Contingency between Historical Substitutions in the Acetylcholine Receptor Pore.

Human adult muscle-type acetylcholine receptors incorporating a reconstructed ancestral β-subunit exhibit reduced single-channel conductance when compared to wild-type. The ancestral and wild-type β-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 β-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 β-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.