Emerging Molecular Mechanisms of Signal Transduction in Pentameric Ligand-Gated Ion Channels  Ákos Nemecz, Marie S. Prevost, Anaïs Menny, Pierre-Jean Corringer  Neuron  Volume 90, Issue 3, Pages 452-470 (May 2016) DOI: 10.1016/j.neuron.2016.03.032 Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 pLGICs (A) Phylogeny of human pLGICs with the inclusion of non-human pLGICs (species listed) that have resolved structures, rooted to split anionic with prokaryotic (prok.) channels (bottom) and cationic channels (top). Aligned using ClustalW (Larkin et al., 2007), with the phylogeny tree generated by PhyML on phylogeny.fr (Dereeper et al., 2008). Subfamilies are color coded, with mammalian anionic (GABAARs, light red; GlyRs, dark red) and cationic (nAChRs, light blue; 5-HT3ARs, dark blue; Zinc-activated channels [ZAC], gray) receptors in solid color, meanwhile invertebrate/prokaryotic (GluClα, purple; GLIC and ELIC, orange; AChBPs, green) homologs are outlined. Starred subunits have structures solved with selected PDB IDs as: AChBP: agonist, PDB: (A.c.) 2BYQ, 2WN9, 2WNJ; (B.t.) 2BJ0; (C.t.) 4AFG, 4AFH; (L.s.) 1UV6, 1UW6, 2ZJU; antagonist, PDB: (A.c.) 2BYR, 2BYP; (L.s.) 1YI5; 5-HT3A, PDB: 4PIR; β3-GABAA, PDB: 4COF; ELIC: closed, PDB: 2VL0, 2YOE; GLIC: open, PDB: 4HFI; closed, PDB: 4NPQ; intermediates, PDB: 4NPP, 3TLT (LC1), 3TLS (LC2), 3TLV (LC3); GluClα: open, PDB: 3RHW (ivermectin), 3RI5 (ivermectin, picrotoxin), 3RIA (ivermectin, iodide), 3RIF (ivermectin, glutamate), closed, PDB: 4TNV, intermediate: 4TNW (POPC); α1-GlyR, open, PDB: 3JAE (glycine), 3JAF (glycine, ivermectin), closed, PDB: 3JAD (strychnine); α3-GlyR, closed, PDB: 5CFB (strychnine). (B) Simplified conformational state schema with the three primary states (Resting, Active, and Desensitized) and an I state that includes fast desensitized and other intermediate states, such as flipped and a primed state that may gate independently. The presence of multiple conformations can exist for each state. (C) Representative whole-cell inward current of a pLGIC upon perfusion of an agonist (bar shows perfusion duration), with plausible state transitions detailed. Neuron 2016 90, 452-470DOI: (10.1016/j.neuron.2016.03.032) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Basic Structure (A) Single subunit side view representation of GLIC and 5-HT3AR. The ECD contains the common β-sandwich fold found in the family with mammalian pLGICs including an N-terminal α-helix; important loops are labeled to give indication of location; the TMD contains four sequential α-helices (labeled M1–M4); and mammalian receptors include an ICD between M3 and M4 (partially visualized in the 5-HT3A structure), where the resolved α-helices are labeled (MA/MX). (B) Side view of the homo-pentamer GluClα (white), with the front subunit removed to show the vestibule and pore of the channel. Surface representation of accessible surface inside the channel is shown in light-cyan, using Mole2 (Berka et al., 2012). Orthosteric (glutamate, green) and allosteric ligands (ivermectin, orange; picrotoxin, magenta) resolved in GluClα structures are also shown bound. Top-down view of ECD (top) and TMD (bottom) with ligands bound, show pentameric form of the receptor and inter-subunit location of the orthosteric pocket, as well as the PAM and channel blocker binding sites. (C) Zoom of orthosteric pocket with loops A–F (magenta, blue, yellow, pink, green, and cyan, respectively) labeled and colored, with the canonical aromatic on loop B involved in ligand binding represented in stick form. Neuron 2016 90, 452-470DOI: (10.1016/j.neuron.2016.03.032) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Pore Radii Channel states represented by two M2 α-helices (prime numbering of residues) from opposite facing subunits with pore radii, calculated using Hole (Smart et al., 1993), shown to the right. The radii of H2O, K+, and hydrated-K+ are shown as dashed lines. Top: Closed/Resting states from GLIC, GluClα, ELIC, α1-GlyR, and 5-HT3AR; middle: Open/Active states from GLIC, GluClα, and α1-GlyR; bottom: Expanded/Desensitized states from β3-GABAAR, α1-GlyR, and GluClα. Receptor subunits are color coded with pore radii traces. A sequence alignment of the M2 α-helices is found underneath. Neuron 2016 90, 452-470DOI: (10.1016/j.neuron.2016.03.032) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 pLGIC Transitions Red, resting-like state conformation; green, active-like state conformation. In each panel, the top-down view of the ECD (top) and TMD (bottom) are shown, along with a three subunit side view of the full receptor (middle). All structures were aligned using the whole pentamer. Left: GLIC transitions (pH7- > pH4); middle: GluClα transitions (apo->ivermectin-bound); right: α1-GlyR transitions (strychnine->glycine/ivermectin-bound). Arrows indicate gate-opening transition motions, with curved arrows representing twisting motions and broad arrows translations/tilts. Neuron 2016 90, 452-470DOI: (10.1016/j.neuron.2016.03.032) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Binding Site Transitions Side view of the orthosteric pocket rotated 45° on the x axis. (A) GluClα glutamate-ivermectin (white cartoon, green lines, glutamate in cyan stick representation) structure. Glutamate polar contacts are shown by dashed yellow lines and residues are labeled. (B) Overlay of GluClα-apo (red cartoon and Cα spheres) and -ivermectin bound (white cartoon and green Cα spheres) structures. Residues (Arg123 and Lys171) that switch conformation are shown in line form for both conformations. (C) Overlay of GLIC-pH7 (red cartoon and Cα spheres) and -pH4 (white cartoon and green Cα spheres) structures. (D) Overlay of α1-GlyR-strychnine (red cartoon and Cα spheres) and -glycine (white cartoon and green Cα spheres) structures. Cyan arrows indicate movement. Neuron 2016 90, 452-470DOI: (10.1016/j.neuron.2016.03.032) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 Allosteric Site Transitions (A) Top-down view of an overlay of the TMD of GluClα-apo (red cartoon/cylinders and Cα atom spheres) with -ivermectin-bound (white cartoon/cylinders and green Cα atom spheres), and α1-GlyR-strychnine (pink Cα atom spheres) with -ivermectin-bound (teal Cα atom spheres) structures. Cylinders of GlyR structures are not shown as they are synonymous with GluClα. Ivermectin from GluClα is shown in green stick representation. (B) Top-down view of an overlay of the TMD of GluClα-apo (red cartoon/cylinders and Cα atom spheres) with -POPC-bound (orange cartoon/cylinders and Cα atom spheres) structures. POPC is shown in orange stick representation. (A and B) Residues of Cα spheres are labeled for GluClα, and respectively colored dashed lines are shown to emphasize change in cavity space. (C) Top-down view, rotated 45° on the x axis, of an overlay of the TMD of GLIC-pH7 (red cartoon/cylinders, Cα spheres, and lines) with -pH4 ethanol-bound Phe14′Ala mutant (white cartoon/cylinders, green Cα spheres and lines). Ethanol is shown as cyan stick representation. Phe14′ of the open GLIC structure is shown in orange line representation. Residues (Asn15′, Glu19′, and Asn200) with polar interaction (shown as yellow dashed lines) to ethanol are labeled and shown as lines with Cα spheres. (A, B, and C) Principal and complementary (with primes) subunit α-helices are labeled. Cyan arrows indicate movement from resting to active or resting to POPC. Neuron 2016 90, 452-470DOI: (10.1016/j.neuron.2016.03.032) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 7 Hypothetical Classification of Known Structures into the Physiological Three State, Resting, Active, and Desensitized, Model Highly simplified structures are represented as two subunit compositions with the ECD as a large rectangle and the TMD as a small rectangle and a cylinder for the M2 α-helix, with color coding representing similar conformations. Resting-like apo conformations show two distinct forms (GluClα-apo and GLIC-pH7), meanwhile antagonist-bound conformations and the locally closed conformation of GLIC would also be considered resting states. The active-like conformations of GLIC-pH4, as well as ivermectin-bound GluClα and α1-GlyR, could be grouped into one synonymous active state, whereas the expanded glycine-bound α1-GlyR conformation would define another. Finally, only one structure has been unequivocally deemed to be in a desensitized-like conformation, which is that of the β3-GABAAR. Assignment of the ELIC, 5-HT3A, and POPC-bound GluClα structures remains elusive and therefore these are not represented. Neuron 2016 90, 452-470DOI: (10.1016/j.neuron.2016.03.032) Copyright © 2016 Elsevier Inc. Terms and Conditions