a Ion permeation pathway generated with HOLE⁶⁰ for GlyR–Apo (salmon red), GlyR–Gly/PTX (deep teal) and GlyR–Gly (slate blue). For clarity, the cartoon representation of only two non-adjacent subunits are shown. Green and purple spheres define radii of 1.8–3.3 Å and >3.3 Å, respectively. The residues located at various pore constrictions are shown as sticks. b The pore radius is plotted as a function of distance along the pore axis. The dotted line indicates the approximate radius of a hydrated chloride ion, which is estimated at 2.26 Å. c A view of M2 helices from the extracellular end for the three GlyR conformations. Positions Leu9′ and Pro-2′ are shown in ball-and-stick representation and the corresponding distances between Cα are given in Å.

a Cryo-EM density segments for neurotransmitter binding site residues and glycine ligand as seen in the GlyR–Gly structure. Residues in the principal subunit and complementary subunit are indicated in black and magenta, respectively. b LigPlot analysis of glycine orientation in the binding pocket and residues within 4 Å distance are displayed in red⁷⁰. c Comparison of the neurotransmitter binding site for the GlyR–Apo and GlyR–Gly conformations. The residues that are involved in neurotransmitter binding are shown in sticks. d Cryo-EM map showing the density for M2 and PTX bound in the pore of GlyR–Gly/PTX. The interacting residues are shown in stick representation. For clarity, M2 for only two diagonal subunits are shown. e LigPlot analysis of PTX and the interacting residues. f A close-up of the M2 conformations is shown upon aligning the three GlyR conformations. Positions Leu9′ and Pro-2′ are shown as sticks.

a A side-view of the ECD interface in GlyR–Apo and GlyR–Gly. The principal subunits are colored (GlyR–Apo: salmon red and GlyR–Gly: slate blue) while the complementary subunits are shown in shades of gray. b A side view of the TMD in GlyR–Apo and GlyR–Gly conformations. c Interactions at the subunit- and domain-interfaces between ECD–ECD, ECD–TMD, and TMD–TMD in GlyR–Apo (left) and GlyR–Gly (right). The residues involved in the interactions are shown in sticks and interactions are highlighted by black dashes. Mutations at positions shown in green are associated with hyperekplexia².

a An overlay of GlyR–Apo and GlyR–Gly. The TMD viewed from the top reveals repositioning of TM helices. The intra-subunit and inter-subunit cavities are indicated by green and gray arrows, respectively. b Extent of conformational change in the ICD formed by the pre-M4 region, the M1–M2 linker, and the post-M3 region. The direction of movement is highlighted by black arrows. c A comparison of the inter-subunit (gray dots) and intra-subunit (green dots) cavities in GlyR–Apo (top) and GlyR–Gly (bottom) predicted using F pocket algorithm⁶¹. The cavities predicted on the surface were removed for clarity. The right and left panels are side and top views, respectively. The residues shown in sticks on the left panel are implicated in binding allosteric ligands⁴⁵.

a TEVC recording of 1 mM glycine-induced currents in the presence and absence of 0.5 μM IVM (left). Representative trace from multiple independent oocyte recordings (n = 5). The traces are normalized to the peak current to show the current decay. Ion permeation pathway for GlyR–Gly/IVM-1 GlyR–Gly/IVM-2 (right). Green and purple spheres define radii of 1.8–3.3 Å and >3.3 Å, respectively. The residues located at various pore constrictions are shown as sticks. b Current recording for 0.1 mM glycine and 0.1 mM glycine and 30 μM IVM (left). Representative trace from multiple independent oocyte recordings (n = 3). The pore profile for GlyR–Gly/IVM-3 structure. c A view of M2 helices from the extracellular end Positions Leu9′ and Pro-2′ and the corresponding distances between Cα in Å. d Alignment of GlyR structures showing the conformational differences at the level of Pro-2′. Dotted arrows indicate the direction of rotation going from the GlyR–Apo (closed) to the GlyR–Gly (desensitized) state.

a Mean pore radius profiles and standard deviations averaged across three independent 30 ns equilibrium simulations for GlyR–Apo (left), GlyR–Gly/PTX (middle), and GlyR–Gly (right) along the central pore axis. The final 20 ns of each 30 ns simulation trajectory was used to evaluate these profiles. The one-standard-deviation range between calculations (n = 3, independent repeats of 30 ns simulations) are shown as a gray band and the thick line is the mean between the triplicate 30 ns simulations. Major constriction sites are indicated and the dotted line denotes the radius of hydrated chloride ion. The black traces are the pore radius profile calculated from the cryo-EM structures. b Corresponding mean water free energy profiles and standard deviations. Peaks in free energy profiles are highlighted. c Trajectories along the pore (z)-axis of water molecules and chloride ion coordinates within 5 Å of the channel axis inside the pore, in the presence of a +500 mV transmembrane potential difference (i.e., with the cytoplasmic side having a positive potential). One of five independent 200 ns replicates is shown for each structure. During these and the preceding simulations, positional restraints were placed on the protein backbone, in order to preserve the experimental conformational state while permitting rotameric flexibility in amino acid side chains. The energetic barriers due to the ring of Leu9′ and Pro-2′ are at z ~0 and −20 Å, respectively.