Ligand-gated Ion Channel Superfamily Research Articles

Cryo-EM structure of the zinc-activated channel (ZAC) in the Cys-loop receptor superfamily

Cys-loop receptors are a large superfamily of pentameric ligand-gated ion channels with various physiological roles, especially in neurotransmission in the central nervous system. Among them, zinc-activated channel (ZAC) is a Zn2+-activated ion channel that is widely expressed in the human body and is conserved among eukaryotes. Due to its gating by extracellular Zn2+, ZAC has been considered a Zn2+ sensor, but it has undergone minimal structural and functional characterization since its molecular cloning. Among the families in the Cys-loop receptor superfamily, only the structure of ZAC has yet to be determined. Here, we determined the cryo-EM structure of ZAC in the apo state and performed structure-based mutation analyses. We identified a few residues in the extracellular domain whose mutations had a mild impact on Zn2+ sensitivity. The constriction site in the ion-conducting pore differs from the one in other Cys-loop receptor structures, and further mutational analysis identified a key residue that is important for ion selectivity. In summary, our work provides a structural framework for understanding the ion-conducting mechanism of ZAC.

Identification and Spatiotemporal Expression of a Putative New GABA Receptor Subunit in the Human Body Louse Pediculus humanus humanus.

The human louse (Pediculus humanus) is an obligatory blood feeding ectoparasite with two ecotypes: the human body louse (Pediculus humanus humanus), a competent vector of several bacterial pathogens, and the human head louse (Pediculus humanus capitis), responsible for pediculosis and affecting millions of people around the globe. GABA (γ-aminobutyric acid) receptors, members of the cys-loop ligand gated ion channel superfamily, are among the main pharmacological targets for insecticides. In insects, there are four subunits of GABA receptors: resistant-to-dieldrin (RDL), glycin-like receptor of drosophila (GRD), ligand-gated chloride channel homologue3 (LCCH3), and 8916 are well described and form distinct phylogenetic clades revealing orthologous relationships. Our previous studies in the human body louse confirmed that subunits Phh-RDL, Phh-GRD, and Phh-LCCH3 are well clustered in their corresponding clades. In the present work, we cloned and characterized a putative new GABA receptor subunit in the human body louse that we named HoCas, for Homologous to Cys-loop α like subunit. Extending our analysis to arthropods, HoCas was found to be conserved and clustered in a new (fifth) phylogenetic clade. Interestingly, the gene encoding this subunit is ancestral and has been lost in some insect orders. Compared to the other studied GABA receptor subunits, HoCas exhibited a relatively higher expression level in all development stages and in different tissues of human body louse. These findings improved our understanding of the complex nature of GABA receptors in Pediculus humanus and more generally in arthropods.

Impact of Ae-GRD on Ivermectin Resistance and Its Regulation by miR-71-5p in Aedes aegypti.

iGABAR, a member of the Cys-loop ligand-gated ion channel superfamily, is a significant target of the insecticide ivermectin (IVM). GRD is the potential subunit of the insect iGABAR. However, little information about GRD in Ae. aegypti has been reported. In this study, we involved cloning and characterizing the iGABAR subunit GRD of Ae. aegypti (Ae-GRD). Sequence analysis indicated that Ae-GRD, as part of the cysteine-loop ligand-gated ion channel family, is similar to other insect GRD. RNA interference (RNAi) was employed to explore IVM resistance in Ae. aegypti, resulting in a significant reduction in Ae-GRD expression (p < 0.05), and the mortality of Ae. aegypti adults with Ae-GRD knockdown was significantly decreased after exposure to ivermectin. Bioinformatics prediction identified miR-71-5p as a potential regulator of Ae-GRD. In vitro, dual-luciferase reporter assays confirmed that Ae-GRD expression was regulated by miR-71-5p. Microinjection of miR-71-5p mimics upregulated miR-71-5p expression and downregulated Ae-GRD gene expression, reducing mortality by 34.52% following IVM treatment. Conversely, microinjection of a miR-71-5p inhibitor decreased miR-71-5p expression but did not affect the susceptibility to IVM despite increased Ae-GRD expression (p < 0.05). In conclusion, Ae-GRD, as one of the iGABA receptor subunits, is a potential target of ivermectin. It may influence ivermectin resistance by modulating the GABA signaling pathway. The inhibition of Ae-GRD expression by miR-71-5p decreased ivermectin resistance and consequently lowered the mortality rate of Ae. aegypti mosquitoes. This finding provides empirical evidence of the relationship between Ae-GRD and its miRNA in modulating insecticide resistance, offering novel perspectives for mosquito control strategies.

Cys-loop ligand-gated ion channel superfamily of Pardosa pseudoannulata: Implication for natural enemy safety

Cys-loop ligand-gated channels mediate neurotransmission in insects and are receptors for many insecticides. Some insecticides acting on cysLGIC also have lethal effects on non-targeting organisms, but the mechanism of this negative effect is unclear due to information absence. The identification and analysis of cysLGIC family in Pardosa pseudoannulata, a pond wolf spider, can deepen the understanding of insecticides for natural enemy safety. Thirty-four cysLGIC genes were identified in P. pseudoannulata genome, including nicotinic acetylcholine receptors, γ-aminobutyric acid gated chloride channels, glutamate-gated chloride channels, histamine-gated chloride channels, and pH-sensitive chloride channels. The expansion of GABACls and HisCls accounts for the large number of cysLGICs in P. pseudoannulata, and the alternative splicing events in nAChR and RDL subunits enriched the diversity of the superfamily. Most cysLGIC genes show the highest expression in brain and lowest expression in the early-egg sac stage. Variable residues (R81, V83, R135, N137, F190, and W197) in P. pseudoannulata nAChR β subunits and critical differences in α6 subunit TM4 region compared with insects would apply for the insensitivity to neonicotinoids and spinosyn. In contrast, avermectin and dieldrin may be lethal to P. pseudoannulata due to the similar drugs binding sites in GluCls compared with insects. These findings will provide a valuable clue for natural enemy protection and environmentally friendly insecticide development.

Modulation of a rapid neurotransmitter receptor-ion channel by membrane lipids.

Membrane lipids modulate the proteins embedded in the bilayer matrix by two non-exclusive mechanisms: direct or indirect. The latter comprise those effects mediated by the physicochemical state of the membrane bilayer, whereas direct modulation entails the more specific regulatory effects transduced via recognition sites on the target membrane protein. The nicotinic acetylcholine receptor (nAChR), the paradigm member of the pentameric ligand-gated ion channel (pLGIC) superfamily of rapid neurotransmitter receptors, is modulated by both mechanisms. Reciprocally, the nAChR protein exerts influence on its surrounding interstitial lipids. Folding, conformational equilibria, ligand binding, ion permeation, topography, and diffusion of the nAChR are modulated by membrane lipids. The knowledge gained from biophysical studies of this prototypic membrane protein can be applied to other neurotransmitter receptors and most other integral membrane proteins.

GABAA receptor-mediated seizure liabilities: a mixed-methods screening approach

GABAA receptors, members of the pentameric ligand-gated ion channel superfamily, are widely expressed in the central nervous system and mediate a broad range of pharmaco-toxicological effects including bidirectional changes to seizure threshold. Thus, detection of GABAA receptor-mediated seizure liabilities is a big, partly unmet need in early preclinical drug development. This is in part due to the plethora of allosteric binding sites that are present on different subtypes of GABAA receptors and the critical lack of screening methods that detect interactions with any of these sites. To improve in silico screening methods, we assembled an inventory of allosteric binding sites based on structural data. Pharmacophore models representing several of the binding sites were constructed. These models from the NeuroDeRisk IL Profiler were used for in silico screening of a compiled collection of drugs with known GABAA receptor interactions to generate testable hypotheses. Amoxapine was one of the hits identified and subjected to an array of in vitro assays to examine molecular and cellular effects on neuronal excitability and in vivo locomotor pattern changes in zebrafish larvae. An additional level of analysis for our compound collection is provided by pharmacovigilance alerts using FAERS data. Inspired by the Adverse Outcome Pathway framework, we postulate several candidate pathways leading from specific binding sites to acute seizure induction. The whole workflow can be utilized for any compound collection and should inform about GABAA receptor-mediated seizure risks more comprehensively compared to standard displacement screens, as it rests chiefly on functional data.

Exploring the effects of nanodiscs on the conformation of a pentameric ligand-gated ion channel.

Recent advances in image acquisition and processing have unleashed a revolution in biomolecular structure determination via cryoelectron microscopy (cryoEM). Membrane proteins, such as the nicotinic acetylcholine receptor from the pentameric ligand-gated ion channel (pLGIC) superfamily, present challenges in structural studies due to their anisotropic and heterogenous lipid environment. In addition to the overall challenges in determining membrane protein structure, both lipid composition and membrane structure significantly affect the pLGIC conformational landscape, favoring some functional states over others. Nanodiscs, a patch of lipid bilayer surrounded by an amphipathic “scaffold”, are a standard reconstitution system for structure determination by single-particle cryoEM. Using an array of scaffolds, including the original apolipoprotein-derived membrane scaffolding protein, different size nanodiscs can be constructed to facilitate structural and biochemical studies in a quasi-native environment. Recent computational and experimental studies have shown, however, that many lipid properties, including membrane thickness, chain order parameters, and lipid diffusion, show position-dependent alterations upon incorporation into the nanodisc scaffold. To date, however, the effect of nanodisc scaffold and altered lipid properties upon a nanodisc-incorporated protein has not been systematically examined. CryoEM studies from our group have demonstrated that nanodisc scaffold choice affects the determined conformation of the pLGIC, ELIC. These nanodiscs differ in size which may be one factor influencing protein structure and dynamics. Here, we use molecular dynamics to examine how nanodisc size (small, 9 nm; medium, 11 nm; large, 13 nm) alters the transmembrane domain (TMD) structure and dynamics of ELIC, a bacterial pLGIC. Our simulation studies show that 1) the nanodisc construct and channel directly interact and 2) lipid properties are affected in a position-dependent manner leading to altered TMD dynamics. Specifically, dynamics in M4, thought to be responsible for lipid-sensing, are altered to the greatest extent.

Breaking brains and barriers: A novel approach to studying neurotransmitter receptor protein-protein interactions.

The Serotonin Receptor 3A (5-HT3A) is a member of the pentameric ligand-gated ion channel (pLGIC) superfamily and plays a role in numerous neuronal signaling pathways. However, relatively little remains known about its regulation. The intracellular domain (ICD) of the 5-HT3A receptor provides an attractive target for regulation studies, as the over 100 amino acid long sequence is exposed to the cell's internal milieu and is poorly conserved compared to that of other superfamily members. In this study, we set off to discover which proteins interact with the ICD of the 5-HT3A receptor, to identify potential regulatory partners. After developing a modified cellular fractionation technique using mouse brains, we were able to show, initially with western blotting, the presence of the 5-HT3A receptor in the endoplasmic reticulum (ER) where the receptor subunits assemble and the plasma membrane (PM) where the pentameric receptor is located. Once cellular localizations were preliminarily confirmed, proteins present in fractionated brain lysate fractions were identified using MALDI-TOF-MS/MS for further validation. Not only did this novel process work for serotonin receptors, it also worked for a variety of other key neurotransmitter receptors. These new techniques and findings provide a potential means of targeting regulatory proteins effecting numerous neuronal processes. This paves the way forward for progress in the study and treatment of neuropsychiatric disorders such as schizophrenia, Alzheimer's, and addiction.

Duplicated binding site for RIC-3 chaperon protein in serotonin type 3A subunits.

Serotonin or 5-hydroxytryptamine type 3A receptors (5-HT3A) belong to the pentameric ligand-gated ion channel super-family, which have been long-standing therapeutic targets for psychiatric disorders and neurological diseases. Due to structural conservation and significant sequence similarities in the extracellular and transmembrane domains of this family, clinical trials for drug candidates targeting these two domains have been hampered by off-subunit modulation. The intracellular domain of this family, in contrast, exhibits significant diversity in length, amino acid composition, and function, and hence positions itself as a potential drug target. We therefore explored the interaction interface of the 5-HT3A intracellular domain (ICD) with its regulator, the resistance to inhibitors of choline esterase (RIC-3) protein. We have previously shown that RIC-3 interacts with the L1-MX segment of the ICD fused to maltose-binding protein. In this study, using synthetic L1-MX-based peptides, Ala-scanning, and a pull-down assay, we identified motif DWLRXX(X)VLDR, as a critical motif for interaction with RIC-3. This motif is repeated twice within the ICD. One site is within the MX-helix, another site is at the MAM4-helix transition. For both sites, triple-Ala substitutions (MX: W347, R349, and L353; MAM4: W447, R449, and L454) disrupt the interactions between 5-HT3A-ICD-peptide and RIC-3. Complementary studies using full-length 5-HT3A subunits confirmed that the Ala substitutions reduced the RIC-3 mediated modulation of 5-HT3A functional surface expression. We thus identified two binding sites for RIC-3 with a shared duplicated motif in 5-HT3A subunits, one in the MX-helix and one at the MAM4-helix transition.

TPC1-Type Channels in Physcomitrium patens: Interaction between EF-Hands and Ca2.

Two-pore channels (TPCs) are members of the superfamily of ligand-gated and voltage-sensitive ion channels in the membranes of intracellular organelles of eukaryotic cells. The evolution of ordinary plant TPC1 essentially followed a very conservative pattern, with no changes in the characteristic structural footprints of these channels, such as the cytosolic and luminal regions involved in Ca2+ sensing. In contrast, the genomes of mosses and liverworts encode also TPC1-like channels with larger variations at these sites (TPC1b channels). In the genome of the model plant Physcomitrium patens we identified nine non-redundant sequences belonging to the TPC1 channel family, two ordinary TPC1-type, and seven TPC1b-type channels. The latter show variations in critical amino acids in their EF-hands essential for Ca2+ sensing. To investigate the impact of these differences between TPC1 and TPC1b channels, we generated structural models of the EF-hands of PpTPC1 and PpTPC1b channels. These models were used in molecular dynamics simulations to determine the frequency with which calcium ions were present in a coordination site and also to estimate the average distance of the ions from the center of this site. Our analyses indicate that the EF-hand domains of PpTPC1b-type channels have a lower capacity to coordinate calcium ions compared with those of common TPC1-like channels.

Aminomethanesulfonic acid illuminates the boundary between full and partial agonists of the pentameric glycine receptor.

To clarify the determinants of agonist efficacy in pentameric ligand-gated ion channels, we examined a new compound, aminomethanesulfonic acid (AMS), a molecule intermediate in structure between glycine and taurine. Despite wide availability, to date there are no reports of AMS action on glycine receptors, perhaps because AMS is unstable at physiological pH. Here, we show that at pH 5, AMS is an efficacious agonist, eliciting in zebrafish α1 glycine receptors a maximum single-channel open probability of 0.85, much greater than that of β-alanine (0.54) or taurine (0.12), and second only to that of glycine itself (0.96). Thermodynamic cycle analysis of the efficacy of these closely related agonists shows supra-additive interaction between changes in the length of the agonist molecule and the size of the anionic moiety. Single particle cryo-electron microscopy structures of AMS-bound glycine receptors show that the AMS-bound agonist pocket is as compact as with glycine, and three-dimensional classification demonstrates that the channel populates the open and the desensitized states, like glycine, but not the closed intermediate state associated with the weaker partial agonists, β-alanine and taurine. Because AMS is on the cusp between full and partial agonists, it provides a new tool to help us understand agonist action in the pentameric superfamily of ligand-gated ion channels.

Probing function in ligand-gated ion channels without measuring ion transport.

Although the functional properties of ion channels are most accurately assessed using electrophysiological approaches, a number of experimental situations call for alternative methods. Here, working on members of the pentameric ligand-gated ion channel (pLGIC) superfamily, we focused on the practical implementation of, and the interpretation of results from, equilibrium-type ligand-binding assays. Ligand-binding studies of pLGICs are by no means new, but the lack of uniformity in published protocols, large disparities between the results obtained for a given parameter by different groups, and a general disregard for constraints placed on the experimental observations by simple theoretical considerations suggested that a thorough analysis of this classic technique was in order. To this end, we present a detailed practical and theoretical study of this type of assay using radiolabeled α-bungarotoxin, unlabeled small-molecule cholinergic ligands, the human homomeric α7-AChR, and extensive calculations in the framework of a realistic five-binding-site reaction scheme. Furthermore, we show examples of the practical application of this method to tackle two longstanding questions in the field: our results suggest that ligand-binding affinities are insensitive to binding-site occupancy and that mutations to amino-acid residues in the transmembrane domain are unlikely to affect the channel's affinities for ligands that bind to the extracellular domain.

Fluorescence Studies of Nicotinic Acetylcholine Receptor and Its Associated Lipid Milieu: The Influence of Erwin London's Methodological Approaches.

Erwin London dedicated considerable effort to understanding lipid interactions with membrane-resident proteins and how these interactions shaped the formation and maintenance of lipid phases and domains. In this endeavor, he developed ad hoc techniques that greatly contributed to advancements in the field. We have employed and/or modified/extended some of his methodological approaches and applied them to investigate lipid interaction with the nicotinic acetylcholine receptor (nAChR) protein, the paradigm member of the superfamily of rapid pentameric ligand-gated ion channels (pLGIC). Our experimental systems ranged from purified receptor protein reconstituted into synthetic lipid membranes having known effects on receptor function, to cellular systems subjected to modification of their lipid content, e.g., varying cholesterol levels. We have often employed fluorescence techniques, including fluorescence quenching of diphenylhexatriene (DPH) extrinsic fluorescence and of nAChR intrinsic fluorescence by nitroxide spin-labeled phospholipids, DPH anisotropy, excimer formation of pyrene-phosphatidylcholine, and Förster resonance energy transfer (FRET) from the protein moiety to the extrinsic probes Laurdan, DPH, or pyrene-phospholipid to characterize various biophysical properties of lipid-receptor interactions. Some of these strategies are revisited in this review. Special attention is devoted to the anionic phospholipid phosphatidic acid (PA), which stabilizes the functional resting form of the nAChR. The receptor protein was shown to organize its PA-containing immediate microenvironment into microdomains with high lateral packing density and rigidity. PA and cholesterol appear to compete for the same binding sites on the nAChR protein.

Roles of Intracellular Second Messengers in Mediation of a7-nAChR-induced Modulations

Nicotinic acetylcholine receptors (nAChRs) are the members of the cys-loop ligandgated ion channel superfamily. They are cation-selective membrane proteins with a homopentameric or heteropentameric structure. The α7 subtype of nAChR (α7-nAChR) belongs to the classical homopentameric structure with the characteristics of relatively low affinity to agonists and high permeability to Ca2+. α7-nAChR plays an important role in modulating learning and memory, cognition, anti-inflammatory, growth and development, and other processes. There are many possible mechanisms underlying α7-nAChR-mediated modulations, including opening the high Ca2+ permeable channels, regulating presynaptic neurotransmitter release, and altering intracellular second messenger-mediated signaling pathways. Here, we summarize the signal pathways associated with α7-nAChR activation involving nucleotides, ions, lipids, and gas molecules. This article provides insights into α7-nAChR-mediated modulations through intracellular signaling systems and helps to understand the α7-nAChR-associated pathogenesis and therapeutics of a variety of diseases and disorders.

Role of Glycine and Glycine Receptors in Vascular Endothelium: A New Perspective for the Management of the Post-Ischemic Injury.

Glycine Receptors (GlyRs) are cell-surface transmembrane proteins that belong to the Cysloop ligand-gated ion channels superfamily (Cys-loop LGICs). Functional glycine receptors are conformed only by α-subunits (homomeric channels) or by α- and β-subunits (heteromeric channels). The role of glycine as a cytoprotective is widely studied. New information about glycine modulation of vascular endothelial cells (ECs) function emerged last year. Glycine and its receptors are recognized to play a role as neurovascular protectors by a mechanism that involves α2GlyRs. Interestingly, the expression of α2GlyRs reduces after stroke injury. However, glycine reverses the inhibition of α2GlyRs by a mechanism involving the VEGF/pSTAT3 signaling. On the other hand, consistent evidence has demonstrated that ECs participate actively in the innate and adaptive immunological response. We recently reported that GlyRs are modulated by interleukin-1β, suggesting new perspectives to explain the immune modulation of vascular function in pathological conditions such as cerebrovascular stroke. In this work, we distinguish the role of glycine and the allosteric modulation of glycine receptors as a new therapeutic target to confront post-ischemic injury.

Enhancing effect of nicotine on electrical field stimulation elicited contractile responses in isolated rabbit bladder straight muscle; the role of cannabinoid and vanilloid receptors.

Nicotine acts as an agonist of nicotinic acetylcholine receptors (nAChR). These receptors belong to a superfamily of ligand-gated ion channels. We previously demonstrated that nicotine increased electrical field stimulation (EFS)-induced contractile or relaxation responses, possibly by facilitating neurotransmitter release from nerve terminals in various rabbit tissues. Studies have shown that there is an interaction between the endocannabinoid and nicotinic systems. This study aimed to investigate the interaction between nicotine and the endocannabinoid system in the rabbit urine bladder and also investigate the enhancing effect of nicotine on EFS-induced contractile responses in rabbit isolated bladder smooth muscle and its interaction with the endocannabinoid system. The New Zealand albino male adult rabbits were used for this study. Following scarification, the urine bladder was rapidly excised, and then uniform strips were prepared. Each strip was mounted under 1 g isometric resting tension in an organ bath containing 20 mL of Krebs-Henseleit solution. After obtaining EFS-induced contractile responses, 10-4 M concentrations of nicotine were applied to the preparations, and EFS was stopped after 5 stimulations. Following washing, the same experimental procedure was performed with the same tissue in the presence of AM251 (a cannabinoid CB1R antagonist, 10-6 M), AM630 (a cannabinoid CB2R antagonist, 10-6 M), and capsazepine (a vanilloid receptor antagonist, 3 × 10-6 M). Nicotine enhanced the EFS-induced contraction responses by 17.16% ± 2.81% at a 4-Hz stimulation frequency. Cannabinoid receptor antagonists AM251 and AM630 reduced this increasing effect of nicotine although it was not significant and vanilloid receptor antagonist capsazepine did not significantly alter the nicotines' effect. These results show that enhancing effect of nicotine in the smooth muscle of the rabbit bladder, even though it was not significant endocannabinoid system possibly have a role in nicotines' effect.

Ligand-gated ion channels as targets of neuroactive insecticides.

The Cys-loop superfamily of ligand-gated ion channels (Cys-loop receptors) is one of the most ubiquitous ion channel families in vertebrates and invertebrates. Despite their ubiquity, they are targeted by several classes of pesticides, including neonicotinoids, phenylpyrazols, and macrolides such as ivermectins. The current commercialized compounds have high target site selectivity, which contributes to the safety of insecticide use. Structural analyses have accelerated progress in this field; notably, the X-ray crystal structures of acetylcholine binding protein and glutamate-gated Cl channels revealed the details of the molecular interactions between insecticides and their targets. Recently, the functional expression of the insect nicotinic acetylcholine receptor (nAChR) has been described, and detailed evaluations using the insect nAChR have emerged. This review discusses the basic concepts and the current insights into the molecular mechanisms of neuroactive insecticides targeting the ligand-gated ion channels, particularly Cys-loop receptors, and presents insights into target-based selectivity, resistance, and future drug design.

Discovery and functional characterization of N-(thiazol-2-yl)-benzamide analogs as the first class of selective antagonists of the Zinc-Activated Channel (ZAC)

The Zinc-Activated Channel (ZAC) is an atypical member of the Cys-loop receptor (CLR) superfamily of pentameric ligand-gated ion channels, with its very different endogenous agonists and signalling properties. In this study, a compound library screening at ZAC resulted in the identification of 2-(5-bromo-2-chlorobenzamido)-4-methylthiazole-5-methyl ester (1) as a novel ZAC antagonist. The structural determinants for ZAC activity in 1 were investigated by functional characterization of 61 analogs at ZAC expressed in Xenopus oocytes by two-electrode voltage clamp electrophysiology, and couple of analogs exerting more potent ZAC inhibition than 1 were identified (IC50 values: 1–3 μM). 1 and N-(4-(tert-butyl)thiazol-2-yl)-3-fluorobenzamide (5a, TTFB) were next applied in studies of the functional properties and the mode of action of this novel class of ZAC antagonists. TTFB was a roughly equipotent antagonist of Zn+- and H+-evoked ZAC signaling and of spontaneous ZAC activity, and the slow on-set of its channel block suggested that its ZAC inhibition is state-dependent. TTFB was found to be a selective ZAC antagonist, exhibiting no significant agonist, antagonist or modulatory activity at 5-HT3A, α3β4 nicotinic acetylcholine, α1β2γ2S GABAA or α1 glycine receptors at 30 μM. 1 displayed largely non-competitive antagonism of Zn2+-induced ZAC signalling, and TTFB was demonstrated to target the transmembrane and/or intracellular domains of the receptor, which collectively suggests that the N-(thiazol-2-yl)-benzamide analog acts a negative allosteric modulator of ZAC. We propose that this first class of selective ZAC antagonists could constitute useful pharmacological tools in future explorations of the presently poorly elucidated physiological functions governed by this CLR.

Therapeutic Targeting of α7 Nicotinic Acetylcholine Receptors.

The α7-type nicotinic acetylcholine receptor is one of the most unique and interesting of all the members of the cys-loop superfamily of ligand-gated ion channels. Since it was first identified initially as a binding site for α-bungarotoxin in mammalian brain and later as a functional homomeric receptor with relatively high calcium permeability, it has been pursued as a potential therapeutic target for numerous indications, from Alzheimer disease to asthma. In this review, we discuss the history and state of the art for targeting α7 receptors, beginning with subtype-selective agonists and the basic pharmacophore for the selective activation of α7 receptors. A key feature of α7 receptors is their rapid desensitization by standard "orthosteric" agonist, and we discuss insights into the conformational landscape of α7 receptors that has been gained by the development of ligands binding to allosteric sites. Some of these sites are targeted by positive allosteric modulators that have a wide range of effects on the activation profile of the receptors. Other sites are targeted by direct allosteric agonist or antagonists. We include a perspective on the potential importance of α7 receptors for metabotropic as well as ionotropic signaling. We outline the challenges that exist for future development of drugs to target this important receptor and approaches that may be considered to address those challenges. SIGNIFICANCE STATEMENT: The α7-type nicotinic acetylcholine receptor (nAChR) is acknowledged as a potentially important therapeutic target with functional properties associated with both ionotropic and metabotropic signaling. The functional properties of α7 nAChR can be regulated in diverse ways with the variety of orthosteric and allosteric ligands described in this review.

Structure and function at the lipid–protein interface of a pentameric ligand-gated ion channel

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.