General anesthetic modulation of a pentameric ligand-gated ion channel explored with simulations.

General anesthetics are known to modulate pentameric ligand-gated ion channels (pLGICs), including GABAA, glycine, serotonin, and glutamate receptors, among others. The bacterial (Gloeobacter) ion channel GLIC captures anesthetic modulation while providing high-resolution structures in each of its distinct functional conformations, allowing for molecular definition of state-dependent binding and quantification of allosteric mechanisms. This study uses molecular dynamics, metadynamics, and free energy simulations to calculate the free energy surfaces of propofol binding to open and closed channels. We observe distinct binding sites in the pore, within and between transmembrane domain subunits, as well as in the extra-cellular domain, that differ in location and free energies between open and closed states. Each transmembrane site was seen to bifurcate into two adjacent sites, with overlapping residues that agree with X-ray crystallography, but with other residues identified as strongly interacting that provide predictions for experimental verification. The investigation revealed distinct pathways between intra-subunit, inter-subunit, and pore sites in the open and closed states. Multiple propofol binding to neighboring subunits was seen to modify the binding surface and lead to a third inter-subunit site within the closed state. Calculated free energy changes are consistent with previous suggestions of bimodal modulation via competing sites that act to inhibit or potentiate the channel. Inhibition via pore binding involves pore occlusion by propofol, with minor changes to pore shape and size, notably tending to close the channel when propofol is bound. Binding to intra-subunit sites was shown to thermodynamically favor the open state, in agreement with experimental evidence for potentiation. Calculations of binding to inter-subunit sites were found to involve opposing effects from the two constituent sites, leading to no clear net shift in equilibrium. These observations offer molecular explanations for the effects of propofol that are likely to apply to other pLGICs, assisting in the development of improved anesthetics with better safety margins in future.