Structural Basis for a Bimodal Allosteric Mechanism of General Anesthetic Modulation in Pentameric Ligand-Gated Ion Channels

Abstract

Ion channel modulation by general anesthetics is a vital pharmacological process with implications for receptor biophysics and drug development. Functional studies have implicated conserved sites of both potentiation and inhibition in pentameric ligand-gated ion channels, but a detailed structural mechanism for these bimodal effects is lacking. The prokaryotic model protein GLIC recapitulates anesthetic modulation of human ion channels, and it is accessible to structure determination in both apparent open and closed states. Here, we report ten X-ray structures and electrophysiological characterization of GLIC variants in the presence and absence of general anesthetics, including the surgical agent propofol. We show that general anesthetics can allosterically favor closed channels by binding in the pore or favor open channels via various subsites in the transmembrane domain. Our results support an integrated, multi-site mechanism for allosteric modulation, and they provide atomic details of both potentiation and inhibition by one of the most common general anesthetics.

Structural evidence for the binding of monocarboxylates and dicarboxylates at pharmacologically relevant extracellular sites of a pentameric ligand-gated ion channel

“…Therefore, thanks to their better disposition for structural studies, prokaryotic pLGICs were used as structural models for the characterization of the transition and dynamics of pLGICs, with the best characterized being that from Gloeobacter violaceus, which is named GLIC (Bocquet et al, 2007). Indeed, the structure of GLIC has been solved in several conformational states, including an apparently open state (Bocquet et al, 2009;Sauguet, Poitevin et al, 2013;Fourati et al, 2015), a resting-like state (Sauguet et al, 2014) and an intermediate locally closed state (Prevost et al, 2012), and in complexes with a range of pharmacological molecules, including general anaesthetics (Fourati et al, 2017(Fourati et al, , 2018Laurent et al, arose as a suitable tool to characterize the structural transition occurring during activation of pLGIC and its modulation at the molecular level.…”

Allosteric potentiation of a ligand-gated ion channel is mediated by access to a deep membrane-facing cavity

“…It remained puzzling, however, why this presumed-inhibited complex remained in an apparent open state. In subsequent work, PFL was found to cocrystallize inside the closed channel pore ( 24 ), indicating an alternative mechanism of pore inhibition ( Fig. 1 ).…”

“…In contrast to the initial association of the intrasubunit site with inhibition, multiple M2 helix variants confer net PFL potentiation rather than inhibition and convert the channel from crystallizing in an apparent closed to open conformation under identical conditions ( 24 ). A more complex profile resulted from mutating the M1 Met-205 to Trp, which produced net potentiation by low to moderate concentrations of PFL, with inhibition restored at high concentrations ( 25 ).…”

“…A more complex profile resulted from mutating the M1 Met-205 to Trp, which produced net potentiation by low to moderate concentrations of PFL, with inhibition restored at high concentrations ( 25 ). Crystallization of Trp-205 showed stabilization of PFL in the intrasubunit cavity ( 24 ). However, static structural data did not explain how dynamic conformational changes influence binding at the protein−lipid interface, nor how such subtle perturbations in structure dramatically alter sensitivity to a modulator with such low apparent affinity, and so few distinctive functional groups, as PFL.…”

Competitive dewetting underlies site-specific binding of general anesthetics to GABA(A) receptors

“…(53,54) Three sevoflurane molecules enter the pore through the ECD vestibule over 1.3 µs, consistent with multiple occupancy of the pore observed in flooding/equilibrium simulations of GLIC and nAChR exposed to isoflurane, (15,55) as well as later crystal structures. (19,20) Since one of the three pore-bound sevoflurane molecules spontaneously shifted from the pore to the ↵ + -inter-subunit site, the pore occupancy was at most two sevoflurane molecules. Unlike GLIC, ELIC, or nAChR, GABA A receptor function is enhanced by sevoflurane, and this result is only consistent with functional results if sevoflurane has lower affinity for the pore than for other TMD binding sites.…”