Structural and Functional Evidence for Multi-Site Allostery Mediated by General Anesthetics in a Model Ligand-Gated Ion Channel

Abstract

General anesthetics act as either positive or negative allosteric modulators of several pentameric ligand-gated ion channels, including physiologically important receptors for γ-aminobutyric acid and acetylcholine. Although functional studies have implicated conserved sites of modulation in this channel family, the limited scope and resolution of structural data for human Cys-loop receptors have hampered mechanistic studies of anesthetic action. We previously showed the prokaryotic homolog GLIC to be a useful model system that recapitulates functional modulation of human ion channels, and enables structure determination both in apparent open and nonconducting states. Specifically, anesthetic inhibition of GLIC can be removed, reversed, or rendered bimodal by site-directed mutations in the transmembrane domain (TMD). In this work, we provide crystallographic and electrophysiological evidence for a multi-site mechanism of bimodal modulation by anesthetizing agents, including the common surgical medication propofol. With the pore in an apparent nonconducting state, propofol bound at the intracellular (lower) end of the channel pore, similar to other inhibitors. Consistent with this result, single hydrophobic substitutions at the lower-pore site enhanced functional inhibition. Conversely, in the apparent open state, anesthetics bound to one or more contiguous sites in the extracellular-facing (upper) end of the TMD, particularly for variants in which anesthetic inhibition was reduced or reversed. In one novel variant exhibiting anesthetic potentiation, propofol binding in the upper-TMD converted the channel from an apparent nonconducting- to open-pore conformation under otherwise identical conditions, providing direct evidence for potentiation via specific sites in the upper TMD. Based on these findings, we propose a structural model for allostery in which anesthetic binding to spatially distinct TMD cavities differentially stabilizes opposing functional states of pentameric ligand-gated ion channels, providing valuable insights into channel modulation and drug development.