Fig. 4: Assessment of conductance state of GlyR conformations.

a Ion permeation pathway generated with HOLE for GlyR-0.1gly structure. For clarity, only two non-adjacent subunits are shown. Colors of the spheres represent the following pore radii: red <1.15 Å, green 1.8-2.3 Å and purple >2.3 Å. Residues lining various pore constrictions are shown as sticks. Gray box is shown to highlight the constriction at Pro-2′ position. b Mean pore radius and one-standard-deviation (from three independent 30 ns equilibrium simulations for the GlyR-0.1gly structure) plotted along the central pore axis (colored lines). The final 20 ns of each 30 ns simulation trajectory was used to evaluate these profiles. Major constriction sites are indicated and the black dotted line denotes the radius of hydrated chloride ions. The gray line is the pore radius profile calculated from the cryo-EM structures. c. A view of Leu9′ and Pro-2′ positions from the extracellular end for the GlyR-0.1gly structure. Positions Leu9′ and Pro-2′ are shown in ball-and-stick representation and the corresponding distances between Cα are given in Å. d Simulation trajectories along the pore (z)-axis of water molecules and chloride ion coordinates within 5 Å of the channel axis inside the pore of GlyR-0.1gly structure, 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. 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. e Ion permeation profile for the GlyR-0.1gly-THC structure. f Pore radius profiles and standard deviations averaged across three independent 30 ns equilibrium simulations for GlyR-0.1gly-THC structure performed using same conditions described above. g A view of Leu9′ and Pro-2′ from the extracellular end for the GlyR-0.1gly-THC structure. h Simulation trajectories along the pore of water molecules and chloride ion coordinates for the GlyR-0.1gly-THC structure.