Pentameric Ligand-gated Channels Research Articles

The modes of action of ion-channel-targeting neurotoxic insecticides: lessons from structural biology.

Insecticides are indispensable tools for plant protection in modern agriculture. Despite having highly heterogeneous structures, many neurotoxic insecticides use similar principles to inhibit or deregulate neuronal ion channels. Insecticides targeting pentameric ligand-gated channels are structural mimetics of neurotransmitters or manipulate and deregulate the proteins. Those binding to (pseudo-)tetrameric voltage-gated(-like) channels, on the other hand, are natural or synthetic compounds that directly block the ion-conducting pore or prevent conformational changes in the transmembrane domain necessary for opening and closing the pore. The use of a limited number of inhibition mechanisms can be problematic when resistances arise and become more widespread. Therefore, there is a rising interest in the development of insecticides with novel mechanisms that evade resistance and are pest-insect-specific. During the last decade, most known insecticide targets, many with bound compounds, have been structurally characterized, bringing the rational design of novel classes of agrochemicals within closer reach than ever before.

Diazepam binding inhibitor positively modulates α5β3 GABA-A receptors

Benzodiazepines (BZDs) exert their antiepileptic, anxiogenic, and somnogenic effects by binding to GABA-A receptors (GABARs). GABARs are inhibitory pentameric ligand-gated channels commonly formed by 2α, 2β and 1γ subunits, though some GABARs are formed by only α and β subunits. The high-affinity binding site for BZDs is located in the extracellular domain between the α and γ subunits and is absent in αβ receptors.

The intracellular domain of homomeric glycine receptors modulates agonist efficacy

Like other pentameric ligand-gated channels, glycine receptors (GlyRs) contain long intracellular domains (ICDs) between transmembrane helices 3 and 4. Structurally characterized GlyRs are generally engineered to have a very short ICD. We show here that for one such construct, zebrafish GlyREM, the agonists glycine, β-alanine, taurine, and GABA have high efficacy and produce maximum single-channel open probabilities greater than 0.9. In contrast, for full-length human α1 GlyR, taurine and GABA were clearly partial agonists, with maximum open probabilities of 0.46 and 0.09, respectively. We found that the elevated open probabilities in GlyREM are not due to the limited sequence differences between the human and zebrafish orthologs, but rather to replacement of the native ICD with a short tripeptide ICD. Consistent with this interpretation, shortening the ICD in the human GlyR increased the maximum open probability produced by taurine and GABA to 0.90 and 0.70, respectively, but further engineering it to resemble GlyREM (by introducing the zebrafish transmembrane helix 4 and C terminus) had no effect. Furthermore, reinstating the native ICD to GlyREM converted taurine and GABA to partial agonists, with maximum open probabilities of 0.66 and 0.40, respectively. Structural comparison of transmembrane helices 3 and 4 in short- and long-ICD GlyR subunits revealed that ICD shortening does not distort the orientation of these helices within each subunit. This suggests that the effects of shortening the ICD stem from removing a modulatory effect of the native ICD on GlyR gating, revealing a new role for the ICD in pentameric ligand-gated channels.

String method solution of the gating pathways for a pentameric ligand-gated ion channel

Pentameric ligand-gated ion channels control synaptic neurotransmission by converting chemical signals into electrical signals. Agonist binding leads to rapid signal transduction via an allosteric mechanism, where global protein conformational changes open a pore across the nerve cell membrane. We use all-atom molecular dynamics with a swarm-based string method to solve for the minimum free-energy gating pathways of the proton-activated bacterial GLIC channel. We describe stable wetted/open and dewetted/closed states, and uncover conformational changes in the agonist-binding extracellular domain, ion-conducting transmembrane domain, and gating interface that control communication between these domains. Transition analysis is used to compute free-energy surfaces that suggest allosteric pathways; stabilization with pH; and intermediates, including states that facilitate channel closing in the presence of an agonist. We describe a switching mechanism that senses proton binding by marked reorganization of subunit interface, altering the packing of β-sheets to induce changes that lead to asynchronous pore-lining M2 helix movements. These results provide molecular details of GLIC gating and insight into the allosteric mechanisms for the superfamily of pentameric ligand-gated channels.

Crystal structure and dynamics of a lipid-induced potential desensitized-state of a pentameric ligand-gated channel.

Desensitization in pentameric ligand-gated ion channels plays an important role in regulating neuronal excitability. Here, we show that docosahexaenoic acid (DHA), a key ω-3 polyunsaturated fatty acid in synaptic membranes, enhances the agonist-induced transition to the desensitized state in the prokaryotic channel GLIC. We determined a 3.25 Å crystal structure of the GLIC-DHA complex in a potentially desensitized conformation. The DHA molecule is bound at the channel-periphery near the M4 helix and exerts a long-range allosteric effect on the pore across domain-interfaces. In this previously unobserved conformation, the extracellular-half of the pore-lining M2 is splayed open, reminiscent of the open conformation, while the intracellular-half is constricted, leading to a loss of both water and permeant ions. These findings, in combination with spin-labeling/EPR spectroscopic measurements in reconstituted-membranes, provide novel mechanistic details of desensitization in pentameric channels.

Toward Understanding Functional Properties and Subunit Arrangement of α4β2δ γ-Aminobutyric Acid, Type A (GABAA) Receptors

GABAA receptors are pentameric ligand-gated channels mediating inhibitory neurotransmission in the CNS. α4βxδ GABAA receptors are extrasynaptic receptors important for tonic inhibition. The functional properties and subunit arrangement of these receptors are controversial. We predefined subunit arrangement by using subunit concatenation. α4, β2, and δ subunits were concatenated to dimeric, trimeric, and, in some cases, pentameric subunits. We constructed in total nine different receptor pentamers in at least two different ways and expressed them in Xenopus oocytes. The δ subunit was substituted in any of the five positions in the α1β2 receptor. In addition, we investigated all receptors with the 2:2:1 subunit stoichiometry for α4, β2, and δ. Several functional receptors were obtained. Interestingly, all of these receptors had very similar EC50 values for GABA in the presence of the neurosteroid 3α, 21-dihydroxy-5α-pregnan-20-one (THDOC). All functional receptors containing δ subunits were sensitive to 4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridin-3-yl]benzamide (DS2). Moreover, none of the receptors was affected by ethanol up to 30 mm These properties recapitulate those of non-concatenated receptors expressed from a cRNA ratio of 1:1:5 coding for α4, β2, and δ subunits. We conclude that the subunit arrangement of α4β2δ GABAA receptors is not strongly predefined but is mostly satisfying the 2:2:1 subunit stoichiometry for α4, β2, and δ subunits and that several subunit arrangements result in receptors with similar functional properties tuned to physiological conditions.

Mechanisms of Gating and Modulation in Pentameric Ligand Gated Channels

Structures of pentameric ligand-gated channels (pLGIC) have now reached atomic resolution, with some members of the family also captured in multiple functional states. However, it is widely known that pLGIC gating is extensively modulated by membrane lipids. Using prokaryotic pLGIC homologues and chimeric channels, we probe the allosteric mechanism underlying these effects. Here we show by a combination of EPR spectroscopy, X-ray crystallography, and electrophysiology how lipids modulate function by altering the dynamics of transmembrane helices at the membrane-lipid interface.

Functional Characterization of a Novel Class of Morantel-Sensitive Acetylcholine Receptors in Nematodes.

Acetylcholine receptors are pentameric ligand–gated channels involved in excitatory neuro-transmission in both vertebrates and invertebrates. In nematodes, they represent major targets for cholinergic agonist or antagonist anthelmintic drugs. Despite the large diversity of acetylcholine-receptor subunit genes present in nematodes, only a few receptor subtypes have been characterized so far. Interestingly, parasitic nematodes affecting human or animal health possess two closely related members of this gene family, acr-26 and acr-27 that are essentially absent in free-living or plant parasitic species. Using the pathogenic parasitic nematode of ruminants, Haemonchus contortus, as a model, we found that Hco-ACR-26 and Hco-ACR-27 are co-expressed in body muscle cells. We demonstrated that co-expression of Hco-ACR-26 and Hco-ACR-27 in Xenopus laevis oocytes led to the functional expression of an acetylcholine-receptor highly sensitive to the anthelmintics morantel and pyrantel. Importantly we also reported that ACR-26 and ACR-27, from the distantly related parasitic nematode of horses, Parascaris equorum, also formed a functional acetylcholine-receptor highly sensitive to these two drugs. In Caenorhabditis elegans, a free-living model nematode, we demonstrated that heterologous expression of the H. contortus and P. equorum receptors drastically increased its sensitivity to morantel and pyrantel, mirroring the pharmacological properties observed in Xenopus oocytes. Our results are the first to describe significant molecular determinants of a novel class of nematode body wall muscle AChR.

Activation Mechanism of Elic by Propylamine

The pentameric ligand-gated channel ELIC has provided us with high resolution structures of its closed state. This prokaryotic channel opens to a high single-channel conductance in response to a variety of amine compounds. Here we report our tests of kinetic models for the activation of recombinant ELIC channels. Outside-out single channel currents elicited by the full agonist propylamine (0.5-50 mM) were analysed by maximum likelihood direct global fitting of kinetic schemes (HJCFIT program; Colquhoun et al J Physiol 547, 699, 2003). The adequacy of a scheme was judged by comparing the predictions of the best fit obtained for each, with the experimental open/shut time distributions and with the time course of macroscopic propylamine-activated currents (fast theta-tube applications, 50-600 ms, 1-50 mM).Other Cys-loop channels, such as glycine receptors, activate via a pre-opening intermediate ('flip' model, Burzomato et al., J Neurosci 24, 10924,2004) and, when fully liganded, open with high efficacy to a single open state. In contrast with that, ELIC open time distributions at saturating propylamine showed more than one component. Thus, more than one open state must be accessible to the fully liganded channel. The ¯primed¯ model (Mukhtasimova et al., Nature 459, 451, 2009) accounts for that by allowing the channel to open from more than one fully liganded intermediate. The best fit of this type of model showed that ELIC maximum open probability (99%) is reached when three molecules of agonist are bound. The overall efficacy of the fully liganded branch was about 130 (c.f. 20 for α1β glycine channels; Burzomato et al., 2004).

Conformational Transitions Underlying Pore Opening and Desensitization in Membrane-Embedded GLIC

Abstract Direct structural insight into the mechanisms underlying activation and desensitization remain unavailable for the pentameric ligand-gated channel family. Here, we report the structural rearrangements underlying gating transitions in membrane-embedded GLIC, a prokaryotic homologue, using site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy. We particularly probed the conformation of pore-lining second transmembrane segment (M2) under conditions that favor the closed and the ligand-bound desensitized states. The spin-label mobility, intersubunit spin-spin proximity and the solvent-accessibility parameters in the two states clearly delineate the underlying protein motions within M2. Our results show that during activation the extracellular hydrophobic region undergoes major changes involving an outward translational movement, away from the pore axis, leading to an increase in the pore diameter, while the lower end of M2 remains relatively immobile. Most notably, during desensitization, the intervening polar residues in the middle of M2 move closer to form a solvent-occluded barrier and thereby reveal the location of a distinct desensitization gate. In comparison to the crystal structure of GLIC, the structural dynamics of the channel in a membrane environment suggest a more loosely packed conformation with water-accessible intrasubunit vestibules penetrating from the extracellular end all the way to the middle of M2 in the closed-state. These regions have been implicated to play a major role in alcohol and drug modulation. Overall, these findings represent a key step towards understanding the fundamentals of gating mechanisms in this class of channels.

Conformational Transitions Underlying Pore Opening and Desensitization in Membrane-embedded Gloeobacter violaceus Ligand-gated Ion Channel (GLIC)

Direct structural insight into the mechanisms underlying activation and desensitization remain unavailable for the pentameric ligand-gated channel family. Here, we report the structural rearrangements underlying gating transitions in membrane-embedded GLIC, a prokaryotic homologue, using site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy. We particularly probed the conformation of pore-lining second transmembrane segment (M2) under conditions that favor the closed and the ligand-bound desensitized states. The spin label mobility, intersubunit spin-spin proximity, and the solvent-accessibility parameters in the two states clearly delineate the underlying protein motions within M2. Our results show that during activation the extracellular hydrophobic region undergoes major changes involving an outward translational movement, away from the pore axis, leading to an increase in the pore diameter, whereas the lower end of M2 remains relatively immobile. Most notably, during desensitization, the intervening polar residues in the middle of M2 move closer to form a solvent-occluded barrier and thereby reveal the location of a distinct desensitization gate. In comparison with the crystal structure of GLIC, the structural dynamics of the channel in a membrane environment suggest a more loosely packed conformation with water-accessible intrasubunit vestibules penetrating from the extracellular end all the way to the middle of M2 in the closed state. These regions have been implicated to play a major role in alcohol and drug modulation. Overall, these findings represent a key step toward understanding the fundamentals of gating mechanisms in this class of channels.

The Molecular Mechanism for the Dual Alcohol Modulation of Cys-Loop Receptors

Despite high sequence identity, pentameric ligand-gated ion channels exhibit remarkable diversity in function with anionic/cationic channels that are either potentiated or inhibited by allosteric ligands. The recently available structures of bacterial homologs of Cys-loop receptors provide an excellent framework for understanding this allosteric modulation and function, but the modeling can be complex; our earlier simulations of the prokaryotic anionic glycine receptor (GlyR) suggest inter-subunit binding for ethanol (Murail, Biophys J 100, 1642, 2011), which at first sight appears to be incompatible with the experimental Gloeobacter violaceus (GLIC) ligand-gated ion channel structure showing binding intra-subunit. Here, we present new simulations of GLIC that confirm the occurrence of multiple binding sites by showing intra-subunit binding for ethanol. By experimentally introducing the single-site F238A mutation in GLIC we can turn it into a highly ethanol-sensitive channel (Howard, PNAS 108, 12149, 2011), similar to GlyR, and simulations of the mutated species confirm the occurrence of multiple binding sites. To critically test the results, we performed extensive docking and free energy calculations to identify alcohol-binding sites and determine their affinity. In the wild-type GLIC, short alcohols preferentially bind intra-subunit, with a very weak binding site inter-subunit. However, with the F238A mutation the inter-subunit site achieves a significantly lower free energy, and even becomes the highest-affinity site in the channel for some alcohols. These results suggest a new model for pentameric ligand-gated channel potentiation and inhibition, where the intra-subunit cavity would control inhibition, and the inter-subunit cavity potentiation. The possibility of allosteric ligands interacting with both cavities - or even a single molecule stretching from one site to the other - offers an attractive explanation for the complex functional dynamics of ligand-gated ion channels as well as an potential explain for the alcohol cutoff effect.

Microsecond Simulations Indicate that Ethanol Binds between Subunits and Could Stabilize an Open-State Model of a Glycine Receptor

Cys-loop receptors constitute a superfamily of ion channels gated by ligands such as acetylcholine, serotonin, glycine, and γ-aminobutyric acid. All of these receptors are thought to share structural characteristics, but due to high sequence variation and limited structure availability, our knowledge about allosteric binding sites is still limited. These sites are frequent targets of anesthetic and alcohol molecules, and are of high pharmacological importance. We used molecular simulations to study ethanol binding and equilibrium exchange for the homomeric α1 glycine receptor (GlyRα1), modeled on the structure of the Gloeobacter violaceus pentameric ligand-gated channel. Ethanol has a well-known potentiating effect and can be used in high concentrations. By performing two microsecond-scale simulations of GlyR with/without ethanol, we were able to observe spontaneous binding in cavities and equilibrium ligand exchange. Of interest, it appears that there are ethanol-binding sites both between and within the GlyR transmembrane subunits, with the intersubunit site having the highest occupancy and slowest exchange (∼200 ns). This model site involves several residues that were previously identified via mutations as being crucial for potentiation. Finally, ethanol appears to stabilize the GlyR model built on a presumably open form of the ligand-gated channel. This stabilization could help explain the effects of allosteric ligand binding in Cys-loop receptors.

Kinetic basis of partial agonism at NMDA receptors

Activation of ligand-gated channels is initiated by the binding of small molecules at extracellular sites and culminates with the opening of a membrane-embedded pore. To investigate how perturbations at ligand-binding domains influence the gating reaction, we examined current traces recorded from individual NMDA receptors in the presence of several subunit-specific partial agonists. Here we show that low-efficacy agonists acting at either the GluN1 or the GluN2A subunit had very similar effects on the receptor’s activation reaction, possibly reflecting a high degree of coupling between the two subunit-types during gating. In addition, we demonstrate that partial agonists increased the height of all energy barriers encountered by NMDA receptors during activation. This result stands in sharp contrast to the localized effects observed for pentameric ligand-gated channels and may represent a novel mechanism by which partial agonists reduce receptor activity.