Abstract
Theories of general anesthesia have shifted in focus from lipid effects to specific interactions with proteins. Target receptors include several pentameric ligand-gated ion channels; however, structures of physiologically relevant proteins have yet to define binding at high resolution. Recent structures of the bacterial protein GLIC provide snapshots of state-dependent binding sites for propofol (PFL), offering a detailed model system for anesthetic modulation. By combining molecular dynamics and oocyte electrophysiology we have been able to reveal differential motion and modulation upon modification of a transmembrane binding site within each subunit. WT channels exhibited net inhibition by PFL, and a contraction of the cavity away from the pore-lining M2 helix in the absence of drug. Conversely, in variants exhibiting net PFL potentiation, the cavity was persistently expanded and proximal to M2. Mutations designed to favor this deepened site enabled sensitivity even to subclinical concentrations of PFL, and a uniquely prolonged mode of potentiation up to ∼30 min after washout. Dependence of these prolonged effects on exposure time implicates the membrane as a reservoir for a lipid-accessible site. However, at the highest measured concentrations, potentiation appeared to be masked by an acute inhibitory effect, consistent with presence of a discrete, water-accessible inhibitory site. This supports a multisite model of transmembrane allosteric modulation, including a possible link between lipid- and receptor-based theories that could inform the development of new anesthetics.
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