pUdOs: Concise Plasmids for Bacterial and Mammalian Cells.

The plasmids from the Université d'Ottawa (pUdOs) are 28 small plasmids each comprising one of four origins of replication and one of seven selection markers, which together afford flexible use in Escherichia coli and several related gram-negative bacteria. The promoterless multicloning site is insulated from upstream spurious promoters by strong transcription terminators and contains type IIP or IIS restriction sites for conventional or Golden Gate cloning. pUdOs can be converted into efficient expression vectors through the insertion of a promoter at the user's discretion. For example, we demonstrate the utility of pUdOs as the backbone for an improved version of a Type III Secretion System reporter in Shigella. In addition, we derive a series of pUdO-based mammalian expression vectors, affording distinct levels of expression and transfection efficiency comparable to commonly used mammalian expression plasmids. Thus, pUdOs could advantageously replace traditional plasmids in a wide variety of cell types and applications.

Derepression may masquerade as activation in ligand-gated ion channels.

Agonists are ligands that bind to receptors and activate them. In the case of ligand-gated ion channels, such as the muscle-type nicotinic acetylcholine receptor, mechanisms of agonist activation have been studied for decades. Taking advantage of a reconstructed ancestral muscle-type ß-subunit that forms spontaneously activating homopentamers, here we show that incorporation of human muscle-type α-subunits appears to repress spontaneous activity, and furthermore that the presence of agonist relieves this apparent α-subunit-dependent repression. Our results demonstrate that rather than provoking channel activation/opening, agonists may instead 'inhibit the inhibition' of intrinsic spontaneous activity. Thus, agonist activation may be the apparent manifestation of agonist-induced derepression. These results provide insight into intermediate states that precede channel opening and have implications for the interpretation of agonism in ligand-gated ion channels.

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

Human adult muscle-type acetylcholine receptors are heteropentameric ion channels formed from two α-subunits, and one each of the ß-, 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 discover that a reconstructed ancestral muscle-type ß-subunit can form homopentameric ion channels. These homopentamers open spontaneously and display single-channel hallmarks of muscle-type acetylcholine receptor activity. Our findings attest to the homopentameric origin of the muscle-type acetylcholine receptor, and demonstrate that signature features of its function are both independent of agonist and do not necessitate the complex heteropentameric architecture of the modern-day protein.

A single historical substitution drives an increase in acetylcholine receptor complexity.

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 ß-subunit can not only replace the extant ß-subunit but can also supplant the neighboring δ-subunit. By forward evolving the ancestral ß-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.

Contingency between Historical Substitutions in the Acetylcholine Receptor Pore.

Human adult muscle-type acetylcholine receptors incorporating a reconstructed ancestral ß-subunit exhibit reduced single-channel conductance when compared to wild-type. The ancestral and wild-type ß-subunits differ by 132 amino acids, including substitution of residues that line the lumen of the channel pore, near its narrowest constriction. Here we show that a single historical substitution in this region of the ancestral ß-subunit accounts for the difference in conductance. Furthermore, the contribution of the substituted residue to conductance is dependent upon its ancestral or wild-type background, and it can be modulated by a neighboring residue that has also evolved throughout the ß-subunit history. Using an expanded molecular phylogeny, we track the order in which these two mutations occurred and then show that the order in which they are installed upon the ancestral, but not the human, background determines their individual contribution to conductance. Our results show how the contribution of amino acids to acetylcholine receptor conductance is contingent upon their evolutionary history and that the order in which substitutions occurred was important for shaping conductance in the modern-day receptor.

Back to the future: Rational maps for exploring acetylcholine receptor space and time.

Global functions of nicotinic acetylcholine receptors, such as subunit cooperativity and compatibility, likely emerge from a network of amino acid residues distributed across the entire pentameric complex. Identification of such networks has stymied traditional approaches to acetylcholine receptor structure and function, likely due to the cryptic interdependency of their underlying amino acid residues. An emerging evolutionary biochemistry approach, which traces the evolutionary history of acetylcholine receptor subunits, allows for rational mapping of acetylcholine receptor sequence space, and offers new hope for uncovering the amino acid origins of these enigmatic properties.

Ancestral Reconstruction Approach to Acetylcholine Receptor Structure and Function.

Acetylcholine receptors (AChRs) are members of a superfamily of proteins called pentameric ligand-gated ion channels, which are found in almost all forms of life and thus have a rich evolutionary history. Muscle-type AChRs are heteropentameric complexes assembled from four related subunits (α, ß, δ, and ɛ). Here we reconstruct the amino acid sequence of a ß subunit ancestor shared by humans and cartilaginous fishes (i.e., Torpedo). Then, by resurrecting this ancestral ß subunit and co-expressing it with human α, δ, and ɛ subunits, we show that despite 132 substitutions, the ancestral subunit is capable of forming human/ancestral hybrid AChRs. Whole-cell currents demonstrate that the agonist acetylcholine has reduced potency for hybrid receptors, while single-channel recordings reveal that hybrid receptors display reduced conductance and open probability. Our results outline a promising strategy for studies of AChR evolution aimed at identifying the amino acid origins of AChR structure and function.