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

Abstract

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 (K1.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 K1.2 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 K1.2 and the established mutant K1.2 G329T. In wild-type K1.2 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 K1.2 G329T. Mutagenesis of Leu317 in WT K1.2 abolished sevoflurane voltage-dependent positive modulation. Azisevoflurane additionally photolabeled a second distinct site at Thr384 near the external selectivity filter in the K1.2 G329T mutant. The identified sevoflurane binding sites are located in critical regions involved in gating of K 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.

Identification of binding sites contributing to volatile anesthetic effects on GABA type A receptors

“…AziSEVO photolabeling of the b 3 -subunit, b 3 -M1 259 W241 and b 3 -M2 29 A249, again supports this a+/b2 interfacial binding cavity. By using mutagenesis and electrophysiology, other investigators have suggested that SEVO and ISO iso have different binding sites in a variety of potassium channels (22,26,49,50), and our results confirm that this is also true in GABA A receptors. Although comparatively less than ISO, a decrease in SEVO-positive modulation was observed within in vitro studies using a 1 -M2 159 S270W or S270I GABA A receptor mutants (8).…”

“…All spectra are provided in Supplemental Figs. [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31]. Photolabeled residues and their locations within either receptor are displayed in Table 2.…”

“…We performed analysis as previously reported (25) with the addition of predicted photolabel modifications (26,27). In brief, we conducted spectral analysis by using Thermo Proteome Discoverer 2.0 (Thermo Fisher Scientific) and the Mascot Daemon search engine using a customized database that contained GABA A receptor protein sequences that were supplied for heterologous expression or the human proteome UniProt database (Uni-ProtKB: P28472; http://www.uniprot.org/uniprot/P28472).…”

“…Maximum torsions were allowed for all investigated ligands-that is, ligands were fully flexible. We performed separate docking simulations using AutoDock Vina (33), as previously reported (26,34), of the entire accessible photolabeled cavities formed by subunit dimers. Grid box dimensions were set at 1Å resolution for ISO and AziISO sites a+/g2/g-intrasubunit (22 3 24 3 22Å), a+/b2 (22 3 22 3 22 A), and b+/a2 (22 3 24 3 42Å) sites.…”

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

“…Its is particularly worth of mention that our findings recapitulate independently very recent photolabeling experiments demonstrating that photoactive analogs of sevoflurane and propofol do interact at the S4S5 linker and the S6P-helix interface of Kv1.2 at the open-activated state 27, 28 . In detail, Leu317 and Thr384 were found to be protected from photoactive analogs, with the former being more protected than the latter.…”