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

Read the full article See related articles


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 show 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 demonstrate that signature features of muscle-type acetylcholine receptor function are independent of agonist, and do not necessitate the complex heteropentameric architecture of the modern-day receptor.

Article activity feed

  1. Evaluation Summary:

    This paper will be of interest to readers interested ligand-gated ion channels and their evolution. The authors show that ancestral AChR beta subunits reconstructed phylogenetically can form homomeric channels that open spontaneously. The work expands our understanding of agonist-independent AChR gating and highlights intriguing aspects of AChR evolution.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)

  2. Reviewer #1 (Public Review):

    (Muscle) acetylcholine receptors are hetero-pentamers incorporating two alpha and one beta, delta and epsilon subunit. This paper closely examines ancestral beta subunits constructed in previous phylogenetic work from this lab using careful experiments and analyses of single channel activity.

    Unlike their previous work on these ancestral subunits, the authors find that the ancestral beta subunit forms pentamers that are active in the absence of the agonist acetylcholine. This shows that ancestral beta subunits, unlike their extant descendants, were probably capable of assembling into homomers and allows subsequent experiments showing that, like their extant hetero-pentameric cousins, these homomeric channels show pre-opening gating steps. Observing homomeric assembly of an ancestral subunit is exciting, as it offers experimental dissection of previous hypotheses on acetylcholine receptor - and the broader pLGIC family - evolution, from homomers into pentamers. It also shows, perhaps not surprisingly but still importantly, that channel gating mechanisms observed in today's receptors was around in those earlier receptors.

  3. Reviewer #2 (Public Review):

    The authors recently found that the phylogenetically reconstructed ancestral beta1 subunit (betaAnc) can insert two copies in the human muscle nAChR, replacing both the (modern) beta and delta subunits.

    The channels thus expressed had heterogeneous behaviour, with a subpopulation of much larger openings (16 vs 10 pA). The larger channels became scarcer when the amount of betaAnc transfected was reduced, and this suggests they are homopentamers of betaAnc.
    This is well supported by the fact that they appear in isolation when only betaAnc is transfected. Their pentameric nature is convincingly confirmed by high quality single channel conductance fingerprinting (mixing high and low conductance variants of the subunit and counting the conductance classes). This is surprising in view of the fact that the muscle nAChR has a robust heteromeric composition.

    Another surprise is that the homomeric betaAnc channels are spontaneously active in the absence of the agonist ACh.

    The betaAnc homopentamers channels display features similar to those seen in muscle nAChRs, including open channel block by ACh and by the blocker QX222. The features of the block (rates, equilibrium constants) are quite similar for the two channels.

    In addition to that, the spontaneous bursts of the homopentamers show more than one closed state, suggesting the presence of activation intermediates (as in other pLGIC channels).

    A point of strength of this study comes from a final set of experiments that shows that an alternative ancestral beta, betaAncS, reconstructed using a different phylogenetic tree, also expressed functional homomeric receptors that open spontaneously. These have a somewhat different kinetics, that allows the detection of more closed primed activation intermediates.

    These results open up many questions on the evolution of muscle nAChR: did muscle nicotinics start as spontaneously opening homomers? When in evolution did they stop opening spontaneously?

  4. Reviewer #3 (Public Review):

    The authors characterize the ion channel function of ancestral beta-subunits of the muscle-type acetylcholine receptor (AchR). They present the striking discovery that these ancestral beta-subunits form spontaneously-active homopentameric channels. Analysis of the single channel currents shows some functional properties similar to the AchR. These include gating kinetics with multiple closed time components suggestive of non-conducting "primed" or "flipped" states previously described in the heteropentameric muscle-type nAchR. The properties of open channel pore block by acetylcholine and QX-222 are also similar to muscle nAchR, indicating a conserved structure in the pore of the ancestral channels. The most notable finding is that these ancestral channels assemble into homopentameric channels that gate in the absence of ligand. This is a unique finding for a nAchR-like channel suggesting the possibility that ligand-gating evolved subsequently to other features of these ion channels.

    The authors achieve their aim in characterizing beta-Anc and beta-AncS by providing high-quality single channel analyses, which definitively confirm the presence of these channels and the fact that they form homopentamers. Analysis of the single channel kinetics indicates multiple closed states. The authors appropriately acknowledge that the single channel kinetics cannot distinguish between multiple gating models, but are consistent with and reminiscent of the presence of "primed" or "flipped" states previously described in other pentameric ligand-gated ion channels. The manuscript is well-written. No significant weaknesses are identified with regards to the methodology and conclusions of the study. However, a broader goal of this study is to gain insight into the structure-function relationships of pentameric ligand-gated ion channels by analyzing reconstructed ancestral beta subunits. To this end, the authors provide minimal insight into the structural determinants underlying distinct aspects of the beta-Anc channel function such as the high-P(open) unliganded gating.