MD simulations of ion permeation and block through the structural model of the glycine-and-ivermectin–bound α1 GlyR. (A) Ion trajectories at −100 mV through an expanded version of the cryo-EM-structure model (PDB ID code 3JAF) obtained using grid-steered MD (Wells et al., 2007). The membrane was bathed by symmetrical 150 mM NaCl, and the temperature was 37°C. Only the trajectories of ions that crossed the membrane are plotted. Gray areas indicate the regions occupied by the membrane in the periodic-simulation system, and upward transitions of the ion trajectories through these regions represent outward crossings. The darker lines are running averages of the data, which are displayed in a lighter shade. The length of the simulation box along the z-axis was 84 Å. (B) Single-channel current–voltage relationships of the (full-length) rat α1 GlyR at ∼22°C. The recordings were obtained in the cell-attached patch-clamp configuration from transiently transfected HEK-293 cells. The pipette solution contained ∼150 mM Cl^–. Taurine is a partial agonist of the α1 GlyR, whereas glycine is a full agonist. (C) Pore-radius profiles (estimated using HOLE [Smart et al., 1996]) of structural models of the glycine-and-ivermectin–bound GlyR. The profile of the cryo-EM-structure model is compared with those computed during MD simulations of ion permeation through a partially expanded version and the expanded version of the 3JAF model. Ion-permeation-MD pore-radius profiles are mean profiles—displayed as the mean (darker lines) ± 1 SD (lighter shade)—calculated from the different frames of each simulation; the vertical axis extends, approximately, between M2 positions −3′ (bottom) and 21′ (top). Side-chain rotamers were optimized using SCWRL4 (Krivov et al., 2009) before the ion-permeation simulations were run. IVM, ivermectin. (D) Cα profiles of the cryo-EM-structure model and the expanded version. For clarity, the Cα profile of the partially expanded model was omitted; it lies somewhere in between those displayed. In the case of the expanded 3JAF model, the profile was calculated for a randomly chosen frame of the ion-permeation MD simulation. The five subunits of each model were averaged. Error bars (omitted if smaller than the symbols) are standard errors. Solid lines are cubic-spline interpolations. The lumen of the pore is to the right of the plot. (E) Snapshot from the ion-permeation MD simulation of the expanded model at −100 mV with a molecule of picrotoxinin placed in the pore. No ion crossings were recorded through this system. The molecule of picrotoxinin is shown in van der Waals representation, and the 30% water-occupancy isosurface is shown as transparent shapes. For clarity, only two nonadjacent chains are shown.