits have retained the ability to self-associate and form α4–α4 or β2–β2 interfaces, meaning that in principle, a variety of subunit stoichiometries and arrangements are possible. While additional factors such as subunit packing, relative expression, trafficking and assembly, and the presence of cellular chaperones provide additional constraints that limit the stoichiometries and arrangements observed (Mazzaferro et al., 2021; Moroni et al., 2006; Nelson et al., 2003; Son et al., 2009; Walsh et al., 2018), the requirement for self-association must remain for at least one of the subunit types. Thus, while the ability to self-associate is not sufficient to permit all possible heteropentameric stoichiometries and arrangements, it is necessary.

Unlike neuronal heteropentameric channels, such as α4β2 above, whose subunit stoichiometries and arrangements exhibit a degree of plasticity, the subunits in the muscle-type AChR are strictly entrenched. In this case, the definition of strict entrenchment neglects the γ- for ε-subunit swap that occurs during development (Mishina et al., 1986), because it occurs at the same location within the heteropentamer, and thus does not alter the stoichiometry and arrangement of the α-, β-, or δ-subunits. The above structural considerations imply that such strict entrenchment should only be expected in heteropentamers composed of three or more subunit types, and where each of the subunits has also lost the ability to self-associate. Muscle-type AChRs fulfill both criteria, in that they are made up of four types of subunits that are each incapable of self-associating and forming homopentamers. The finding that reconstructed ancestral β-subunits retain the ability to self-associate and form homomers, suggests that, unlike their extant human and Torpedo counterparts, the ancestral β-subunits were not strictly entrenched, and that their entrenchment occurred in parallel lineages after the last common ancestor shared by humans and Torpediniformes (i.e. Gnathostomata).

A degree of uncertainty is inherent in the reconstruction of any ancestral protein, and to solidify evolutionary conclusions it is important to assess whether the functions of putative ancestors are robust to these uncertainties. We have shown that an alternate ancestral β-subunit (β_AncS), with 67 substitutions and 6 indels relative to β_Anc, was still able to form homomers that opened spontaneously. Furthermore, spontaneous openings of β_AncS homomers also occurred in bursts that contained brief closings (i.e. ‘nachschlag shuttings’) and had an open probability that exceeded 0.90. These findings demonstrate that the ability of β_Anc to form spontaneously opening homomers is robust to substantial variations in the inferred ancestral β-subunit amino acid sequence. Thus, the capacity of these reconstructed ancestral β-subunits to form spontaneously opening homomers is deeply embedded in their structure and evolutionary history.

The kinetic behaviour of β_AncS homomers was more complex than originally observed with β_Anc. This increased complexity necessitated the expansion of the original scheme used to fit β_Anc to include two additional priming steps. Both the original β_Anc and expanded β_AncS schemes parallel the scheme used to describe the muscle-type AChR, which included two priming steps (i.e. ‘singly’ and ‘doubly’ primed) that each correlated with conformational changes around the two agonist-binding sites (Mukhtasimova et al., 2009). In the case of β_AncS, which lacks agonist-binding sites, the simplest interpretation is that the different levels of priming correlate with conformational changes occurring within individual subunits. Although the states in Scheme 5 are labelled as ‘singly’, ‘doubly’, and ‘triply’ primed, the three priming steps could represent the three most terminal priming steps, where (assuming that β_AncS also forms homopentamers) three, four, or five β_AncS subunits are primed. Within this framework, openings from β_AncS channels with zero, one, or two primed subunits are presumably unstable, and thus not observed in our single-channel recordings. In an alternate scenario, ‘singly’ and ‘doubly’ primed could refer to β_AncS channels with one or two primed subunits, respectively. While ‘triply’ primed could refer to channels with three or more primed subunits, but where openings from β_AncS channels with three, four, or five primed subunits are indistinguishable. Applied to β_Anc, these interpretations suggest that either (1) openings from only the terminal priming step, where all five β_Anc subunits are primed, are stable enough to be observed in β_Anc homopentamers, or (2) openings can occur with fewer than five primed subunits, but these openings are kinetically indistinguishable. Regardless of the interpretation, priming is an important step in the activation of both β_AncS and β_Anc homopentamers.

Modern mechanisms of AChR activation include intermediate closed states, which place the roots of agonism at a stage in the activation process that precedes channel opening (Lape et al., 2008; Mukhtasimova et al., 2016; Mukhtasimova et al., 2009). A consequence of these mechanisms is that the ultimate opening and closing rates of the channel are independent of agonist. Here, we have shown that additional single-channel hallmarks of AChR function are independent of agonist, as they occur in spontaneously opening homopentameric channels that are devoid of agonist-binding sites, and which are formed from reconstructed ancestral AChR β-subunits. Often overlooked, the β-subunit is the least conserved of the four AChR subunits, and is the only subunit that does not contribute residues to the two AChR agonist-binding sites. Despite these considerations, hallmarks of AChR function remain deeply embedded in β-subunit sequence, structure, and evolutionary history. Given that these functional hallmarks are independent of agonist, it is tempting to speculate that they predate agonism, and thus that agonism evolved subsequently as an additional layer of regulation in this family of pentameric ion channels.

All data generated and analysed during this study are included in the manuscript and as source data.

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Author details

1. Christian JG Tessier

   1. Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Canada
   2. Centre for Chemical and Synthetic Biology, University of Ottawa, Ottawa, Canada

   Contribution

   CJGT acquired and analyzed all electrophysiological data, except the data for the electrical fingerprinting, which was recorded and analyzed by RMS, CJGT, and CJBdC. CJGT and CJBdC interpreted the data and wrote the manuscript., Conceptualization, Formal analysis, Investigation, Writing – original draft, Writing – review and editing, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6006-7755

2. Raymond M Sturgeon

   1. Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Canada
   2. Centre for Chemical and Synthetic Biology, University of Ottawa, Ottawa, Canada

   Contribution

   Conceptualization, Formal analysis, Investigation, Methodology, RMS recorded and analyzed electrophysiological data for the electrical fingerprinting experiments., Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0043-0241

3. Johnathon R Emlaw

   1. Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Canada
   2. Centre for Chemical and Synthetic Biology, University of Ottawa, Ottawa, Canada

   Contribution

   Conceptualization, JRE acquired some of the initial recordings containing β_Anc homopentamers., Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7255-182X

4. Gregory D McCluskey

   1. Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Canada
   2. Centre for Chemical and Synthetic Biology, University of Ottawa, Ottawa, Canada

   Contribution

   Conceptualization, Formal analysis, GDM reconstructed β_AncS and acquired β_AncS electrophysiological data., Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6594-4665

5. F Javier Pérez-Areales

   1. Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Canada
   2. Centre for Chemical and Synthetic Biology, University of Ottawa, Ottawa, Canada

   Present address

   Yusuf Hamied Department of Chemistry, University of Cambridge, Cambridge, United Kingdom

   Contribution

   FJPA acquired some of the initial recordings containing β_Anc homopentamers., Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9525-9346

6. Corrie JB daCosta

   1. Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Canada
   2. Centre for Chemical and Synthetic Biology, University of Ottawa, Ottawa, Canada

   Contribution

   CJBdC interpreted the data and wrote the manuscript, and supervised the project., Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Writing – original draft, Writing – review and editing

   For correspondence

   cdacosta@uottawa.ca

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9546-5331

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