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Allosteric potentiation of a ligand-gated ion channel is mediated by access to a deep membrane-facing cavity

Significance Molecular mechanisms of general anesthetic modulation in pentameric ligand-gated ion channels remain controversial. Here we present molecular simulations and functional data that reveal correlations between dynamic differences in a membrane-accessible cavity and dramatic anesthetic effects, separate inhibitory and potentiating effects within the same electrophysiology recordings, and support a model for communication between the lipid bilayer and ion channel pore. In particular, enhanced electrostatic interactions in the membrane-accessible site were associated with a unique mode of anesthetic potentiation, persisting tens of minutes after washout. These results offer a bridge between lipid- and receptor-based theories of anesthesia, with the potential to inform both mechanistic understanding and drug development.

virtual site hydrogens were added to PFL, and a 5 fs time step was used. All apo simulations used a 5 fs time step.

Analysis of molecular dynamics simulations
Gromacs was used to calculate RMSDs. For these calculations, the first frame of each trajectory was used as a reference, and RMSDs were calculated from alignments of backbone α-carbons in each subsequent frame.
For cavity volume calculations, MDpocket (17) was used to identify all cavities in a starting model for each of the systems (holo and apo forms of Met, Trp, Gly and Pro variants). Points corresponding to intrasubunit PFL pockets were then selected and tracked throughout the trajectories to yield pocket volume vs. time. Propofol molecular volume was estimated from the supplier-reported density and molar mass (Sigma-Aldrich Sweden AB, Stockholm, Sweden) VMD (18) was used for calculating distances. Pocket depth was tracked by the shortest distance between the cavity perimeter and residue Val-242. Distance between the hydroxyl oxygen of PFL and the backbone carbonyl of Ile-202 was used to evaluate possible hydrogen bond formation.
The percentage of frames during which the measured PFL-Ile-202 distance was within a cut-off of 3.5 Å was calculated. For the other propertied vs. time, averages were calculated. Error estimates for these averages were calculated with the Gromacs gmx analysis tool using block averaging, the details of which have been described previously (19). Subunits where the function used for error estimates could not be adequately fitted to the autocorrelation function were excluded from the averages and error estimates. For the pocket volume estimates from crystal structures, the standard error of the mean ( ! = / ) was calculated, the five subunits giving n=5.
Pore hydration was tracked using VMD (18). The number of waters within ±2 Å along the pore axis from the center of mass of Ile-233 (9') was counted in each frame and plotted versus time. A running average over fifty points was added to guide the eye.

Molecular docking
Docking of PFL to X-ray structures was carried out using AutoDock Vina (20), reporting the top nine clusters of binding poses in each of two search volumes, ranked by mean energy score. Ligand atoms representing PFL (PDB ID 5MUO), and protein atoms representing apo-closed (4NPQ, chains A-E) andopen (4HFI) and holo-open WT (3P50) and Trp-205 (5MVN) GLIC structures, were extracted from their respective PDB files. Side-chain positions were selected and/or completed, and hydrogens and charges added, using the Dock Prep tool in UCSF Chimera (21). To dock to the intrasubunit site, search volumes of 20 x 15 x 20 Å were placed to enclose the M1-M4 helices of a single (A) subunit of each structure, extending ~4 helical registers (~50% of the conduction pathway length) from the M2-M3 loop towards the intracellular side. To dock to the channel pore, 19 x 19 x 31 Å volumes were placed parallel to the conduction pathway, enclosing residues Thr-226 (2') through Ile-240 (16') of each structure; resulting clusters were categorized by proximity to either residue Ser-230 (6') or Ala-237 (13').
Briefly, nuclei of Xenopus oocytes were injected with 0.5-6.0 ng cDNA and stored in incubation medium for 2-7 days prior to recordings. 100-mM stock solutions of PFL in dimethylsulfoxide (DMSO) were stored at 4°C. All modulators were freshly diluted in running buffer; PFL and bromoform solutions were sonicated for 2 min prior to application.
For each mutant, EC10 conditions were first determined as previously described (23) and as calculated below. Modulation was measured by activating the channel in EC10 buffer for 2 min (referred to as the pretreatment peak), then washing out for 5 min; preapplying the drug in running buffer for 1 min, coapplying in EC10 buffer for 2 min (cotreatment peak), then washing out for 5 min; then activating again at EC10 for 2 min (posttreatment peak). To measure time dependent persistence in Fig. 3E and SI Appendix Fig S7B, 100 μM PFL in running buffer (pH 7.5) was applied for 0.5, 1, 3 or 6 min instead of the usual cotreatment. To avoid artifacts from persistent drug effects, each oocytes was discarded after a single modulation protocol.

Analysis of electrophysiology data
Each recording was collected using 4-11 oocytes from at least two frogs. To determine EC10 conditions (the agonist concentration producing 10% maximal response), proton concentrationdependence curves were determined by nonlinear regression fit using the equation R = Rmax/(1+10^(logEC10-C)*nH)) for which R is the peak response, Rmax the maximal activation, C the proton concentration, and nH the Hill coefficient. Acute modulation in Fig. 2F, S6A-C, and S8A-B represents mean % ± standard error of the mean (s.e.m.) values for ((Rco-Rpre)/Rpre)*100, where Rpre is the peak response prior to drug exposure (pretreatment) and Rco the response in the presence of modulator (cotreatment).