Gating mechanism of a pentameric channel receptor – Pentagate

The program aims at integrating structural data at high resolution (mainly X-ray crystallography), with local structural data (via fluorescent sensors allowing following the inter-residues distance in real time), and with functional data of channel opening by electrophysiology and of ligand binding by surface plasmon resonance, together with molecular dynamic simulations of the full length protein embedded at the membrane. The synthesis of labeled molecules (with fluorescence or labeled with selenium), come in support of the expeimental approaches, and also aims at developping specific ligands targetting selected regions of the protein.

The work allowed following the activation transition of GLIC in real time with fluorescence, thereby identifying and characterising an intermediate conformation in the transition activation pathway. Several classes of compounds have been identified as modulators of GLIC, among which carboxylic acids, general anesthetics such as barbiturates, propofol and Xenon are currently used in clinics. Their binding site has been identified by X-ray crystallography and their mechanism of action studied by molecular dynamic simulations. Photoisomerizable molecular tweeser have been synthetized with the aim to controle the activity of GLIC by light. A systematic analysis of GLIC has been carried out, identifying key residues involved in proton sensing and in the desensitization.

The work provides the basis of the molecular mechanisms that regulate the activity of this important class of channel receptors. The technological approaches, notably by fluorescence and molecular dynamics, will be valuable when studying brain receptors. The structure of the desensitized state remains elusive, and work will continue for its structural resolution.

Beside the many communications in international congres, the work yielded 8 original publications, and 2 publications are currently submitted.

Ligand-gated ion channels (LGIC) are key players of cell excitability, neuronal communication and brain computation. However, these integral membrane proteins are inherently difficult to produce and handle biochemically and their molecular mechanisms of function remain poorly understood. While the three-dimensional structures of the major classes of LGICs have been solved by X-ray crystallography, further work is needed to understand how LGICs from our brain perform signal transduction, that is conversion of neurotransmitter binding into opening of their ion channel. Our project focuses on the family of pentameric LGICs that mediates neurotransmission by acetylcholine, serotonin, GABA and glycine. pLGICs are major players of both the inhibitory neurotransmission and of excitatory neuromodulation in the brain. Accordingly, they are major drug targets for anxiolytics, sedatives, general anesthetics, cognitive enhancers, neuroprotective (anti-Alzheimer and anti-Parkinson diseases) and anti-smoking compounds. Over the last years, our group has been at the forefront in the field of pLGICs with the identification of bacterial pLGICs, allowing the X-ray resolution of a pLGIC in an open-channel conformation and identification of key pharmacological sites for general anesthetics and ethanol. We now aim at tackling the next challenges in the field: understanding the mechanisms of gating and desensitization. Indeed, pLGICs are allosteric proteins in constant interconversion between several conformations, a resting state, an active state carrying an open channel, and one or several desensitized states.
To this aim, we will focus our study on the bacterial homolog from Gloeobacter violaceus named GLIC. We will use GLIC as a prototype of the whole superfamily. We have extensive knowledge of this protein, and we notably showed that it functions as a proton-gated ion channel, and solved its structure in two conformations at the highest resolution among available X-ray structures from the family. We will develop a broad multidisciplinary approach. We will perform an extensive search of ligands and mutations that stabilize the various allosteric conformations, by combining chemical synthesis, mutagenesis, surface plasmon resonance and electrophysiology. We will also engineer de novo agonist binding sites for zinc and amino acids. Key GLIC mutants and GLIC-ligand complexes will be expressed in E. coli and engaged in crystallization for structural resolution of new allosteric conformations. The contribution of these conformations to the GLIC function will be assessed on GLIC reconstituted in liposomes and tagged with fluorescent reporters. Both steady state and time-resolved assays will aim at assigning to particular X-ray conformations specific allosteric states, either resting, desensitized, or intermediates. Finally, the mechanisms underlying the allosteric reorganizations will be explored by molecular dynamic simulations of GLIC embedded in an explicit lipid membrane.
Our approach, from X-ray crystallography to electrophysiology in cell lines, aims at understanding the fundamental mechanisms of signal transduction operating in pLGICs. This knowledge will allow the identification of new putative binding pockets, as well as their reorganization during gating, paving the way for the design of new classes of drugs targeting these important neurotransmitter receptors.