Volume 25, Issue 8, Pages 1295-1302.e3 (August 2017) Ancestral Reconstruction Approach to Acetylcholine Receptor Structure and Function  Jethro E. Prinston, Johnathon R. Emlaw, Mathieu F. Dextraze, Christian J.G. Tessier, F. Javier Pérez-Areales, Melissa S. McNulty, Corrie J.B. daCosta  Structure  Volume 25, Issue 8, Pages 1295-1302.e3 (August 2017) DOI: 10.1016/j.str.2017.06.005 Copyright © 2017 Elsevier Ltd Terms and Conditions

Structure 2017 25, 1295-1302.e3DOI: (10.1016/j.str.2017.06.005) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Reconstruction of an Ancestral AChR β Subunit (A) Muscle-type AChRs are heteropentamers formed from five homologous subunits (α2βδɛ) arranged around a central ion-conducting pore (PDB: 2BG9). Agonist and competitive antagonist binding sites are located at the interface between α-ɛ and α-δ subunits (asterisks). (B) Maximum-likelihood molecular phylogeny based on muscle-type AChR β subunit amino acid sequences. The location of Torpedo marmorata and Homo sapiens are highlighted in blue and orange, while the location of their most recent common ancestor (βAnc) is highlighted in purple. The tree is rooted to nematode homologs (see Figure S1), and the scale bar represents the average number of substitutions per site. (C) Mapping the substitutions between the human β subunit and βAnc onto the structure of a β subunit (PDB: 2BG9) shows that the substituted residues (black spheres) are delocalized over the entire protein. The cytoplasmic region comprising ∼100 amino acids, for which structural information is absent, is indicated by the dashed line. (D) βAnc rescues cell-surface expression of human α, δ, and ɛ subunits, while co-expression with a Torpedo β subunit (βTorp.) does not. Relative expression was determined by measuring binding of the radiolabeled competitive antagonist, α-bungarotoxin (see STAR Methods). Error bars represent ± 1SD from the mean (n = 2). (E) Sequence alignment of the H. sapiens and Torpedo marmorata β subunits alongside the reconstructed ancestral β subunit sequence (βAnc). Amino acids shared with H. sapiens, Torpedo marmorata, and unique to the ancestor are highlighted in orange, blue, and purple, respectively. Annotations above the alignment depict secondary structure elements, as well as the location of the eponymous “Cys loop.” Structure 2017 25, 1295-1302.e3DOI: (10.1016/j.str.2017.06.005) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 βAnc-Containing AChRs Are Functional but Exhibit Reduced Sensitivity to Acetylcholine (A) Representative ensemble whole-cell currents from cells heterologously expressing wild-type human AChR subunits (left), or cells expressing human α, δ, and ɛ subunits with the ancestral β subunit (βAnc; right). Acetylcholine (ACh) was applied for a 2-s pulse (bar above traces) at the concentrations (in μM) indicated on the left. (B) Dose-response relationships for wild-type AChRs (orange circles, n = 5), βAnc-containing AChRs (purple squares, n = 5), human α, δ, and ɛ subunits co-expressed without a β subunit (gray upward-facing triangles, n = 16), and the ancestral β subunit expressed by itself (white downward-facing triangles, n = 16). In each case, error bars represent ±1 SD from the mean. Structure 2017 25, 1295-1302.e3DOI: (10.1016/j.str.2017.06.005) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 βAnc-Containing AChRs Display Reduced Single-Channel Conductance (A) Single-channel activity of wild-type human (top) and βAnc-containing (bottom) AChRs. Shown are representative bursts of single-channel activity, where openings are upward deflections. (B) All-points histograms from the bursts shown in (A) where the baseline and open-channel currents are each fit with a Gaussian function to determine the indicated single-channel current amplitude. (C) Mean single-channel amplitudes for wild-type human AChRs (orange; 18 measurements from 3 recordings) and βAnc-containing AChRs (purple; 12 measurements from 2 recordings). Recordings were acquired at −120 mV and digitally filtered with a 10-kHz Gaussian filter. Error bars in “C” represent ± 1 SD from the mean. Structure 2017 25, 1295-1302.e3DOI: (10.1016/j.str.2017.06.005) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 βAnc-Containing AChRs Have Altered Single-Channel Kinetic Activity (A and B) Bursts of single-channel activity elicited by 30 μM acetylcholine for (A) wild-type human AChRs and (B) βAnc-containing AChRs. In each case, the boxed regions in the top two traces are expanded directly below to reveal the fine kinetic structure of single-channel openings and closings. Recordings were acquired at −120 mV and digitally filtered with a 25-kHz Gaussian filter. The scale bars in (B) also apply to (A). (C and D) Open and closed dwell time histograms for (C) wild-type human AChRs, and (D) βAnc-containing AChRs. Each histogram was fit (solid line) by a sum of individual exponentials (dashed lines). Structure 2017 25, 1295-1302.e3DOI: (10.1016/j.str.2017.06.005) Copyright © 2017 Elsevier Ltd Terms and Conditions