It has been proposed that general anesthesia results from direct multisite interactions with multiple and diverse ion channels in the brain. An understanding of the mechanisms by which general anesthetics modulate ion channels is essential to clarify their underlying behavior and their role in reversible immobilization and amnesia. Despite the fact that volatile general anesthetics are drugs that primarily induce insensitivity to pain, they have been reported to sensitize and active the vanilloid-1 receptor, TRPV1, which is known to mediate the response of the nervous system to certain harmful stimuli and which plays a crucial role in the pain pathway. Currently, the mechanism of action of anesthetics is unknown and the precise molecular sites of interaction have not been identified. Here, using ∼2.5 μs of classical molecular dynamics simulations and metadynamics, we explore these enigmas. Binding sites are identified and the strength of the association is further characterized using alchemical free-energy calculations. Anesthetic binding/unbinding proceeds primarily through a membrane-embedded pathway, and subsequently, a complex scenario is established involving multiple binding sites featuring single or multiple occupancy states of two small volatile drugs. One of the five anesthetic binding sites reported was previously identified experimentally, and another one, importantly, is identical to that of capsaicin, one of the chemical stimuli that activate TRPV1. However, in contrast to capsaicin, isoflurane and chloroform binding free-energies render modest to no association compared to capsaicin, suggesting a different activation mechanism. Uncovering chloroform and isoflurane modulatory sites will further our understanding of the TRPV1 molecular machinery and open the possibility of developing site-specific drugs.
Keywords: TRP channels; anesthesia; docking; funnel metadynamics; molecular dynamics simulations.