A lipid site shapes the agonist response of a pentameric ligand-gated ion channel.
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ABSTRACT:
SUBMITTER: Henault CM
PROVIDER: S-EPMC8423587 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Pentameric ligand-gated ion channels (pLGICs) are crucial mediators of electrochemical signal transduction in various organisms from bacteria to humans. Lipids play an important role in regulating pLGIC function, yet the structural bases for specific pLGIC-lipid interactions remain poorly understood. The bacterial channel ELIC recapitulates several properties of eukaryotic pLGICs, including activation by the neurotransmitter GABA and binding and modulation by lipids, offering a simplified model system for structure-function relationship studies. In this study, functional effects of noncanonical amino acid substitution of a potential lipid-interacting residue (W206) at the top of the M1-helix, combined with detergent interactions observed in recent X-ray structures, are consistent with this region being the location of a lipid-binding site on the outward face of the ELIC transmembrane domain. Coarse-grained and atomistic molecular dynamics simulations revealed preferential binding of lipids containing a positive charge, particularly involving interactions with residue W206, consistent with cation-π binding. Polar contacts from other regions of the protein, particularly M3 residue Q264, further support lipid binding via headgroup ester linkages. Aromatic residues were identified at analogous sites in a handful of eukaryotic family members, including the human GABAA receptor ε subunit, suggesting conservation of relevant interactions in other evolutionary branches. Further mutagenesis experiments indicated that mutations at this site in ε-containing GABAA receptors can change the apparent affinity of the agonist response to GABA, suggesting a potential role of this site in channel gating. In conclusion, this work details type-specific lipid interactions, which adds to our growing understanding of how lipids modulate pLGICs.
| S-EPMC8327344 | biostudies-literature
Project description:Pentameric ligand-gated ion channels (pLGICs) are neurotransmitter-activated receptors that mediate fast synaptic transmission. In pLGICs, binding of agonist to the extracellular domain triggers a structural rearrangement that leads to the opening of an ion-conducting pore in the transmembrane domain and, in the continued presence of neurotransmitter, the channels desensitize (close). The flexible loops in each subunit that connect the extracellular binding domain (loops 2, 7, and 9) to the transmembrane channel domain (M2-M3 loop) are essential for coupling ligand binding to channel gating. Comparing the crystal structures of two bacterial pLGIC homologues, ELIC and the proton-activated GLIC, suggests channel gating is associated with rearrangements in these loops, but whether these motions accurately predict the motions in functional lipid-embedded pLGICs is unknown. Here, using site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy and functional GLIC channels reconstituted into liposomes, we examined if, and how far, the loops at the ECD/TMD gating interface move during proton-dependent gating transitions from the resting to desensitized state. Loop 9 moves ∼9 Å inward toward the channel lumen in response to proton-induced desensitization. Loop 9 motions were not observed when GLIC was in detergent micelles, suggesting detergent solubilization traps the protein in a nonactivatable state and lipids are required for functional gating transitions. Proton-induced desensitization immobilizes loop 2 with little change in position. Proton-induced motion of the M2-M3 loop was not observed, suggesting its conformation is nearly identical in closed and desensitized states. Our experimentally derived distance measurements of spin-labeled GLIC suggest ELIC is not a good model for the functional resting state of GLIC, and that the crystal structure of GLIC does not correspond to a desensitized state. These findings advance our understanding of the molecular mechanisms underlying pLGIC gating.
| S-EPMC3833874 | biostudies-literature
Project description:The proton-gated ion channel from Gloeobacter violaceus (GLIC) is a prokaryotic homolog of the eukaryotic nicotinic acetylcholine receptor that responds to the binding of neurotransmitter acetylcholine and mediates fast signal transmission. Recent emergence of a high-resolution crystal structure of GLIC captured in a potentially open state allowed detailed, atomic-level insight into ion conduction and selectivity mechanisms in these channels. Herein, we have examined the barriers to ion conduction and origins of ion selectivity in the GLIC channel by the construction of potential-of-mean-force profiles for sodium and chloride ions inside the transmembrane region. Our calculations reveal that the GLIC channel is open for a sodium ion to transport, but presents a ?11 kcal/mol free energy barrier for a chloride ion. Our collective findings identify three distinct contributions to the observed preference for the permeant ions. First, there is a substantial contribution due to a ring of negatively charged glutamate residues (E-2') at the narrow intracellular end of the channel. The negative electrostatics of this region and the ability of the glutamate side chains to directly bind cations would strongly favor the passage of sodium ions while hindering translocation of chloride ions. Second, our results imply a significant hydrophobic contribution to selectivity linked to differences in the desolvation penalty for the sodium versus chloride ions in the central hydrophobic region of the pore. This hydrophobic contribution is evidenced by the large free energy barriers experienced by Cl? in the middle of the pore for both GLIC and the E-2'A mutant. Finally, there is a distinct contribution arising from the overall negative electrostatics of the channel.
| S-EPMC3021669 | biostudies-literature
Project description:While the pentameric ligand-gated ion channel ELIC has recently provided first insight into the architecture of the family at high resolution, its detailed investigation was so far prevented by the fact that activating ligands were unknown. Here we describe a study on the functional characterization of ELIC by electrophysiology and X-ray crystallography. ELIC is activated by a class of primary amines that include the neurotransmitter GABA at high micro- to millimolar concentrations. The ligands bind to a conserved site and evoke currents that slowly desensitize over time. The protein forms cation selective channels with properties that resemble the nicotinic acetylcholine receptor. The high single channel conductance and the comparably simple functional behavior make ELIC an attractive model system to study general mechanisms of ion conduction and gating in this important family of neurotransmitter receptors.
| S-EPMC3119659 | biostudies-literature
Project description:Ketamine inhibits pentameric ligand-gated ion channels (pLGICs), including the bacterial pLGIC from Gloeobacter violaceus (GLIC). The crystal structure of GLIC shows R-ketamine bound to an extracellular intersubunit cavity. Here, we performed molecular dynamics simulations of GLIC in the absence and presence of R- or S-ketamine. No stable binding of S-ketamine in the original cavity was observed in the simulations, largely due to its unfavorable access to residue D154, which provides important electrostatic interactions to stabilize R-ketamine binding. Contrary to the symmetric binding shown in the crystal structure, R-ketamine moved away from some of the binding sites and was bound to GLIC asymmetrically at the end of simulations. The asymmetric binding is consistent with the experimentally measured negative cooperativity of ketamine binding to GLIC. In the presence of R-ketamine, all subunits showed changes in structure and dynamics, irrespective of binding stability; the extracellular intersubunit cavity expanded and intersubunit electrostatic interactions involved in channel activation were altered. R-ketamine binding promoted a conformational shift toward closed GLIC. Conformational changes near the ketamine-binding site were propagated to the interface between the extracellular and transmembrane domains, and further to the pore-lining TM2 through two pathways: pre-TM1 and the ?1-?2 loop. Both signaling pathways have been predicted previously using the perturbation-based Markovian transmission model. The study provides a structural and dynamics basis for the inhibitory modulation of ketamine on pLGICs.
| S-EPMC5550297 | biostudies-literature
Project description:Cys-loop receptors are molecular targets of general anesthetics, but the knowledge of anesthetic binding to these proteins remains limited. Here we investigate anesthetic binding to the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), a structural homolog of cys-loop receptors, using an experimental and computational hybrid approach. Tryptophan fluorescence quenching experiments showed halothane and thiopental binding at three tryptophan-associated sites in the extracellular (EC) domain, transmembrane (TM) domain, and EC-TM interface of GLIC. An additional binding site at the EC-TM interface was predicted by docking analysis and validated by quenching experiments on the N200W GLIC mutant. The binding affinities (K(D)) of 2.3 ± 0.1 mM and 0.10 ± 0.01 mM were derived from the fluorescence quenching data of halothane and thiopental, respectively. Docking these anesthetics to the original GLIC crystal structure and the structures relaxed by molecular dynamics simulations revealed intrasubunit sites for most halothane binding and intersubunit sites for thiopental binding. Tryptophans were within reach of both intra- and intersubunit binding sites. Multiple molecular dynamics simulations on GLIC in the presence of halothane at different sites suggested that anesthetic binding at the EC-TM interface disrupted the critical interactions for channel gating, altered motion of the TM23 linker, and destabilized the open-channel conformation that can lead to inhibition of GLIC channel current. The study has not only provided insights into anesthetic binding in GLIC, but also demonstrated a successful fusion of experiments and computations for understanding anesthetic actions in complex proteins.
| S-EPMC2941008 | biostudies-literature
Project description:Although it has long been proposed that membrane proteins may contain tightly bound lipids, their identity, the structure of their binding sites, and their functional and structural relevance have remained elusive. To some extent, this is because tightly bound lipids are often located at the periphery of proteins, where the quality of density maps is usually poorer, and because they may be outcompeted by detergent molecules used during standard purification procedures. As a step toward characterizing natively bound lipids in the superfamily of pentameric ligand-gated ion channels (pLGICs), we applied single-particle cryogenic electron microscopy to fragments of native membrane obtained in the complete absence of detergent-solubilization steps. Because of the heterogeneous lipid composition of membranes in the secretory pathway of eukaryotic cells, we chose to study a bacterial pLGIC (ELIC) expressed in Escherichia coli's inner membrane. We obtained a three-dimensional reconstruction of unliganded ELIC (2.5-Å resolution) that shows clear evidence for two types of tightly bound lipid at the protein-bulk-membrane interface. One of them was consistent with a "regular" diacylated phospholipid, in the cytoplasmic leaflet, whereas the other one was consistent with the tetra-acylated structure of cardiolipin, in the periplasmic leaflet. Upon reconstitution in E. coli polar-lipid bilayers, ELIC retained the functional properties characteristic of members of this superfamily, and thus, the fitted atomic model is expected to represent the (long-debated) unliganded-closed, "resting" conformation of this ion channel. Notably, the addition of cardiolipin to phosphatidylcholine membranes restored the ion-channel activity that is largely lost in phosphatidylcholine-only bilayers.
| S-EPMC8201805 | biostudies-literature
Project description:Pentameric ligand-gated ion channels (pLGICs) are receptor proteins that are sensitive to their membrane environment, but the mechanism for how lipids modulate function under physiological conditions in a state dependent manner is not known. The glycine receptor is a pLGIC whose structure has been resolved in different functional states. Using a realistic model of a neuronal membrane coupled with coarse-grained molecular dynamics simulations, we demonstrate that some key lipid-protein interactions are dependent on the receptor state, suggesting that lipids may regulate the receptor's conformational dynamics. Comparison with existing structural data confirms known lipid binding sites, but we also predict further protein-lipid interactions including a site at the communication interface between the extracellular and transmembrane domain. Moreover, in the active state, cholesterol can bind to the binding site of the positive allosteric modulator ivermectin. These protein-lipid interaction sites could in future be exploited for the rational design of lipid-like allosteric drugs.
| S-EPMC7904231 | biostudies-literature
Project description:Nerve signaling in humans and chemical sensing in bacteria both rely on the controlled opening and closing of the ion-conducting pore in pentameric ligand-gated ion channels. With the help of a multiscale simulation approach that combines mixed elastic network model calculations with molecular dynamics simulations, we study the opening and closing of the pore in Gloeobacter violaceus channel GLIC at atomic resolution. In our simulations of the GLIC transmembrane domain, we first verify that the two endpoints of the transition are open and closed to sodium ion conduction, respectively. We then show that a two-stage tilting of the pore-lining helices induces cooperative drying and iris-like closing of the channel pore. From the free energy profile of the gating transition and from unrestrained simulations, we conclude that the pore of the isolated GLIC transmembrane domain closes spontaneously. The mechanical work of opening the pore is performed primarily on the M2-M3 loop. Strong interactions of this short and conserved loop with the extracellular domain are therefore crucial to couple ligand binding to channel opening.
| S-EPMC2993413 | biostudies-literature
Project description:Pentameric ligand-gated ion channels (pLGICs), which mediate chemo-electric signal transduction in animals, have been recently found in bacteria. Despite clear sequence and 3D structure homology, the phylogenetic distance between prokaryotic and eukaryotic homologs suggests significant structural divergences, especially at the interface between the extracellular (ECD) and the transmembrane (TMD) domains. To challenge this possibility, we constructed a chimera in which the ECD of the bacterial protein GLIC is fused to the TMD of the human α1 glycine receptor (α1GlyR). Electrophysiology in Xenopus oocytes shows that it functions as a proton-gated ion channel, thereby locating the proton activation site(s) of GLIC in its ECD. Patch-clamp experiments in BHK cells show that the ion channel displays an anionic selectivity with a unitary conductance identical to that of the α1GlyR. In addition, pharmacological investigations result in transmembrane allosteric modulation similar to the one observed on α1GlyR. Indeed, the clinically active drugs propofol, four volatile general anesthetics, alcohols, and ivermectin all potentiate the chimera while they inhibit GLIC. Collectively, this work shows the compatibility between GLIC and α1GlyR domains and points to conservation of the ion channel and transmembrane allosteric regulatory sites in the chimera. This provides evidence that GLIC and α1GlyR share a highly homologous 3D structure. GLIC is thus a relevant model of eukaryotic pLGICs, at least from the anionic type. In addition, the chimera is a good candidate for mass production in Escherichia coli, opening the way for investigations of "druggable" eukaryotic allosteric sites by X-ray crystallography.
| S-EPMC3141993 | biostudies-literature