Utilizing Non-Equilibrium Methods to Probe the Effects of General Anesthetics on Ligand-Gated Ion Channel Dynamics

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Although anesthetics have been in use for over 150 years, the molecular mechanism behind their action is still poorly understood. Ligand-gated ion channels have been identified as the targets of these drugs, but it is unknown how anesthetics exert their effects. Previous simulations from our group have uncovered a membrane-dependent entrance mechanism, whereby the anesthetic partitions to the membrane prior to binding a transmembrane site identified in crystal structures. To probe for additional binding sites unbiasedly and corroborate the membrane-dependent entrance tunnel previously discovered, we have used the flooding technique where the system is flooded with multiple copies of the drug to increase the probability of observing binding events. Our simulations elucidated not only the binding region indicated in crystal structures, but also a novel second transmembrane binding site (TM2) observed consistently across all subunits. The TM2 site is directly between the crystal structure site (TM1) and the entrance tunnel elucidated in our simulations. Free energy calculations show binding to the TM2 site increases the affinity of anesthetic for the TM1 site in addition to acting as a physical barrier blocking the exit of anesthetic from the protein. Moreover, anesthetic binding to the TM1/TM2 sites induces an iris-like tilting of the M2 helix, as observed in our previous simulations, leading to constriction and dehydration of the conduction pore. Utilizing umbrella sampling, this conformational change significantly increases the barrier to conduction, prohibiting ion conduction in the dehydrated state. Lastly, measurements of non-equilibrium work show that desflurane only minimally perturbs the work required to induce constriction of the pore, while greatly increasing the work needed for the dehydrated to open state transition. This suggests that anesthetics stabilize the dehydrated state, but do not destabilize the open state.