Photoaffinity Ligand for the Inhalational Anesthetic Sevoflurane Allows Mechanistic Insight into Potassium Channel Modulation

Sevoflurane is a commonly used inhaled general anesthetic. Despite this, its mechanism of action remains largely elusive. Compared to other anesthetics, sevoflurane exhibits distinct functional activity. In particular, sevoflurane is a positive modulator of voltage-gated Shaker-related potassium channels (K(v)1.x), which are key regulators of action potentials. Here, we report the synthesis and validation of azisevoflurane, a photoaffinity ligand for the direct identification of sevoflurane binding sites in the K(v)1.2 channel. Azisevoflurane retains major sevoflurane protein binding interactions and pharmacological properties within in vivo models. Photoactivation of azisevoflurane induces adduction to amino acid residues that accurately reported sevoflurane protein binding sites in model proteins. Pharmacologically relevant concentrations of azisevoflurane analogously potentiated wild-type K(v)1.2 and the established mutant K(v)1.2 G329T. In wild-type K(v)1.2 channels, azisevoflurane photolabeled Leu317 within the internal S4-S5 linker, a vital helix that couples the voltage sensor to the pore region. A residue lining the same binding cavity was photolabeled by azisevoflurane and protected by sevoflurane in the K(v)1.2 G329T. Mutagenesis of Leu317 in WT K(v)1.2 abolished sevoflurane voltage-dependent positive modulation. Azisevoflurane additionally photolabeled a second distinct site at Thr384 near the external selectivity filter in the K(v)1.2 G329T mutant. The identified sevoflurane binding sites are located in critical regions involved in gating of K-v channels and related ion channels. Azisevoflurane has thus emerged as a new tool to discover inhaled anesthetic targets and binding sites and investigate contributions of these targets to general anesthesia.