Flexibility of a Transmembrane Helix Underlies Dramatic Reversal of Net Anesthetic Effects in a Pentameric Ligand-Gated Ion Channel

Abstract

Mechanisms of action for general anesthetics have evolved substantially from purely lipid-based theories to models involving specific protein interfaces, with the capacity to allosterically inhibit as well as potentiate human Cys-loop receptors. However, we still lack a structurally detailed mechanism for these bimodal allosteric effects. Recently, we mapped an allosteric model for anesthetic modulation involving multiple state-dependent binding sites, based on new co-crystal structures of the bacterial homolog GLIC and the general anesthetic propofol. Notably, a point mutation (M205W) in the first transmembrane helix facilitated propofol binding in a membrane-accessible intrasubunit site, and conferred primarily potentiating rather than inhibitory net anesthetic effects. Molecular dynamics simulations correlated the potentiated phenotype with changes in backbone flexibility of the first transmembrane helix. Here, we used mutagenesis and two-electrode voltage-clamp electrophysiology to reveal that helix-perturbing residues at the same transmembrane position lead to potent potentiation even by subclinical concentrations of propofol. Interestingly, these variants were so sensitive to propofol that they exhibited persistent potentiation for up to 40 minutes after exposure to moderate concentrations of propofol, in contrast to an almost instantaneous washout in wild-type channels. The extent of this effect depended on propofol exposure time, suggesting involvement of the membrane as a reservoir for a lipid-accessible binding site. Persistent potentiation also appeared to be independent of an inhibitory effect observed at high acute doses, consistent with the presence of a discrete, water-accessible site for inhibition. The resulting mechanism for general anesthetic modulation could bridge the lipid-based Meyer-Overton theory, postulated more than a century ago, with more complex receptor-based models, informing both our understanding of allostery and the development of new anesthetics.