Binding of the general anesthetic sevoflurane to ion channels.

The direct-site hypothesis assumes general anesthetics bind ion channels to impact protein equilibrium and function, inducing anesthesia. Despite advancements in the field, a first principle all-atom demonstration of this structure-function premise is still missing. We focus on the clinically used sevoflurane interaction to anesthetic-sensitive Kv1.2 mammalian channel to resolve if sevoflurane binds protein's well-characterized open and closed structures in a conformation-dependent manner to shift channel equilibrium. We employ an innovative approach relying on extensive docking calculations and free-energy perturbation of all potential binding sites revealed by the latter, and find sevoflurane binds open and closed structures at multiple sites under complex saturation and concentration effects. Results point to a non-trivial interplay of site and conformation-dependent modes of action involving distinct binding sites that increase channel open-probability at diluted ligand concentrations. Given the challenge in exploring more complex processes potentially impacting channel-anesthetic interaction, the result is revealing as it demonstrates the process of multiple anesthetic binding events alone may account for open-probability shifts recorded in measurements.

Concentration-Dependent Binding of Small Ligands to Multiple Saturable Sites in Membrane Proteins.

Membrane proteins are primary targets for most therapeutic indications in cancer and neurological diseases, binding over 50% of all known small molecule drugs. Understanding how such ligands impact membrane proteins requires knowledge on the molecular structure of ligand binding, a reasoning that has driven relentless efforts in drug discovery and translational research. Binding of small ligands appears however highly complex involving interaction to multiple transmembrane protein sites featuring single or multiple occupancy states. Within this scenario, looking for new developments in the field, we investigate the concentration-dependent binding of ligands to multiple saturable sites in membrane proteins. The study relying on docking and free-energy perturbation provides us with an extensive description of the probability density of protein-ligand states that allows for computation of thermodynamic properties of interest. It also provides one- and three-dimensional spatial descriptions for the ligand density across the protein-membrane system which can be of interest for structural purposes. Illustration and discussion of the results are shown for binding of the general anesthetic sevoflurane against Kv1.2, a mammalian ion channel for which experimental data are available.

Positive Allosteric Modulation of Kv Channels by Sevoflurane: Insights into the Structural Basis of Inhaled Anesthetic Action.

Inhalational general anesthesia results from the poorly understood interactions of haloethers with multiple protein targets, which prominently includes ion channels in the nervous system. Previously, we reported that the commonly used inhaled anesthetic sevoflurane potentiates the activity of voltage-gated K+ (Kv) channels, specifically, several mammalian Kv1 channels and the Drosophila K-Shaw2 channel. Also, previous work suggested that the S4-S5 linker of K-Shaw2 plays a role in the inhibition of this Kv channel by n-alcohols and inhaled anesthetics. Here, we hypothesized that the S4-S5 linker is also a determinant of the potentiation of Kv1.2 and K-Shaw2 by sevoflurane. Following functional expression of these Kv channels in Xenopus oocytes, we found that converse mutations in Kv1.2 (G329T) and K-Shaw2 (T330G) dramatically enhance and inhibit the potentiation of the corresponding conductances by sevoflurane, respectively. Additionally, Kv1.2-G329T impairs voltage-dependent gating, which suggests that Kv1.2 modulation by sevoflurane is tied to gating in a state-dependent manner. Toward creating a minimal Kv1.2 structural model displaying the putative sevoflurane binding sites, we also found that the positive modulations of Kv1.2 and Kv1.2-G329T by sevoflurane and other general anesthetics are T1-independent. In contrast, the positive sevoflurane modulation of K-Shaw2 is T1-dependent. In silico docking and molecular dynamics-based free-energy calculations suggest that sevoflurane occupies distinct sites near the S4-S5 linker, the pore domain and around the external selectivity filter. We conclude that the positive allosteric modulation of the Kv channels by sevoflurane involves separable processes and multiple sites within regions intimately involved in channel gating.