Mutational analysis to explore long-range allosteric couplings involved in a pentameric channel receptor pre-activation and activation

Abstract

Pentameric ligand-gated ion channels (pLGICs) mediate chemical signaling through a succession of allosteric transitions that are yet not completely understood as intermediate states remain poorly characterized by structural approaches. In a previous study on the prototypic bacterial proton-gated channel GLIC, we generated several fluorescent sensors of the protein conformation that report a fast transition to a pre-active state, which precedes the slower process of activation with pore opening. Here, we explored the phenotype of a series of allosteric mutations, using paralleled steady-state fluorescence and electrophysiological measurements over a broad pH range. Our data, fitted to a 3-states Monod-Wyman-Changeux (MWC) model, show that mutations at the subunit interface in the extracellular domain (ECD) principally alter pre-activation, while mutations in the lower ECD and in the transmembrane domain principally alter activation. We also show that propofol alters both transitions. Data are discussed in the framework of transition pathways generated by normal mode analysis (iModFit) that suggest collective protein motions concerted with pore opening. It further supports that pre-activation involves major quaternary compaction of the ECD, and suggests that activation involves principally a re-organization of a 'central gating region' involving a contraction of the ECD β-sandwich and the tilt of the channel lining M2 helix.

Open-channel structure of a pentameric ligand-gated ion channel reveals a mechanism of leaflet-specific phospholipid modulation

“…These include a significant outward blooming of the TM helices and expansion of the ECD β-sheets (β1-β6), indicating that the transition from pre-active to open involves global conformational changes spanning the ECD and TMD. Such an observation is consistent with a model that long-range global structural rearrangements underlie the transitions between functional states in pLGICs 58 .…”

Illumination of a progressive allosteric mechanism mediating the glycine receptor activation

“…Interestingly, phenotypes resembling the one presented here have been already described for the bacterial channel GLIC and other cationic pLGICs. With GLIC, we reported a “pre-activation” phenotype for a series of quenching sensors at the ECD interface and M2-M3 loop 14,15 . They all report fluorescence variations at non-activating agonist (proton) concentrations and the fluorescence variation was faster (ms range measured by stopped-flow) than the onset of activation (10-100 of ms measured by patch-clamp) 48 .…”

“…In order to monitor this reorganization, we used the tryptophan quenching technique that can sense the relative place of two residues 34,35 . This technique is particularly relevant to monitor distance changes of 5-15 Å range 14,15 . We introduced a cysteine at position Q219 in the pre-M1 region previously used for labeling with fluorescent dyes 28 , together with a tryptophan in Cys-loop at position K143, in order to quench the fluorophore in a conformation dependent manner (Figure 1 A,B).…”

“…At the structural level, fluorescence experiments provided evidence that the bacterial pLGIC homologue GLIC undergoes a progressive reorganization, with a first pre-activation step involving a quaternary compaction of the ECD 14 followed by pore opening associated with a structural reorganization of the TMD and ECD-TMD interface 15 . However, direct evidence for a progressive signal transduction in eukaryotic receptors is essentially lacking, despite recent cryo-EM structures of pLGICs revealing that the binding of orthosteric ligands is often accompanied by a significant reorganization of the extracellular domain (ECD), while the channel remains closed at the transmembrane domain (TMD), e.g.…”

Illumination of a progressive allosteric mechanism mediating the glycine receptor activation

“…At the structural level, fluorescence experiments provided evidence that the bacterial pLGIC homolog GLIC undergoes a progressive reorganization, with a first pre-activation step involving quaternary compaction of the ECD 14 followed by pore opening associated with a structural reorganization of the TMD and ECD-TMD interface 15 . However, direct evidence for progressive signal transduction in eukaryotic receptors is essentially lacking, despite recent cryo-EM structures of pLGICs revealing that the binding of orthosteric ligands is often accompanied by a significant reorganization of the extracellular domain (ECD), while the channel remains closed at the transmembrane domain (TMD), e.g.…”