Ligand-gated Ion Channel GLIC Research Articles

Cryo-EM structures of prokaryotic ligand-gated ion channel GLIC provide insights into gating in a lipid environment

GLIC, a proton-activated prokaryotic ligand-gated ion channel, served as a model system for understanding the eukaryotic counterparts due to their structural and functional similarities. Despite extensive studies conducted on GLIC, the molecular mechanism of channel gating in the lipid environment requires further investigation. Here, we present the cryo-EM structures of nanodisc-reconstituted GLIC at neutral and acidic pH in the resolution range of 2.6 – 3.4 Å. In our apo state at pH 7.5, the extracellular domain (ECD) displays conformational variations compared to the existing apo structures. At pH 4.0, three distinct conformational states (C1, C2 and O states) are identified. The protonated structures exhibit a compacted and counter-clockwise rotated ECD compared with our apo state. A gradual widening of the pore in the TMD is observed upon reducing the pH, with the widest pore in O state, accompanied by several layers of water pentagons. The pore radius and molecular dynamics (MD) simulations suggest that the O state represents an open conductive state. We also observe state-dependent interactions between several lipids and proteins that may be involved in the regulation of channel gating. Our results provide comprehensive insights into the importance of lipids impact on gating.

Cryo-EM structures of nanodisc reconstituted ligand-gated ion channel GLIC in various conformations reveal state-dependent protein-lipid interactions and gating mechanism

Cryo-EM structures of nanodisc reconstituted ligand-gated ion channel GLIC in various conformations reveal state-dependent protein-lipid interactions and gating mechanism

Discovery of lipid binding sites in the ligand-gated ion channel GLIC through simulations and Cryo-EM.

Ligand-gated ion channels play an important role in electrochemical signal transduction in neurons and other excitable cells. Aside from canonical ligands, phospholipids have been shown to bind specifically and sometimes modulate several types of channels, but the structural details of such protein-lipid interactions remain unclear. Here, we present multiple lipid interaction sites in the pentameric ligand-gated ion channel, GLIC, by integrating cryo-electron microscopy and large-scale atomistic molecular dynamics simulations together with Markov state modeling. We identified 25 lipids in the closed state of GLIC - a conformation in which, to our knowledge, lipids have not been previously resolved. Both simulations and cryo-EM data predicted, for each subunit, three lipids in the inner leaflet and two in the outer leaflet. Further, we used simulations to make predictions about lipid interactions in the open state, resulting in six sites - three in each leaflet. Interactions of particular interest include one slow-exchanging interaction with a deeply buried lipid tail in the intersubunit space, where tail saturation levels played a role in the binding pose, and where the interaction could be broken due to a single mutation. An additional lipid interaction was preferential to the open state, potentially playing a role in stabilization of this state. This work provides molecular details of GLIC-lipid contacts, especially in the understudied closed state; a testable hypotheses for state-dependent binding; and a multidisciplinary strategy for modeling protein-lipid interactions.

Probing solution structure of the pentameric ligand-gated ion channel GLIC by small-angle neutron scattering

Pentameric ligand-gated ion channels undergo subtle conformational cycling to control electrochemical signal transduction in many kingdoms of life. Several crystal structures have now been reported in this family, but the functional relevance of such models remains unclear. Here, we used small-angle neutron scattering (SANS) to probe ambient solution-phase properties of the pH-gated bacterial ion channel GLIC under resting and activating conditions. Data collection was optimized by inline paused-flow size-exclusion chromatography, and exchanging into deuterated detergent to hide the micelle contribution. Resting-state GLIC was the best-fit crystal structure to SANS curves, with no evidence for divergent mechanisms. Moreover, enhanced-sampling molecular-dynamics simulations enabled differential modeling in resting versus activating conditions, with the latter corresponding to an intermediate ensemble of both the extracellular and transmembrane domains. This work demonstrates state-dependent changes in a pentameric ion channel by SANS, an increasingly accessible method for macromolecular characterization with the coming generation of neutron sources.

Conformational Landscapes of Ligand-Gated Ion Channel GLIC from Markov State Modeling

Ligand-gated ion channels (LGICs) are responsible for propagating electrochemical signals in neurons and other excitable cells, by rapidly cycling between functional states upon appropriate chemical stimuli. However, few LGICs have been characterized in both open and closed conformations, and the timescales of the gating transitions, initiated by ligand-binding, are typically too slow for classical molecular dynamics simulations. Consequently, the precise landscape of LGIC transitions is poorly understood. We have utilized enhanced sampling to run unbiased molecular dynamics simulations in a massively parallel fashion, and construct Markov state models of a structurally rich, pH-gated bacterial LGIC, GLIC from Gloeobacter violaceus. We then tested the ability of these models to recapitulate functional effects of protonation and mutagenesis on channel opening. Consistent with functional recordings, our models reveal deepening of the open state free energy well upon protonation in the extracellular domain or mutation (I233T) of the transmembrane gate, with relatively low maximal open probability. Analysis of states along the main reaction coordinate confirms multiple previously suggested mechanisms, but we note that the blooming and twisting motions of the extracellular domain mainly depend on pH and not the conformational state of the channel. Additionally, analysis of the conformational symmetries reveals a highly asymmetric intermediate and symmetric open state, while the closed states display mixed asymmetric extracellular and symmetric transmembrane domains. This work demonstrates the construction of plausible Markov state models from limited sampling of LGIC variants, capable of predicting allosteric effects from chemical perturbation, and identifying geometric variables in functional transitions. Such statistical tools constitute a promising approach to elucidate LGIC gating mechanisms, with practical implications for modeling neuronal function and the development of state-selective drugs.

Cryo-EM Structure Determination and Model Fitting of the Proton-Gated Ligand-Gated Ion Channel glic at Multiple pH States

Cryo-EM Structure Determination and Model Fitting of the Proton-Gated Ligand-Gated Ion Channel glic at Multiple pH States

Deciphering Anesthetic Action of Noble Gases through their Modulation of Membrane Protein and Lipid Bilayer Properties

The molecular mechanism of action underlying general anesthesia remains a matter of controversy. One proposed mode of action involves binding of anesthetic molecules to membrane proteins such as ligand gated ion channels, thereby modulating their function. An alternative mode of action discussed in the literature is through binding to the lipid bilayer. We chose to explore both modes of action at the molecular level by studying two types of targets: pure lipid bilayer systems such as POPC, and a lipid bilayer with the pentameric ligand-gated ion channel GLIC inserted into it. Sauguet et al. recently solved the structure of GLIC in complex with the general anesthetic Xenon [1], describing a multitude of binding sites. Here, we studied the effect of a series of three Noble gases: Argon, Krypton and Xenon. We have performed molecular dynamics (MD) simulations to gain key insights on the Noble gases binding location and affinity. Different gas:lipid ratios were used to characterize their effect on the physical properties of the membrane, as well as their affinity for the core of the lipid bilayer. Microsecond-long flooding simulations, combined with free energy calculations, were run to characterize access to binding sites and quantify binding affinities of Nobles Gases on the GLIC channel. This work extends and generalizes our previous study on bromoform action on GLIC, that revealed a complex network of interconnected binding sites, possibly all contributing in concert to the anesthetic effect [2]. We go beyond previous work by considering the measurable effect on lipid bilayer properties induced by the Noble gases therefore providing a better description of key pathways for anesthetic action. [1] Sauguet et al. Plos One. 2016. doi: 10.1371/ journal.pone.0149795 [2] Laurent, Murail et al. Structure. 2016. doi: 10.1016/j.str.2016.02.014

P-54PHYSICOCHEMICAL DETERMINANTS OF ALCOHOL BINDING IN A MODEL LIGAND-GATED ION CHANNEL

Pentameric ligand-gated ion channels are heavily implicated in neurological effects of alcohol, yet high-resolution structural data in this family of receptors are limited. The prokaryotic ligand-gated ion channel GLIC is a potentially valuable model system whose structure …

Genuine open form of the pentameric ligand-gated ion channel GLIC.

Pentameric ligand-gated ion channels (pLGICs) mediate fast chemical neurotransmission of nerve signalling in the central and peripheral nervous systems. GLIC is a bacterial homologue of eukaryotic pLGIC, the X-ray structure of which has been determined in three different conformations. GLIC is thus widely used as a model to study the activation and the allosteric transition of this family of receptors. The recently solved high-resolution structure of GLIC (2.4 Å resolution) in the active state revealed two bound acetate molecules in the extracellular domain (ECD). Here, it is shown that these two acetates exactly overlap with known sites of pharmacological importance in pLGICs, and their potential influence on the structure of the open state is studied in detail. Firstly, experimental evidence is presented for the correct assignment of these acetate molecules by using the anomalous dispersion signal of bromoacetate. Secondly, the crystal structure of GLIC in the absence of acetate was solved and it is shown that acetate binding induces local conformational changes that occur in strategic sites of the ECD. It is expected that this acetate-free structure will be useful in future computational studies of the gating transition in GLIC and other pLGICs.

Structure based computational assessment of channel properties of assembled ORF-8a from SARS-CoV.

ABSTRACTORF 8a is a short 39 amino acid bitopic membrane protein encoded by severe acute respiratory syndrome causing corona virus (SARS‐CoV). It has been identified to increase permeability of the lipid membrane for cations. Permeability is suggested to occur due to the assembly of helical bundles. Computational models of a pentameric assembly of 8a peptides are generated using the first 22 amino acids, which include the transmembrane domain. Low energy structures reveal a hydrophilic pore mantled by residues Thr‐8, and −18, Ser‐11, Cys‐13, and Arg‐22. Potential of mean force (PMF) profiles for mono (Na+, K+, Cl−) and divalent (Ca2+) ions along the pore are calculated. The data support experimental findings of a weak cation selectivity of the channel. Calculations on 8a are compared to data derived for a pentameric bundle consisting of the M2 helices of the bacterial pentameric ligand gated ion channel GLIC (3EHZ). PMF curves of both, bundles 8a and M2, show sigmoidal shaped profiles. In comparison to the data for the M2‐GLIC model, data of the 8a bundle show lower amplitude of the PMF values between maximum and minimum and less discrimination amongst ions. Proteins 2015; 83:300–308. © 2014 Wiley Periodicals, Inc.

Structural Requirements in the Transmembrane Domain of GLIC Revealed by Incorporation of Noncanonical Histidine Analogs

The cyanobacterial pentameric ligand-gated ion channel GLIC, a homolog of the Cys-loop receptor superfamily, has provided useful structural and functional information about its eukaryotic counterparts. X-ray diffraction data and site-directed mutagenesis have previously implicated a transmembrane histidine residue (His234) as essential for channel function. Here, we investigated the role of His234 via synthesis and incorporation of histidine analogs and α-hydroxy acids using invivo nonsense suppression. Receptors were expressed heterologously in Xenopus laevis oocytes, and whole-cell voltage-clamp electrophysiology was used to monitor channel activity. We show that an interhelix hydrogen bond involving His234 is important for stabilization of the open state, and that the shape and basicity of its side chain are highly sensitive to perturbations. In contrast, our data show that two other His residues are not involved in the acid-sensing mechanism.

Probing Pentameric Ligand-Gated Ion Channels with Bromoform Reveals Many Interconnected Anesthetic Binding Sites

Sauguet et al. recently solved the structure of the pentameric ligand-gated ion channel GLIC in complex with the general anesthetic bromoform [1]. This structure highlights three binding sites in an intra-subunit cavity close to the propofol and desflurane binding sites previously described by Nury et al. [2]. Additionally, a F238A (F14'A) mutant reveals a supplementary inter-subunit binding site.In this work we characterize these and additional sites by computational methods. We combine several approaches to address three key questions: (i) are the crystal binding sites spontaneously accessible? (ii) can bromoform travel from one site to another? (iii) what is the bromoform affinity for each binding site? Molecular dynamics (MD) simulations of flooding the receptor with bromoform recover most of the experimentally observed sites, with a modulated occupancy between the open and the locally closed conformations. Hundreds of short MD simulations were carried out to extensively explore the binding pockets, providing data on possible routes connecting them. These simulations furthermore highlight residues that may play key roles in controlling the interaction between anesthetic and receptor molecules. Free energy of binding calculations indicate significant affinity for all crystallographic binding sites in open and locally closed conformations, in some cases modulated by pH.[1] Sauguet et al. 2013, Nature communications. 4:1697.[2] Nury et al. 2011, Nature. 469:428-31.

Asymmetric Ligand Binding Facilitates Conformational Transitions in Pentameric Ligand-Gated Ion Channels

The anesthetic propofol inhibits the currents of the homopentameric ligand-gated ion channel GLIC, yet the crystal structure of GLIC with five propofol molecules bound symmetrically shows an open-channel conformation. To address this dilemma and determine if the symmetry of propofol binding sites affects the channel conformational transition, we performed a total of 1.5 μs of molecular dynamics simulations for different GLIC systems with propofol occupancies of 0, 1, 2, 3, and 5. GLIC without propofol binding or with five propofol molecules bound symmetrically, showed similar channel conformation and hydration status over multiple replicates of 100-ns simulations. In contrast, asymmetric binding to one, two or three equivalent sites in different subunits accelerated the channel dehydration, increased the conformational heterogeneity of the pore-lining TM2 helices, and shifted the lateral and radial tilting angles of TM2 toward a closed-channel conformation. The results differentiate two groups of systems based on the propofol binding symmetry. The difference between symmetric and asymmetric groups is correlated with the variance in the propofol-binding cavity adjacent to the hydrophobic gate and the force imposed by the bound propofol. Asymmetrically bound propofol produced greater variance in the cavity size that could further elevate the conformation heterogeneity. The force trajectory generated by propofol in each subunit over the course of a simulation exhibits an ellipsoidal shape, which has the larger component tangential to the pore. Asymmetric propofol binding creates an unbalanced force that expedites the channel conformation transitions. The findings from this study not only suggest that asymmetric binding underlies the propofol functional inhibition of GLIC, but also advocate for the role of symmetry breaking in facilitating channel conformational transitions.

Reversal of ion-charge selectivity renders the pentameric ligand-gated ion channel GLIC insensitive to anaesthetics.

pLGICs (pentameric ligand-gated ion channels) are a family of structurally homologous cation and anion channels involved in neurotransmission. Cation-selective members of the pLGIC family are typically inhibited by general anaesthetics, whereas anion-selective members are potentiated. GLIC is a prokaryotic cation pLGIC and can be inhibited by clinical concentrations of general anaesthetics. The introduction of three mutations, Y221A (Y-3'A), E222P (E-2'P) and N224R (N0'R), at the selectivity filter and one, A237T (A13'T), at the hydrophobic gate, converted GLIC into an anion channel. The mutated GLIC (GLIC4) became insensitive to the anaesthetics propofol and etomidate, as well as the channel blocker picrotoxin. MD (molecular dynamics) simulations revealed changes in the structure and dynamics of GLIC4 in comparison with GLIC, particularly in the tilting angles of the pore-lining helix [TM2 (transmembrane helix 2)] that consequently resulted in different pore radius and hydration profiles. Propofol binding to an intra-subunit site of GLIC shifted the tilting angles of TM2 towards closure at the hydrophobic gate region, consistent with propofol inhibition of GLIC. In contrast, the pore of GLIC4 was much more resilient to perturbation from propofol binding. The present study underscores the importance of pore dynamics and conformation to anaesthetic effects on channel functions.

Structure of the Pentameric Ligand-Gated Ion Channel GLIC Bound with Anesthetic Ketamine

Pentameric ligand-gated ion channels (pLGICs) are targets of general anesthetics, but a structural understanding of anesthetic action on pLGICs remains elusive. GLIC, a prokaryotic pLGIC, can be inhibited by anesthetics, including ketamine. The ketamine concentration leading to half-maximal inhibition of GLIC (58μM) is comparable to that on neuronal nicotinic acetylcholine receptors. A 2.99Å resolution X-ray structure of GLIC bound with ketamine revealed ketamine binding to an intersubunit cavity that partially overlaps with the homologous antagonist-binding site in pLGICs. The functional relevance of the identified ketamine site was highlighted by profound changes in GLIC activation upon cysteine substitution of the cavity-lining residue N152. The relevance is also evidenced by changes in ketamine inhibition upon the subsequent chemical labeling of N152C. The results provide structural insight into the molecular recognition of ketamine and are valuable for understanding the actions of anesthetics and other allosteric modulators on pLGICs.

Mutational Analysis of the Prokaryotic Pentameric Ligand-Gated Ion Channel GLIC

For pentameric ligand-gated ion channels (pLGICs), the movements involved in coupling neurotransmitter binding to channel activation are unclear. The crystal structures of the prokaryotic pLGICs homologs from E. chrysanthemi (ELIC) and G. violaceus (GLIC) in presumed closed and open channel conformations provide unparalleled opportunities to explore how ligand binding triggers channel opening. GLIC is a proton-gated channel. To test the hypothesis that the proton binding site in GLIC is located in an analogous position as the agonist binding sites of eukaryotic pLGICs and is formed, in part, by acidic amino acids, we neutralized acidic residues in Loops A (E74Q), C (E176Q, D177N, E180Q), and F (D144N, E146Q). Two-electrode voltage clamp recordings from wild-type and mutant GLIC channels expressed in Xenopus oocytes showed that the mutations had no effect on proton-mediated channel gating (pH50, pHmax) suggesting that titration of these carboxylates is not involved in the proton-dependent gating of GLIC and that the proton binding site is located elsewhere.We are using fluorescence recording of site-specific labels in GLIC expressed in Xenopus oocytes combined with two-electrode voltage clamping to monitor the motions in GLIC associated with channel opening.We made cysteine mutations in a cys-less GLIC (C26A) background in loop 2 (K32C), loop 9 (T157C), M2 helix (E242C, T243C, N244C) and M2-M3 loop (K247C, P249C). The cysteine mutant receptors were labeled with the sulfhydryl-reactive, environmentally-sensitive fluorescent probe Alexafluor 546 C5-maleimide. Combined voltage clamp and fluorometry monitor proton-induced channel activity and local protein movements simultaneously. Application of pH 5 buffer decreased fluorescence at T157C, K247C and P249C suggesting that these residues become more exposed to a hydrophilic or aqueous environment consistent with a movement of the M2-M3 loop upwards and outward on channel activation.