Probing Conformational Motions Underlying Anesthetic Drug Actions in a Ligand-Gated Ion Channel

Abstract

General anesthetics and ethanol exert their functional effects on pentameric ligand-gated ion channels (pLGICs) by binding to distinct allosteric sites that are far from the agonist binding sites. While recent high-resolution structures of prokaryotic and eukaryotic pLGICs, solved in the presence of these drugs have illuminated their binding sites, the structural mechanisms underlying how these drugs enhance or inhibit pLGIC function are largely unknown. Recently, a single residue variant in the bacterial pLGIC, GLIC, was identified that is potentiated by ethanol (PNAS 108:12149-12154, 2011). Taking advantage of this GLIC ethanol-sensitive mutant, we are using site directed spin labeling electron paramagnetic resonance (SDSL EPR) spectroscopy to study motions underlying ethanol potentiation of GLIC currents. Previous studies have shown that agonist-mediated channel opening is associated with an inward tilting of the extracellular binding domain (ECD). Here, we examined if, and how, the ECD moves in response to ethanol binding in the transmembrane channel domain. Individual cysteine residues were introduced in the ECD of an ethanol-sensitive GLIC construct (F14’A) at the top of β-strands (β1, 2, 5, 6, 8), labeled with the spin probe MTSL and ethanol-induced motions were measured using double electron-electron resonance (DEER) spectroscopy and compared to agonist (proton)-induced gating motions. DEER distance distributions for spin labels attached to N19C (β1), S93C (β5) and Q101C (β6) revealed that the attached probes moved closer together at pH 3.0, indicating that the inner β-sheet of GLIC ECD tilts inwards upon agonist activation. Interestingly, DEER distance distributions for N19 (β1) and S93 (β5) showed no significant changes in the presence of ethanol, indicating that structural mechanisms underlying ethanol allosteric drug modulation of pLGIC function are different than agonist-mediated motions that lead to channel activation.