Qualitative inspection of the single channel traces presente
Qualitative inspection of the single-channel traces presented in both Figures 3 and 4 shows that within bursts of activity, βAnc-containing AChRs display a marked reduction in open probability. To quantify these differences in single-channel behavior, we performed dwell-time analysis under high-resolution conditions (Mukhtasimova et al., 2016) (Figures 4C and 4D). Whereas open duration histograms for wild-type AChRs are best fit by a single exponential component, open histograms for βAnc-containing AChRs exhibit a minimum of two components with briefer durations, indicative of multiple open states at this concentration (30 μM) of acetylcholine. Analysis of closed duration histograms reveals that the majority of single-channel closings for wild-type AChRs are brief in duration, while βAnc-containing AChRs exhibit an increased proportion of longer-lived closings. This dwell-time analysis confirms that, although βAnc-containing AChRs are functional, they display quantifiable differences in their single-channel behavior, providing insight into AChR mechanism and subunit cooperativity.
Discussion To assess the utility of an ancestral sequence reconstruction approach for examining AChR structure-function relationships, we have reconstructed the most likely sequence of an ancestral AChR β subunit. Based on a maximum-likelihood molecular phylogeny, this subunit was ancestral to both the human (H. sapiens) and Torpedo (Torpedo californica and Torpedo marmorata) AChR β subunits. Despite 132 substitutions, and phylogenetic uncertainty in the precise placement of this ancestor, our resurrected βAnc can rescue cell-surface dextromethorphan hydrobromide of human α, δ, and ɛ subunits co-expressed without a human β subunit, demonstrating that βAnc is able to substitute for its human counterpart and form hybrid human/ancestral AChRs. Hybrid AChRs incorporating βAnc display altered single-channel conductance and kinetic behavior, testifying to the potential of an ancestral sequence reconstruction approach for studying AChR subunit evolution, and uncovering AChR structure-function relationships. Human AChRs are finely tuned molecular machines, evolved to respond efficiently to brief pulses of the agonist acetylcholine, and it is well established that substitution of individual amino acids in AChR subunits can cause loss-of-function phenotypes that disrupt synaptic signaling (Sine, 2012). It is therefore surprising that βAnc, which bears 132 substitutions delocalized across its entire structure, is able to substitute for the human β subunit and recover AChR function. Furthermore, although βAnc-containing AChRs display a loss-of-function phenotype, the magnitude of this phenotype is similar to that observed when individual amino acids are substituted. In particular, the observed decrease in open probability for βAnc-containing AChRs is reminiscent of phenotypes causing rare congenital myasthenic syndromes in humans (Engel et al., 2010, Shen et al., 2008). While many of these syndromes result from a single amino acid substitution, in βAnc-containing AChRs 132 substitutions conspire for an equivalent phenotype. While it is thus tempting to conclude that these 132 substitutions are tolerated because they are each innocuous, it is important to consider that residues within proteins do not evolve independently of one another (Lunzer et al., 2010). Just as how residues between AChR subunits may have co-evolved, so too may have those within each subunit. Thus, cryptic epistatic interactions between co-evolving residues may allow for apparently neutral substitutions to accrue, when in fact some of these substitutions may be deleterious in isolation (Lunzer et al., 2010). The general sensitivity of AChRs to mutation, and the fact that we have been able to introduce 132 substitutions, altering ∼30% of the β subunit, and still recover a β subunit that expresses, assembles, and is functional, speaks to the power of modern algorithms for estimating viable ancestral protein sequences and accounting for this epistasis.