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

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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.

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Table 1 included in the manuscript correspond to a summary table for figures 4 to 8.

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Solène N Lefebvre

Neuroscience Department, Institut Pasteur, Paris, France

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-1333-2042
Antoine Taly

Institut de Biologie Physico-chimique, Paris, France

For correspondence
antoine.taly@ibpc.fr

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0001-5109-0091
Anaïs Menny

Life Sciences, Imperial College London, London, United Kingdom

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-6044-4119
Karima Medjebeur

Neuroscience Department, Institut Pasteur, Paris, France

Competing interests
The authors declare that no competing interests exist.
Pierre-Jean Corringer

Neuroscience Department, Institut Pasteur, Paris, France

For correspondence
pjcorrin@pasteur.fr

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-4770-430X

Funding

Agence Nationale de la Recherche (ANR-13-BSV-0020)

Solène N Lefebvre
Anaïs Menny
Karima Medjebeur
Pierre-Jean Corringer

Agence Nationale de la Recherche (ANR-11-LABX-0011)

Antoine Taly

European Research Council (grant No. 788974)

Pierre-Jean Corringer

Sorbonne University - Doctoral school ED3C (PhD fellowship)

Solène N Lefebvre

Fondation pour la Recherche Médicale (PhD fellowship complement)

Solène N Lefebvre

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

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This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.