Multisite Binding of Anesthetics to GLIC, a Pentameric Ligand-Gated Ion Channel

Abstract

Volatile and intravenous anesthetics inhibit channel function of nicotinic acetylcholine receptors (nAChRs).Here we report the putative general anesthetic binding sites in Gloeobacter vioaceus pentameric ligand-gated ion channel (GLIC), a bacterial homolog of nAChR, using fluorescence quenching, multi-ns molecular dynamics (MD), and docking analysis. Fluorescence quenching results of halothane (Qmax∼ 85%, Kd ∼ 2 mM−1) and thiopental (Qmax∼ 100%, Kd ∼ 0.1 mM−1) suggest these two anesthetics bind to almost all TRPs. However, ketamine and etomidate only quenched about 20 ∼ 30% with nonspecific binding, indicating other non-TRP binding sites may exist. X-ray structure of GLIC (PDB: 3EAM) reveals three tryptophan (TRP) sites: A) W47 and W72 in the extracellular domain (ECD); B) W160 at the interface between ECD and transmembrane domain (TMD); and C) W213 and W217 in the TMD. Fluorescence quenching of three site-directed mutants, where A, B, or C site was singly remained, demonstrated similar binding affinities to all three sites for both halothane (Qmax: 84 ∼ 90%, Kd : 1.3 ∼ 2.1 mM−1) and thiopental (Qmax: 94 ∼ 100%, Kd : 0.07 ∼ 0.1 mM−1). Of these three sites, halothane prefers W47 and W72 (Qmax = 90 ± 2%, Kd = 1.3 ± 0.2 mM−1) while thiopental prefers W160 (Qmax = 94 ± 2%, Kd = 0.067 ± 0.005 mM−1). In consistent with fluorescence quenching experiments, our theoretical study also finds that both anesthetics bind to all TRPs though both prefer W160. Furthermore, non-TRP sites are discovered by our theoretical study: one near the TM2-3 loop and the other near D86 in the ECD. Collectively, This investigation will elucidate where volatile and intravenous anesthetics interact with pLGICs and reveal how the binding may lead channel functional changes. (Supported by NIH R01GM056257, R01GM069766, R37GM049202, R01GM066358).