Periodic Reporting for period 3 - DYNACOTINE (Signal transduction and allosteric modulation of nicotinic acetylcholine receptors:from ion channel electrophysiology to atomic 3D structures)
Berichtszeitraum: 2022-01-01 bis 2023-06-30
Pentameric ligand-gated ion channels are a major class of neurotransmitter receptors mediating synaptic transmission in the brain and at the periphery. The family includes Nicotinic acetylcholine receptors (nAChRs), serotonin 5HT3 receptors, Glycine (GlyRs) and GABA (GABAAR) receptors. They participate in a wide range of physiological functions (voluntary motion, memory, sleep, anxiety, reward, pain, ...) are involved in numerous pathologies (epilepsy, Parkinson and Alzheimer diseases, ...) and are major targets for addictive and therapeutic drugs.
The recent resolution of their high-resolution structures, notably by X-ray crystallography and cryo-electron microscopy, have revealed that these receptors display an unanticipated conformational plasticity. They adopt multiple conformations depending on the ligand bound and the way these integral membrane proteins are extracted from the plasma membrane and stabilized in solution. However, understanding the gating cycle of these receptors requires to monitor their conformational changes in physiological conditions, in a living cell at room temperature.
To address this challenge, we propose to develop a top-down approach starting from the study of the conformational transitions of pLGICs functionally expressed in cells, and then dissecting the molecular mechanisms on purified proteins.
Aim 1: develop innovative fluorescence quenching approaches to follow the protein motions concomitant with channel opening at the cell membrane, using the Voltage-clamp fluorometry (VCF) technique. We currently implement this technique on nAChRs, GlyRs and 5HT3Rs.
Aim 2: exploit the fluorescence quenching technique on purified proteins, along with other techniques, to study the role/requirement of lipids, and their pharmacological crosstalk with allosteric modulators acting at the transmembrane domain. We implement this technique on the nAChRs that are major brain receptors contributing to higher brain functions such as cognition and reward.
Aim 3: Exploit the gained knowledge to open original routes to solve 3D structures of nAChRs, in novel conformations and in complex with lipids and allosteric modulators.
This multidisciplinary project combines electrophysiology, fluorescence, pharmacology, membrane protein biochemistry and structural biology. The results will provide fundamental insights into the allosteric mechanisms underlying both pLGIC function and their modulation by allosteric modulators that hold promises in therapeutics.
The recent resolution of their high-resolution structures, notably by X-ray crystallography and cryo-electron microscopy, have revealed that these receptors display an unanticipated conformational plasticity. They adopt multiple conformations depending on the ligand bound and the way these integral membrane proteins are extracted from the plasma membrane and stabilized in solution. However, understanding the gating cycle of these receptors requires to monitor their conformational changes in physiological conditions, in a living cell at room temperature.
To address this challenge, we propose to develop a top-down approach starting from the study of the conformational transitions of pLGICs functionally expressed in cells, and then dissecting the molecular mechanisms on purified proteins.
Aim 1: develop innovative fluorescence quenching approaches to follow the protein motions concomitant with channel opening at the cell membrane, using the Voltage-clamp fluorometry (VCF) technique. We currently implement this technique on nAChRs, GlyRs and 5HT3Rs.
Aim 2: exploit the fluorescence quenching technique on purified proteins, along with other techniques, to study the role/requirement of lipids, and their pharmacological crosstalk with allosteric modulators acting at the transmembrane domain. We implement this technique on the nAChRs that are major brain receptors contributing to higher brain functions such as cognition and reward.
Aim 3: Exploit the gained knowledge to open original routes to solve 3D structures of nAChRs, in novel conformations and in complex with lipids and allosteric modulators.
This multidisciplinary project combines electrophysiology, fluorescence, pharmacology, membrane protein biochemistry and structural biology. The results will provide fundamental insights into the allosteric mechanisms underlying both pLGIC function and their modulation by allosteric modulators that hold promises in therapeutics.
Key advances have been made on the following topics:
1/ Conformational pathway of activation of the GlyR:
pLGICs are expressed at the plasma membrane. They contain an intrinsic ion channel that is closed in resting conditions, and that opens upon binding of the neurotransmitter. This activation triggers cell excitation or inhibition, which is the key event mediating neuronal communication at chemical synapses. Using VCF on the GlyR, we found that the pathway of activation is not direct, but involves first a conformational change to an intermediate conformation, followed by a second conformational change where the channel opens. We also found that glycine, the natural neurotransmitter of the GlyR, produces robust activation, while a class of compound named “partial agonists” trigger the full transition to the intermediate state but only partially trigger the downward transition of activation. The general anesthetic propofol is also shown to substantially stabilize the intermediate state, without producing activation. These observations have profound consequences concerning our knowledge of the molecular mechanisms of GlyR activation, unraveling sequential movements. In addition, they allow deciphering at which step partial agonists fail to elicit full activation, a feature that will be useful for drug-design purpose. Finally, the data will be of particular interest to help annotating known high-resolution structures to actual allosteric states occurring in physiological conditions.
2/ Conformational pathway of desensitization of the GlyRs and GABAARs
In addition to triggering activation, neurotransmitters trigger the desensitization of the channel upon prolonged exposure. Desensitization, that corresponds to the closure of the channel in the presence of the agonist, contributes to shape the time-course of the synaptic currents. We found by VCF that desensitization involves a reorganization of the extracellular domain of the GlyR, a feature that was not anticipated from known high-resolution structures. This work will allow a novel way to analyze both the mechanisms of desensitization, and the effect of key allosteric effectors on this process. For instance, general anesthetics are known to potentiate the GlyR, contributing to anesthesia, but our preliminary data clearly show that they do strongly fasten desensitization, a feature that may have important implications in clinics. In parallel, we analyzed concatemeric GABAA receptors, recording and modelling of a series of 26 mutants where the kinetics of desensitization are altered. Data suggests a pathway where subunits move independently during the desensitization, the transition of two subunits being required to occlude the pore. This work hints towards a very diverse and labile conformational landscape during desensitization, with potential implications in physiology and pharmacology.
3/ Access of ions to the channel of the GlyR through lateral portals
In collaboration with computational scientists, we also established, using single channel patch-clamp recordings, that extracellular chlorides ions access to the channel through lateral portals. Ion translocation is the key function of ligands gated ion channels, this demonstration is thus of primary importance and will be valuable in understanding pathological mutations of this family of channels that cause hyperekplexia and also to understand the ion permeation pathway in other LGICs.
1/ Conformational pathway of activation of the GlyR:
pLGICs are expressed at the plasma membrane. They contain an intrinsic ion channel that is closed in resting conditions, and that opens upon binding of the neurotransmitter. This activation triggers cell excitation or inhibition, which is the key event mediating neuronal communication at chemical synapses. Using VCF on the GlyR, we found that the pathway of activation is not direct, but involves first a conformational change to an intermediate conformation, followed by a second conformational change where the channel opens. We also found that glycine, the natural neurotransmitter of the GlyR, produces robust activation, while a class of compound named “partial agonists” trigger the full transition to the intermediate state but only partially trigger the downward transition of activation. The general anesthetic propofol is also shown to substantially stabilize the intermediate state, without producing activation. These observations have profound consequences concerning our knowledge of the molecular mechanisms of GlyR activation, unraveling sequential movements. In addition, they allow deciphering at which step partial agonists fail to elicit full activation, a feature that will be useful for drug-design purpose. Finally, the data will be of particular interest to help annotating known high-resolution structures to actual allosteric states occurring in physiological conditions.
2/ Conformational pathway of desensitization of the GlyRs and GABAARs
In addition to triggering activation, neurotransmitters trigger the desensitization of the channel upon prolonged exposure. Desensitization, that corresponds to the closure of the channel in the presence of the agonist, contributes to shape the time-course of the synaptic currents. We found by VCF that desensitization involves a reorganization of the extracellular domain of the GlyR, a feature that was not anticipated from known high-resolution structures. This work will allow a novel way to analyze both the mechanisms of desensitization, and the effect of key allosteric effectors on this process. For instance, general anesthetics are known to potentiate the GlyR, contributing to anesthesia, but our preliminary data clearly show that they do strongly fasten desensitization, a feature that may have important implications in clinics. In parallel, we analyzed concatemeric GABAA receptors, recording and modelling of a series of 26 mutants where the kinetics of desensitization are altered. Data suggests a pathway where subunits move independently during the desensitization, the transition of two subunits being required to occlude the pore. This work hints towards a very diverse and labile conformational landscape during desensitization, with potential implications in physiology and pharmacology.
3/ Access of ions to the channel of the GlyR through lateral portals
In collaboration with computational scientists, we also established, using single channel patch-clamp recordings, that extracellular chlorides ions access to the channel through lateral portals. Ion translocation is the key function of ligands gated ion channels, this demonstration is thus of primary importance and will be valuable in understanding pathological mutations of this family of channels that cause hyperekplexia and also to understand the ion permeation pathway in other LGICs.
A major aim of the project was to investigate the mechanisms of pLGIC activation and desensitization on living cells in physiological conditions. Indeed, X-ray and cryo-EM experiments, while yielding high-resolution structures, are performed in non-physiological condition at very low temperature and outside from the cell membrane. Using either VCF or concatemers, we found a number of novel findings that could not be deduced from high-resolution structural work. The demonstration of an intermediate state during activation, the mechanisms of desensitization that involves the extracellular domain and strongly asymmetric motions, as well as the identification of an unexpected permeation pathway of chloride ions in the GlyR are novel findings. Beside they interest in our fundamental knowledge of pLGIC molecular mechanisms, they will contribute to help annotating know high resolution structures to physiological allosteric states, and to characterize and design ligands with therapeutic potential on the important family of neurotransmitter receptors.