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Inflammatory pain sensitization is initiated by prostaglandin-induced phosphorylation of α3 glycine receptors (GlyRs) that are specifically located in inhibitory synapses on spinal pain sensory neurons. Phosphorylation reduces the magnitude of glycinergic synaptic currents, thereby disinhibiting nociceptive neurons. Although α1 and α3 subunits are both expressed on spinal nociceptive neurons, α3 is a more promising therapeutic target as its sparse expression elsewhere implies a reduced risk of side-effects. Here we compared glycine-mediated conformational changes in α1 and α3 GlyRs to identify structural differences that might be exploited in designing α3-specific analgesics. Using voltage-clamp fluorometry, we show that glycine-mediated conformational changes in the extracellular M2-M3 domain were significantly different between the two GlyR isoforms. Using a chimeric approach, we found that structural variations in the intracellular M3-M4 domain were responsible for this difference. This prompted us to test the hypothesis that phosphorylation of S346 in α3 GlyR might also induce extracellular conformation changes. We show using both voltage-clamp fluorometry and pharmacology that Ser346 phosphorylation elicits structural changes in the α3 glycine-binding site. These results provide the first direct evidence for phosphorylation-mediated extracellular conformational changes in pentameric ligand-gated ion channels, and thus suggest new loci for investigating how phosphorylation modulates structure and function in this receptor family. More importantly, by demonstrating that phosphorylation alters α3 GlyR glycine-binding site structure, they raise the possibility of developing analgesics that selectively target inflammation-modulated GlyRs.

The 5-HT3A receptor homology model, based on the partial structure of the nicotinic acetylcholine receptor from Torpedo marmorata, reveals an asymmetric ion channel with five portals framed by adjacent helical amphipathic (HA) stretches within the 114-residue loop between the M3 and M4 membrane-spanning domains. The positive charge of Arg-436, located within the HA stretch, is a rate-limiting determinant of single channel conductance (γ). Further analysis reveals that positive charge and volume of residue 436 are determinants of 5-HT3A receptor inward rectification, exposing an additional role for portals. A structurally unresolved stretch of 85 residues constitutes the bulk of the M3-M4 loop, leaving a >45-A gap in the model between M3 and the HA stretch. There are no additional structural data for this loop, which is vestigial in bacterial pentameric ligand-gated ion channels and was largely removed for crystallization of the Caenorhabditis elegans glutamate-activated pentameric ligand-gated ion channels. We created 5-HT3A subunit loop truncation mutants, in which sequences framing the putative portals were retained, to determine the minimum number of residues required to maintain their functional integrity. Truncation to between 90 and 75 amino acids produced 5-HT3A receptors with unaltered rectification. Truncation to 70 residues abolished rectification and increased γ. These findings reveal a critical M3-M4 loop length required for functions attributable to cytoplasmic portals. Examination of all 44 subunits of the human neurotransmitter-activated Cys-loop receptors reveals that, despite considerable variability in their sequences and lengths, all M3-M4 loops exceed 70 residues, suggesting a fundamental requirement for portal integrity.

General anesthetics exert many of their CNS actions by binding to and modulating membrane-embedded pentameric ligand-gated ion channels (pLGICs). The structural mechanisms underlying how anesthetics modulate pLGIC function remain largely unknown. GLIC, a prokaryotic pLGIC homologue, is inhibited by general anesthetics, suggesting anesthetics stabilize a closed channel state, but in anesthetic-bound GLIC crystal structures the channel appears open. Here, using functional GLIC channels expressed in oocytes, we examined whether propofol induces structural rearrangements in the GLIC transmembrane domain (TMD). Residues in the GLIC TMD that frame intrasubunit and intersubunit water-accessible cavities were individually mutated to cysteine. We measured and compared the rates of modification of the introduced cysteines by sulfhydryl-reactive reagents in the absence and presence of propofol. Propofol slowed the rate of modification of L240C (intersubunit) and increased the rate of modification of T254C (intrasubunit), indicating that propofol binding induces structural rearrangements in these cavities that alter the local environment near these residues. Propofol acceleration of T254C modification suggests that in the resting state propofol does not bind in the TMD intrasubunit cavity as observed in the crystal structure of GLIC with bound propofol (Nury, H., Van Renterghem, C., Weng, Y., Tran, A., Baaden, M., Dufresne, V., Changeux, J. P., Sonner, J. M., Delarue, M., and Corringer, P. J. (2011) Nature 469, 428-431). In silico docking using a GLIC closed channel homology model suggests propofol binds to intersubunit sites in the TMD in the resting state. Propofol-induced motions in the intersubunit cavity were distinct from motions associated with channel activation, indicating propofol stabilizes a novel closed state.

2 arachidonyl glycerol (2 AG) allosterically potentiates GABA(A) receptors via a binding site located in transmembrane segment M4 of the ß2 subunit. Two amino acid residues have been described that are essential for this effect. With the aim to further describe this potential drug target we performed a cysteine scanning of the entire M4 and part of M3. All four residues in M4 affecting the potentiation here and the two already identified residues locate to the same side of the a helix. This side is exposed to M3 where further residues were identified. From the fact that the important residues span > 18 Å we conclude that the hydrophobic tail of the bound 2 AG molecule must be near linear and that the site mainly locates to the inner leaflet but stretches far into the membrane. The influence of the structure of the head group of the ligand molecule on the activity of the molecule was also investigated. We present a model of 2 AG docked to the GABA(A) receptor.

This review focuses on a very broad topic, so in fact it is a minireview. The first task is to briefly present the current state of nicotinic acetylcholine receptors and, even more briefly, some information about other members of the Cys-loop receptor family. The second task is to show studies in this field performed either independently in the author’s laboratory or in collaboration with foreign institutions.

N-methyl-d-aspartate (NMDA) receptors are heteromultimeric ion channels that contain an essential GluN1 subunit and two or more GluN2 (GluN2A–GluN2D) subunits. The biophysical properties and physiological roles of synaptic NMDA receptors are dependent on their subunit composition. In the basolateral amygdala (BLA), it has been suggested that the plasticity that underlies fear learning requires activation of heterodimeric receptors composed of GluN1/GluN2B subunits. In this study, we investigated the subunit composition of NMDA receptors present at synapses on principal neurons in the BLA. Purification of the synaptic fraction showed that both GluN2A and GluN2B subunits are present at synapses, and co-immunoprecipitation revealed the presence of receptors containing both GluN2A and GluN2B subunits. The kinetics of NMDA receptor-mediated synaptic currents and pharmacological blockade indicate that heterodimeric GluN1/GluN2B receptors are unlikely to be present at glutamatergic synapses on BLA principal neurons. Selective RNA interference-mediated knockdown of GluN2A subunits converted synaptic receptors to a GluN1/GluN2B phenotype, whereas knockdown of GluN2B subunits had no effect on the kinetics of the synaptically evoked NMDA current. Blockade of GluN1/GluN2B heterodimers with ifenprodil had no effect, but knockdown of GluN2B disrupted the induction of CaMKII-dependent long-term potentiation at these synapses. These results suggest that, on BLA principal neurons, GluN2B subunits are only present as GluN1/GluN2A/GluN2B heterotrimeric NMDA receptors. The GluN2B subunit has little impact on the kinetics of the receptor, but is essential for the recruitment of signaling molecules essential for synaptic plasticity.

Background and Purpose Most GABAA receptor subtypes comprise 2α, 2β and 1γ subunit, although for some isoforms, a δ replaces a γ-subunit. Extrasynaptic δ-GABAA receptors are important therapeutic targets, but there are few suitable pharmacological tools. We profiled DS2, the purported positive allosteric modulator (PAM) of δ-GABAA receptors to better understand subtype selectivity, mechanism/site of action and activity at native δ-GABAA receptors. Experimental Approach Subunit specificity of DS2 was determined using electrophysiological recordings of Xenopus laevis oocytes expressing human recombinant GABAA receptor isoforms. Effects of DS2 on GABA concentration–response curves were assessed to define mechanisms of action. Radioligand binding and electrophysiology utilising mutant receptors and pharmacology were used to define site of action. Using brain-slice electrophysiology, we assessed the influence of DS2 on thalamic inhibition in wild-type and δ0/0 mice. Key Results Actions of DS2 were primarily determined by the δ-subunit but were additionally influenced by the α, but not the β, subunit (α4/6βxδ > α1βxδ >> γ2-GABAA receptors > α4β3). For δ-GABAA receptors, DS2 enhanced maximum responses to GABA, with minimal influence on GABA potency. (iii) DS2 did not act via the orthosteric, or known modulatory sites on GABAA receptors. (iv) DS2 enhanced tonic currents of thalamocortical neurones from wild-type but not δ0/0 mice. Conclusions and Implications DS2 is the first PAM selective for α4/6βxδ receptors, providing a novel tool to investigate extrasynaptic δ-GABAA receptors. The effects of DS2 are mediated by an unknown site leading to GABAA receptor isoform selectivity.

GABAA receptors are members of the ligand-gated ion channel superfamily that mediate inhibitory neurotransmission in the central nervous system. They are thought to be composed of 2 alpha (α), 2 beta (β) subunits and one other such as a gamma (γ) or delta (δ) subunit. The potency of GABA is influenced by the subunit composition. However, there are no reported systematic studies that evaluate GABA potency on a comprehensive number of subunit combinations expressed in Xenopus oocytes, despite the wide use of this heterologous expression system in structure-function studies and drug discovery. Thus, the aim of this study was to conduct a systematic characterization of the potency of GABA at 43 human recombinant GABA(A) receptor combinations expressed in Xenopus oocytes using the two-electrode voltage clamp technique. The results show that the α-subunits and to a lesser extent, the β-subunits influence GABA potency. Of the binary and ternary combinations with and without the γ2L subunit, the α6/γ2L-containing receptors were the most sensitive to GABA, while the β2- or β3-subunit conferred higher sensitivity to GABA than receptors containing the β1-subunit with the exception of the α2β1γ2L and α6β1γ2L subtypes. Of the δ-subunit containing GABA(A) receptors, α4/δ-containing GABA(A) receptors displayed highest GABA sensitivity, with mid-nanomolar concentrations activating α4β1δ and α4β3δ receptors. At α4β2δ, GABA had low micromolar activity.

G protein–coupled receptors and ion channels couple a wide range of external stimuli to cellular growth and division, metabolism, motility, and a myriad of intra- and intercellular signaling pathways. G protein–coupled receptors initiate complex, interrelated downstream signaling cascades, whereas rapid ionic flux through channels directly supports membrane excitability and mediates cellular functions through second messengers. Because of these characteristics, these ubiquitous transmembrane proteins are valuable therapeutic targets and have provided fertile ground for the development of leading-edge synthetic and chemical biological approaches. Here we summarize recent advances in the use of site-directed incorporation of unnatural amino acids and chemical probes to study ligand-receptor interactions, determine the location of binding sites, and examine the downstream conformational consequences of ligand binding in G protein–coupled receptors and ion channels.

Background: Pentameric ligand-gated ion channels are modulated by general anesthetics. Results: The crystal structure of ELIC in complex with bromoform reveals anesthetic binding in the channel pore and in novel sites in the transmembrane and extracellular domain. Conclusion: General anesthetics allosterically modulate channel function via multisite binding. Significance: Our data reveal detailed insight into multisite recognition of general anesthetics at the structural level., Pentameric ligand-gated ion channels (pLGICs), such as nicotinic acetylcholine, glycine, γ-aminobutyric acid GABAA/C receptors, and the Gloeobacter violaceus ligand-gated ion channel (GLIC), are receptors that contain multiple allosteric binding sites for a variety of therapeutics, including general anesthetics. Here, we report the x-ray crystal structure of the Erwinia chrysanthemi ligand-gated ion channel (ELIC) in complex with a derivative of chloroform, which reveals important features of anesthetic recognition, involving multiple binding at three different sites. One site is located in the channel pore and equates with a noncompetitive inhibitor site found in many pLGICs. A second transmembrane site is novel and is located in the lower part of the transmembrane domain, at an interface formed between adjacent subunits. A third site is also novel and is located in the extracellular domain in a hydrophobic pocket between the β7–β10 strands. Together, these results extend our understanding of pLGIC modulation and reveal several specific binding interactions that may contribute to modulator recognition, further substantiating a multisite model of allosteric modulation in this family of ion channels.

Human LYNX1, belonging to the Ly6/neurotoxin family of three-finger proteins, is membrane-tethered with a glycosylphosphatidylinositol anchor and modulates the activity of nicotinic acetylcholine receptors (nAChR). Recent preparation of LYNX1 as an individual protein in the form of water-soluble domain lacking glycosylphosphatidylinositol anchor (ws-LYNX1; Lyukmanova, E. N., Shenkarev, Z. O., Shulepko, M. A., Mineev, K. S., D'Hoedt, D., Kasheverov, I. E., Filkin, S. Y., Krivolapova, A. P., Janickova, H., Dolezal, V., Dolgikh, D. A., Arseniev, A. S., Bertrand, D., Tsetlin, V. I., and Kirpichnikov, M. P. (2011) NMR structure and action on nicotinic acetylcholine receptors of water-soluble domain of human LYNX1. J. Biol. Chem. 286, 10618-10627) revealed the attachment at the agonist-binding site in the acetylcholine-binding protein (AChBP) and muscle nAChR but outside it, in the neuronal nAChRs. Here, we obtained a series of ws-LYNX1 mutants (T35A, P36A, T37A, R38A, K40A, Y54A, Y57A, K59A) and examined by radioligand analysis or patch clamp technique their interaction with the AChBP, Torpedo californica nAChR and chimeric receptor composed of the α7 nAChR extracellular ligand-binding domain and the transmembrane domain of α1 glycine receptor (α7-GlyR). Against AChBP, there was either no change in activity (T35A, T37A), slight decrease (K40A, K59A), and even enhancement for the rest mutants (most pronounced for P36A and R38A). With both receptors, many mutants lost inhibitory activity, but the increased inhibition was observed for P36A at α7-GlyR. Thus, there are subtype-specific and common ws-LYNX1 residues recognizing distinct targets. Because ws-LYNX1 was inactive against glycine receptor, its "non-classical" binding sites on α7 nAChR should be within the extracellular domain. Micromolar affinities and fast washout rates measured for ws-LYNX1 and its mutants are in contrast to nanomolar affinities and irreversibility of binding for α-bungarotoxin and similar snake α-neurotoxins also targeting α7 nAChR. This distinction may underlie their different actions, i.e. nAChRs modulation versus irreversible inhibition, for these two types of three-finger proteins.

In this study we report the X-ray crystal structure of the extracellular domain (ECD) of the human neuronal α2 nicotinic acetylcholine receptor (nAChR) subunit in complex with the agonist epibatidine at 3.2 A. Interestingly, α2 was crystallized as a pentamer, revealing the intersubunit interactions in a wild type neuronal nAChR ECD and the full ligand binding pocket conferred by two adjacent α subunits. The pentameric assembly presents the conserved structural scaffold observed in homologous proteins, as well as distinctive features, providing unique structural information of the binding site between principal and complementary faces. Structure-guided mutagenesis and electrophysiological data confirmed the presence of the α2(+)/α2(-) binding site on the heteromeric low sensitivity α2β2 nAChR and validated the functional importance of specific residues in α2 and β2 nAChR subunits. Given the pathological importance of the α2 nAChR subunit and the high sequence identity with α4 (78%) and other neuronal nAChR subunits, our findings offer valuable information for modeling several nAChRs and ultimately for structure-based design of subtype specific drugs against the nAChR associated diseases.

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.

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. Fundamental to these transitions is the intrinsic flexibility of the protein, which is critically modulated by membrane lipid-composition. To better understand the structural basis of channel function, it is necessary to study protein dynamics in a physiological membrane environment. Electron Paramagnetic Resonance (EPR) spectroscopy is an important tool to characterize conformational transitions between functional states. In comparison to NMR and X-ray crystallography, the information obtained from EPR is intrinsically of lower resolution. However, unlike in other techniques, in EPR there is no upper-limit to the molecular weight of the protein, the sample requirements are significantly lower, and more importantly the protein is not constrained by the crystal lattice forces. Therefore, EPR is uniquely suited for studying large protein complexes and proteins in reconstituted systems. In this article, we will discuss general protocols for site-directed spin labeling and membrane reconstitution using a prokaryotic proton-gated pentameric Ligand-Gated Ion Channel (pLGIC) from Gloeobacter violaceus (GLIC) as an example. A combination of steady-state Continuous Wave (CW) and Pulsed (Double Electron Electron Resonance-DEER) EPR approaches will be described that will enable a complete quantitative characterization of channel dynamics.

GABAA receptors are the main inhibitory neurotransmitter receptors in the brain and are targets for numerous clinically important drugs such as benzodiazepines, anxiolytics and anesthetics. We previously identified novel ligands of the classical benzodiazepine binding pocket in α1β2γ2 GABAA receptors using an experiment-guided virtual screening (EGVS) method. This screen also identified novel ligands for intramembrane low affinity diazepam site(s). In the current study we have further characterized compounds 31 and 132 identified with EGVS as well as 4-O-methylhonokiol. We investigated the site of action of these compounds in α1β2γ2 GABAA receptors expressed in Xenopus laevis oocytes using voltage-clamp electrophysiology combined with a benzodiazepine site antagonist and transmembrane domain mutations. All three compounds act mainly through the two β+/α− subunit transmembrane interfaces of the GABAA receptors. We then used concatenated receptors to dissect the involvement of individual β+/α− interfaces. We further demonstrated that these compounds have anesthetic activity in a small aquatic animal model, Xenopus laevis tadpoles. The newly identified compounds may serve as scaffolds for the development of novel anesthetics.

International audience; With the use of surface plasmon resonance (SPR) it was shown that ws-Lynx1, a water-soluble analog of the three-finger membrane-bound protein Lynx1, that modulates the activity of brain nicotinic acetylcholine receptors (nAChRs), interacts with the acetylcholine-binding protein (AChBP) with high affinity, K D = 62 nM. This result agrees with the earlier demonstrated competition of ws-Lynx1 with radioiodinated α-bungarotoxin for binding to AChBP. For the first time it was shown that ws-Lynx1 binds to GLIC, prokaryotic Cys-loop receptor (K D = 1.3 μM). On the contrary, SPR revealed that α-cobratoxin, a three-finger protein from cobra venom, does not bind to GLIC. Obtained results indicate that SPR is a promising method for analysis of topography of ws-Lynx1 binding sites using its mutants and those of AChBP and GLIC.

Nicotinic acetylcholine receptors (nAChRs) are neuron receptor proteins that provide a transmission of nerve impulse through the synapses. They are composed of a pentametric assembly of five homologous subunits (5 α7 subunits for α7nAChR, for example), oriented around the central pore. These receptors might be found in the chemical synapses of central and peripheral nervous system, and also in the neuromuscular synapses. Transmembrane domain of the one of such receptors constitutes ion channel. The conductive properties of ion channel strongly depend on the receptor conformation changes in the response of binding with some molecule, f.e. acetylcholine. Investigation of interaction between ligands and acetylcholine receptor is important for drug design. In this work we investigate theoretically the interaction between the set of different ligands (such as vanillin, thymoquinone, etc.) and the nicotinic acetylcholine receptor (primarily with subunit of the α7nAChR) by different methods and packages (AutodockVina, GROMACS, KVAZAR, HARLEM, VMD). We calculate interaction energy between different ligands in the subunit using molecular dynamics. On the base of obtained calculation results and using molecular docking we found an optimal location of different ligands in the subunit.

Cation-π interactions are common in biological systems, in particular ligand-gated ion channels (LGICs). Our own studies, in addition to more recent structural studies, have revealed the aromatic box as a common motif for binding cationic ligands. With the aim of understanding the nature of the aromatic box, several computational methods were evaluated for their ability to reproduce experimental cation-π binding energies. We find the DFT method M06 with the 6-31G(d,p) basis set performs best of several methods tested. The binding of benzene to a number of different cations (sodium, potassium, ammonium, tetramethylammonium, and guanidinium) was studied. In addition, the binding of the organic cations ammonium and tetramethylammonium to ab initio generated aromatic boxes as well as examples of aromatic boxes from a number of protein crystal structures was investigated. In addition, we have performed a study of the intrinsic distance dependence of the cation-π interaction. We find that multiple aromatic residues can contribute to cation binding in a variety of LGICs including ELIC, the glycine receptor, the GABA(A) receptor, and a model of the nicotinic acetylcholine receptor. Progressive fluorination of benzene and indole was studied as well, and binding energies obtained were used to reaffirm the validity of the “fluorination strategy” to study cation-π interactions in vivo.

The Erwinia ligand-gated ion channel (ELIC) is a bacterial pentameric ligand ion channel (pLGIC) originally identified in the enterobacterium Erwinia chrysanthemii. ELIC is closely related to a large class of neurotransmitter-gated ion channels (Cys-loop receptors) that underpin fast synaptic transmission in vertebrates and whose malfunction underlies a range of neurological disorders. As structures of Cys-loop receptors have lagged well behind those from prokaryotic pLGICs, ELIC, whose structure was solved in 2008, has proved to be an attractive model system. Here we explore molecular interactions in ELIC revealed by its structure. Of particular interest are intra-protein cation-π interactions, of which there are potentially six in each ELIC subunit: R48-W66; R99-F95; R105-Y38; R141-F142; R199-W160; and R301-W224. To probe these, the amino acids in each pair were swopped, using site directed mutagenesis, in addition to creating mutants with single amino acid changes to Ala and its pair. ELIC function was then probed following expression in HEKT cells using a membrane potential sensitive dye in a Flexstation. WT ELIC revealed GABA concentration dependent increases in fluorescence with a pEC50 of 2.97 + 0.01 (M, n > 6). The mutations had a range of effects from ablation of responses with all substituted amino acids (e.g. R105-Y38) to wild type responses with all substituted amino acids (e.g. R48-W66). The nature and importance of the interactions formed by these amino acids in ELIC, and whether the data can be extrapolated to better understand vertebrate Cys loop receptors, will be discussed.

The invertebrate glutamate-gated chloride-selective receptors (GluClRs) are ion channels serving as targets for ivermectin (IVM), a broad-spectrum anthelmintic drug used to treat human parasitic diseases like river blindness and lymphatic filariasis. The native GluClR is a heteropentamer consisting of α and β subunit types, with yet unknown subunit stoichiometry and arrangement. Based on the recent crystal structure of a homomeric GluClαR, we introduced mutations at the intersubunit interfaces where Glu (the neurotransmitter) binds. By electrophysiological characterization of these mutants, we found heteromeric assemblies with two equivalent Glu-binding sites at β/α intersubunit interfaces, where the GluClβ and GluClα subunits, respectively, contribute the "principal" and "complementary" components of the putative Glu-binding pockets. We identified a mutation in the IVM-binding site (far away from the Glu-binding sites), which significantly increased the sensitivity of the heteromeric mutant receptor to both Glu and IVM, and improved the receptor subunits' cooperativity. We further characterized this heteromeric GluClR mutant as a receptor having a third Glu-binding site at an α/α intersubunit interface. Altogether, our data unveil heteromeric GluClR assemblies having three α and two β subunits arranged in a counterclockwise β-α-β-α-α fashion, as viewed from the extracellular side, with either two or three Glu-binding site interfaces.

Cys-loop receptors mediate fast synaptic transmission in the nervous system, and their dysfunction is associated with a number of diseases. While some sequence variability is essential to ensure specific recognition of a chemically diverse set of ligands, other parts of the underlying amino acid sequences show a high degree of conservation, possibly to preserve the overall structural fold across the protein family. In this study, we focus on the only two absolutely conserved residues across the Cys-loop receptor family, two Trp side chains in the WXD motif of Loop D and in the WXPD motif of Loop A. Using a combination of conventional mutagenesis, unnatural amino acid incorporation, immunohistochemistry and MD simulations, we demonstrate the crucial contributions of these two Trp residues to receptor expression and function in two prototypical Cys-loop receptors, the anion-selective GlyR α1 and the cation-selective nAChR α7. Specifically, our results rule out possible electrostatic contributions of these Trp side chains and instead suggest that the overall size and shape of this aromatic pair is required in stabilizing the Cys-loop receptor extracellular domain.

The Cys-loop receptors play prominent roles in the nervous system. They include γ-aminobutyric acid type A receptors, nicotinic acetylcholine receptors, 5-hydroxytryptamine type-3 receptors, and glycine receptors. Proteostasis represents an optimal state of the cellular proteome in normal physiology. The proteostasis network regulates the folding, assembly, degradation, and trafficking of the Cys-loop receptors, ensuring their efficient functional cell surface expressions. Here, we summarize current advances about the protein biogenesis process of the Cys-loop receptors. Because operating on individual biogenesis steps influences the receptor cell surface level, manipulating the proteostasis network components can regulate the function of the receptors, representing an emerging therapeutic strategy for corresponding channelopathies.

5-Hydroxytryptamine type 3 (5-HT(3)) receptors are cation-selective Cys loop receptors found in both the central and peripheral nervous systems. There are five 5-HT(3) receptor subunits (A-E), and all functional receptors require at least one A subunit. Regions from noncontiguous parts of the subunit sequence contribute to the agonist-binding site, and the roles of a range of amino acid residues that form the binding pocket have been identified. Drugs that selectively antagonize 5-HT(3) receptors (the "setrons") are the current gold standard for treatment of chemotherapy-induced and postoperative nausea and vomiting and have potential for the treatment of a range of other conditions.

Endogenous neurosteroids are among the most potent and efficacious potentiators of activation of GABA(A) receptors. It has been proposed that a conserved glutamine residue in the first membrane-spanning region (TM1 region) of the α subunits is required for binding of potentiating neurosteroids. Mutations of this residue can reduce or remove the ability of steroids to potentiate function. However, it is not known whether potentiation requires that a steroid interact with the α subunit, or not. To examine this question we mutated the homologous residue in the β2 and γ2L subunits to glutamine, and found that these mutations could not confer potentiation by allopregnanolone (3α5αP) when expressed in receptors containing ineffective α1 subunits. However, potentiation is restored when the entire TM1 region from the α1 subunit is transferred to the β2 or γ2L subunit. Mutations in the TM1 region that affect potentiation when made in the α1 subunit have similar effects when made in transferred TM1 region. Further, the effects of 3α5αP on single-channel kinetics are similar for wild-type receptors and receptors with moved TM1 regions. These results support the idea that steroids bind in the transmembrane regions of the receptor. The observations are consistent with previous work indicating that neurosteroid potentiation is mediated by an action that affects the receptor as a whole, rather than an individual subunit or pair of subunits, and in addition demonstrate that the mechanism is independent of the nature of the subunit that interacts with steroid.

Pentameric ligand-gated ion channel (pLGIC) receptors exhibit desensitization, the progressive reduction in ionic flux in the prolonged presence of agonist. Despite its pathophysiological importance and the fact that it was first described over half a century ago, surprisingly little is known about the structural basis of desensitization in this receptor family. Here, we explain how desensitization is defined using functional criteria. We then review recent progress into reconciling the structural and functional basis of this phenomenon. The extracellular-transmembrane domain interface is a key locus. Activation is well known to involve conformational changes at this interface, and several lines of evidence suggest that desensitization involves a distinct conformational change here that is incompatible with activation. However, major questions remain unresolved, including the structural basis of the desensitization-induced agonist affinity increase and the mechanism of pore closure during desensitization.

We provide a theory for employing Förster resonance energy transfer (FRET) measurements to determine altered heteropentameric ion channel stoichiometries in intracellular compartments of living cells. We simulate FRET within nicotinic receptors (nAChRs) whose α4 and β2 subunits contain acceptor and donor fluorescent protein moieties, respectively, within the cytoplasmic loops. We predict FRET and normalized FRET (NFRET) for the two predominant stoichiometries, (α4)3(β2)2 vs. (α4)2(β2)3. Studying the ratio between FRET or NFRET for the two stoichiometries, minimizes distortions due to various photophysical uncertainties. Within a range of assumptions concerning the distance between fluorophores, deviations from plane pentameric geometry, and other asymmetries, the predicted FRET and NFRET for (α4)3(β2)2 exceeds that of (α4)2(β2)3. The simulations account for published data on transfected Neuro2a cells in which α4β2 stoichiometries were manipulated by varying fluorescent subunit cDNA ratios: NFRET decreased monotonically from (α4)3(β2)2 stoichiometry to mostly (α4)2(β2)3. The simulations also account for previous macroscopic and single-channel observations that pharmacological chaperoning by nicotine and cytisine increase the (α4)2(β2)3 and (α4)3(β2)2 populations, respectively. We also analyze sources of variability. NFRET-based monitoring of changes in subunit stoichiometry can contribute usefully to studies on Cys-loop receptors.

Background: Venoms from rare snake species may contain toxins of new structural or/and pharmacological types. Results: Amino acid sequence of the new polypeptide azemiopsin isolated from Azemiops feae viper venom was established, and its biological activity was determined. Conclusion: Azemiopsin is the first natural toxin that blocks nicotinic acetylcholine receptors and does not contain disulfide bridges. Significance: Azemiopsin is the first member of a new toxin group., Azemiopsin, a novel polypeptide, was isolated from the Azemiops feae viper venom by combination of gel filtration and reverse-phase HPLC. Its amino acid sequence (DNWWPKPPHQGPRPPRPRPKP) was determined by means of Edman degradation and mass spectrometry. It consists of 21 residues and, unlike similar venom isolates, does not contain cysteine residues. According to circular dichroism measurements, this peptide adopts a β-structure. Peptide synthesis was used to verify the determined sequence and to prepare peptide in sufficient amounts to study its biological activity. Azemiopsin efficiently competed with α-bungarotoxin for binding to Torpedo nicotinic acetylcholine receptor (nAChR) (IC50 0.18 ± 0.03 μm) and with lower efficiency to human α7 nAChR (IC50 22 ± 2 μm). It dose-dependently blocked acetylcholine-induced currents in Xenopus oocytes heterologously expressing human muscle-type nAChR and was more potent against the adult form (α1β1ϵδ) than the fetal form (α1β1γδ), EC50 being 0.44 ± 0.1 μm and 1.56 ± 0.37 μm, respectively. The peptide had no effect on GABAA (α1β3γ2 or α2β3γ2) receptors at a concentration up to 100 μm or on 5-HT3 receptors at a concentration up to 10 μm. Ala scanning showed that amino acid residues at positions 3–6, 8–11, and 13–14 are essential for binding to Torpedo nAChR. In biological activity azemiopsin resembles waglerin, a disulfide-containing peptide from the Tropidechis wagleri venom, shares with it a homologous C-terminal hexapeptide, but is the first natural toxin that blocks nAChRs and does not possess disulfide bridges.

A series of arylidene anabaseines were synthesized to probe the functional impact of hydrogen bonding on human α7 nicotinic acetylcholine receptor (nAChR) activation and desensitization. The aryl groups were either hydrogen bond acceptors (furans), donors (pyrroles), or neither (thiophenes). These compounds were tested against a series of point mutants of the ligand-binding domain residue Gln-57, a residue hypothesized to be proximate to the aryl group of the bound agonist and a putative hydrogen bonding partner. Q57K, Q57D, Q57E, and Q57L were chosen to remove the dual hydrogen bonding donor/acceptor ability of Gln-57 and replace it with hydrogen bond donating, hydrogen bond accepting, or nonhydrogen bonding ability. Activation of the receptor was compromised with hydrogen bonding mismatches, for example, pairing a pyrrole with Q57K or Q57L, or a furan anabaseine with Q57D or Q57E. Ligand co-applications with the positive allosteric modulator PNU-120596 produced significantly enhanced currents whose degree of enhancement was greater for 2-furans or -pyrroles than for their 3-substituted isomers, whereas the nonhydrogen bonding thiophenes failed to show this correlation. Interestingly, the PNU-120596 agonist co-application data revealed that for wild-type α7 nAChR, the 3-furan desensitized state was relatively stabilized compared with that of 2-furan, a reversal of the relationship observed with respect to the barrier for entry into the desensitized state. These data highlight the importance of hydrogen bonding on the receptor-ligand state, and suggest that it may be possible to fine-tune features of agonists that mediate state selection in the nAChR.

GABAA receptors are the major inhibitory transmitter receptors in the brain. They are ligand-gated chloride channels composed of five subunits that can belong to different subunit classes. The existence of six α, three β, three γ, one δ, one e, one θ, one π and three ρ subunits gives rise to an enormous diversity of GABAA receptor subtypes with different subunit composition and different pharmacological properties (Olsen and Sieghart, 2008; Sieghart, 1995). The majority of GABAA receptors, however, are composed of two α, two β and one γ subunit (Olsen and Sieghart, 2008). GABAA receptors are the site of action of a variety of pharmacologically and clinically important drugs such as benzodiazepines, barbiturates, neuroactive steroids, anaesthetics and convulsants, which allosterically modulate GABA-induced currents (Sieghart, 1995). Due to the action of these drugs, it is now clear that GABAA receptors are modulating anxiety, the excitability of the brain, muscle tonus, vigilance, circadian rhythms, learning and memory (Sieghart, 1995). Here, we are briefly reviewing what is known on the structure and pharmacology of GABAA receptors and their heterogeneity. We then shortly discuss some drugs that selectively interact with some of the GABAA receptor subtypes and finally describe the identification and importance of drugs mediating their effects via a novel drug binding site at the α+β− interface of GABAA receptors.

Nicotinic acetylcholine receptors (nAChRs), which are responsible for mediating key physiological functions, are ubiquitous in the central and peripheral nervous systems. As members of the Cys loop ligand-gated ion channel family, neuronal nAChRs are pentameric, composed of various permutations of α (α2 to α10) and β (β2 to β4) subunits forming functional heteromeric or homomeric receptors. Diversity in nAChR subunit composition complicates the development of selective ligands for specific subtypes, since the five binding sites reside at the subunit interfaces. The acetylcholine binding protein (AChBP), a soluble extracellular domain homologue secreted by mollusks, serves as a general structural surrogate for the nAChRs. In this work, homomeric AChBPs from Lymnaea and Aplysia snails were used as in situ templates for the generation of novel and potent ligands that selectively bind to these proteins. The cycloaddition reaction between building-block azides and alkynes to form stable 1,2,3-triazoles was used to generate the leads. The extent of triazole formation on the AChBP template correlated with the affinity of the triazole product for the nicotinic ligand binding site. Instead of the in situ protein-templated azide-alkyne cycloaddition reaction occurring at a localized, sequestered enzyme active center as previously shown, we demonstrate that the in situ reaction can take place at the subunit interfaces of an oligomeric protein and can thus be used as a tool for identifying novel candidate nAChR ligands. The crystal structure of one of the in situ-formed triazole-AChBP complexes shows binding poses and molecular determinants of interactions predicted from structures of known agonists and antagonists. Hence, the click chemistry approach with an in situ template of a receptor provides a novel synthetic avenue for generating candidate agonists and antagonists for ligand-gated ion channels.

A GABA(A) receptor β3 subunit mutation, G32R, has been associated with childhood absence epilepsy. We evaluated the possibility that this mutation, which is located adjacent to the most N-terminal of three β3 subunit N-glycosylation sites, might reduce GABAergic inhibition by increasing glycosylation of β3 subunits. The mutation had three major effects on GABA(A) receptors. First, coexpression of β3(G32R) subunits with α1 or α3 and γ2L subunits in HEK293T cells reduced surface expression of γ2L subunits and increased surface expression of β3 subunits, suggesting a partial shift from ternary αβ3γ2L receptors to binary αβ3 and homomeric β3 receptors. Second, β3(G32R) subunits were more likely than β3 subunits to be N-glycosylated at Asn-33, but increases in glycosylation were not responsible for changes in subunit surface expression. Rather, both phenomena could be attributed to the presence of a basic residue at position 32. Finally, α1β3(G32R)γ2L receptors had significantly reduced macroscopic current density. This reduction could not be explained fully by changes in subunit expression levels (because γ2L levels decreased only slightly) or glycosylation (because reduction persisted in the absence of glycosylation at Asn-33). Single channel recording revealed that α1β3(G32R)γ2L receptors had impaired gating with shorter mean open time. Homology modeling indicated that the mutation altered salt bridges at subunit interfaces, including regions important for subunit oligomerization. Our results suggest both a mechanism for mutation-induced hyperexcitability and a novel role for the β3 subunit N-terminal α-helix in receptor assembly and gating.

γ-Aminobutyric acid type A (GABA(A)) receptors are pentameric ligand-gated ion channels that mediate fast inhibition in the central nervous system. Depending on their subunit composition, these receptors exhibit distinct pharmacological properties and differ in their ability to interact with proteins involved in receptor anchoring at synaptic or extra-synaptic sites. Whereas GABA(A) receptors containing α1, α2, or α3 subunits are mainly located synaptically where they interact with the submembranous scaffolding protein gephyrin, receptors containing α5 subunits are predominantly found extra-synaptically and seem to interact with radixin for anchorage. Neuroplastin is a cell adhesion molecule of the immunoglobulin superfamily that is involved in hippocampal synaptic plasticity. Our results reveal that neuroplastin and GABA(A) receptors can be co-purified from rat brain and exhibit a direct physical interaction as demonstrated by co-precipitation and Förster resonance energy transfer (FRET) analysis in a heterologous expression system. The brain-specific isoform neuroplastin-65 co-localizes with GABA(A) receptors as shown in brain sections as well as in neuronal cultures, and such complexes can either contain gephyrin or be devoid of gephyrin. Neuroplastin-65 specifically co-localizes with α1 or α2 but not with α3 subunits at GABAergic synapses. In addition, neuroplastin-65 also co-localizes with GABA(A) receptor α5 subunits at extra-synaptic sites. Down-regulation of neuroplastin-65 by shRNA causes a loss of GABA(A) receptor α2 subunits at GABAergic synapses. These results suggest that neuroplastin-65 can co-localize with a subset of GABA(A) receptor subtypes and might contribute to anchoring and/or confining GABA(A) receptors to particular synaptic or extra-synaptic sites, thus affecting receptor mobility and synaptic strength.

The nicotinic acetylcholine receptor (nAChR) β3 subunit is thought to serve an accessory role in nAChR subtypes expressed in dopaminergic regions implicated in drug dependence and reward. When β3 subunits are expressed in excess, they have a dominant-negative effect on function of selected nAChR subtypes. In this study, we show, in Xenopus oocytes expressing α2, α3 or α4 plus either β2 or β4 subunits, that in the presumed presence of similar amounts of each nAChR subunit, co-expression with wild-type β3 subunits generally (except for α3*-nAChR) lowers amplitudes of agonist-evoked, inward peak currents by 20-50% without having dramatic effects (≤ 2-fold) on agonist potencies. By contrast, co-expression with mutant β3(V9'S) subunits generally (except for α4β2*-nAChR) increases agonist potencies, consistent with an expected gain-of-function effect. This most dramatically demonstrates formation of complexes containing three kinds of subunit. Moreover, for oocytes expressing nAChR containing any α subunit plus β4 and β3(V9'S) subunits, there is spontaneous channel opening sensitive to blockade by the open channel blocker, atropine. Collectively, the results indicate that β3 subunits integrate into all of the studied receptor assemblies and suggest that natural co-expression with β3 subunits can influence levels of expression and agonist sensitivities of several nAChR subtypes.

BACKGROUND AND PURPOSE GABAA receptors mediate both synaptic and extrasynaptic actions of GABA. In several neuronal populations, α4 and δ subunits are key components of extrasynaptic GABAA receptors that strongly influence neuronal excitability and could mediate the effects of neuroactive agents including neurosteroids and ethanol. However, these receptors can be difficult to study in native cells and recombinant δ subunits can be difficult to express in heterologous systems. EXPERIMENTAL APPROACH We engineered concatemeric (fused) subunits to ensure δ and α4 subunit expression. We tested the pharmacology of the concatemeric receptors, compared with a common synaptic-like receptor subunit combination (α1 +β2 +γ2L), and with free-subunit α4/δ receptors, expressed in Xenopus oocytes. KEY RESULTS δ-β2 −α4 +β2-α4 cRNA co-injected into Xenopus oocytes resulted in GABA-gated currents with the expected pharmacological properties of α4/δ-containing receptors. Criteria included sensitivity to agonists of different efficacy, sensitivity to the allosteric activator pentobarbital, and modulation of agonist responses by DS2 (4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridine-3-yl benzamide; a δ-selective positive modulator), furosemide, and Zn2+. We used the concatemers to examine neurosteroid sensitivity of extrasynaptic-like, δ-containing receptors. We found no qualitative differences between extrasynaptic-like receptors and synaptic-like receptors in the actions of either negative or positive neurosteroid modulators of receptor function. Quantitative differences were explained by the partial agonist effects of the natural agonist GABA and by a mildly increased sensitivity to low steroid concentrations. CONCLUSIONS AND IMPLICATIONS The neurosteroid structure-activity profile for α4/δ-containing extrasynaptic receptors is unlikely to differ from that of synaptic-like receptors such as α1/β2/γ2-containing receptors.

The α4β2 subtype of the nicotinic acetylcholine receptor has been pursued as a drug target for treatment of psychiatric and neurodegenerative disorders and smoking cessation aids for decades. Still, a thorough understanding of structure-function relationships of α4β2 agonists is lacking. Using binding experiments, electrophysiology and x-ray crystallography we have investigated a consecutive series of five prototypical pyridine-containing agonists derived from 1-(pyridin-3-yl)-1,4-diazepane. A correlation between binding affinities at α4β2 and the acetylcholine-binding protein from Lymnaea stagnalis (Ls-AChBP) confirms Ls-AChBP as structural surrogate for α4β2 receptors. Crystal structures of five agonists with efficacies at α4β2 from 21–76% were determined in complex with Ls-AChBP. No variation in closure of loop C is observed despite large efficacy variations. Instead, the efficacy of a compound appears tightly coupled to its ability to form a strong intersubunit bridge linking the primary and complementary binding interfaces. For the tested agonists, a specific halogen bond was observed to play a large role in establishing such strong intersubunit anchoring.

Prokaryotic orthologs of eukaryotic Cys-loop receptor channels recently emerged as structural and mechanistic surrogates to investigate this superfamily of intercellular signaling proteins. Here, we examine proton activation of the prokaryotic ortholog GLIC using patch clamp electrophysiology, mutagenesis, and molecular dynamics (MD) simulations. Whole-cell current recordings from human embryonic kidney (HEK) 293 cells expressing GLIC show half-maximal activation at pH 6, close to the pK(a) of histidine, implicating the three native His residues in proton sensing linked to activation. The mutation H235F abolishes proton activation, H277Y is without effect, and all nine mutations of His-127 prevent expression on the cell surface. In the GLIC crystal structure, His-235 on transmembrane (TM) α-helix 2, hydrogen bonds to the main chain carbonyl oxygen of Ile-259 on TM α-helix 3. MD simulations show that when His-235 is protonated, the hydrogen bond persists, and the channel remains in the open conformation, whereas when His-235 is deprotonated, the hydrogen bond dissociates, and the channel closes. Mutations of the proximal Tyr-263, which also links TM α-helices 2 and 3 via a hydrogen bond, alter proton sensitivity over a 1.5 pH unit range. MD simulations show that mutations of Tyr-263 alter the hydrogen bonding capacity of His-235. The overall findings show that His-235 in the TM region of GLIC is a novel proton binding site linked to channel activation.

BACKGROUND AND PURPOSE Morphine is an antagonist at 5-HT3A receptors. 5-HT3 and opioid receptors are expressed in many of the same neuronal pathways where they modulate gut motility, pain and reinforcement. There is increasing interest in the 5-HT3B subunit, which confers altered pharmacology to 5-HT3 receptors. We investigated the mechanisms of inhibition by morphine of 5-HT3 receptors and the influence of the 5-HT3B subunit. EXPERIMENTAL APPROACH 5-HT-evoked currents were recorded from voltage-clamped HEK293 cells expressing human 5-HT3A subunits alone or in combination with 5-HT3B subunits. The affinity of morphine for the orthosteric site of 5-HT3A or 5-HT3AB receptors was assessed using radioligand binding with the antagonist [3H]GR65630. KEY RESULTS When pre-applied, morphine potently inhibited 5-HT-evoked currents mediated by 5-HT3A receptors. The 5-HT3B subunit reduced the potency of morphine fourfold and increased the rates of inhibition and recovery. Inhibition by pre-applied morphine was insurmountable by 5-HT, was voltage-independent and occurred through a site outside the second membrane-spanning domain. When applied simultaneously with 5-HT, morphine caused a lower potency, surmountable inhibition of 5-HT3A and 5-HT3AB receptors. Morphine also fully displaced [3H]GR65630 from 5-HT3A and 5-HT3AB receptors with similar potency. CONCLUSIONS AND IMPLICATIONS These findings suggest that morphine has two sites of action, a low-affinity, competitive site and a high-affinity, non-competitive site that is not available when the channel is activated. The affinity of morphine for the latter is reduced by the 5-HT3B subunit. Our results reveal that morphine causes a high-affinity, insurmountable and subunit-dependent inhibition of human 5-HT3 receptors.

Background: Cytisine and varenicline are smoking cessation drugs binding to nicotinic receptors (nAChRs). Results: We studied crystal structures of cytisine and varenicline with AChBP and analyzed binding of α4β2-like or α7-like AChBP mutants to cytisine. Conclusion: Ligand selectivity relies on residues beyond the binding site primary shell. Significance: These structures will contribute to designing novel compounds targeting specific nAChR subtypes., Smoking cessation is an important aim in public health worldwide as tobacco smoking causes many preventable deaths. Addiction to tobacco smoking results from the binding of nicotine to nicotinic acetylcholine receptors (nAChRs) in the brain, in particular the α4β2 receptor. One way to aid smoking cessation is by the use of nicotine replacement therapies or partial nAChR agonists like cytisine or varenicline. Here we present the co-crystal structures of cytisine and varenicline in complex with Aplysia californica acetylcholine-binding protein and use these as models to investigate binding of these ligands binding to nAChRs. This analysis of the binding properties of these two partial agonists provides insight into differences with nicotine binding to nAChRs. A mutational analysis reveals that the residues conveying subtype selectivity in nAChRs reside on the binding site complementary face and include features extending beyond the first shell of contacting residues.

In plants heterotrimeric complexes of G proteins (consisting of alpha, beta, and gamma subunits) regulate several signaling pathways including seed germination, seedling development, organ shape and size determination. The alpha subunit has GTP binding and hydrolysis activity and the beta- gamma subunits interact with downstream effectors as a heterodimer. Some structural homology among the plant and mammalian subunits have led to early assumptions about similarities in the activation and transduction mechanisms in the two systems. However, recent evidence on the lack of membrane receptors in plants, the constitutively active state of the plant alpha subunit and the existence of a large family of gamma subunits indicate that the mechanisms involving the plant proteins may be significantly different from those in their mammalian counterparts.In our group the alpha subunit from A. thaliana (AtGPA1), an N-terminal mutant (GPA1t), the gamma subunits (AGG1, AGG2), and the rice gamma subunits (RGG1 and RGG2) were expressed in yeast and bacteria. Absorbance spectroscopy, circular dichroism spectropolarimetry, and dynamic light scattering (DLS) analyses show the structural and stability differences between AtGPA1 and GPA1t as well as among all gamma subunits. DLS, native-PAGE and small angle X-ray scattering measurements reveal the stable oligomeric forms of the proteins in solution indicating possible functional roles for the oligomers. Results also demonstrate the high level of the flexibility in the structures of all subunits. Models for possible roles of different subunits in G protein signaling in plants will be presented.Supported by Turkish Atomic Energy Commission and Instruct, a Landmark ESFRI project.

Determining the structure of the ligand-binding domain of the nicotinic acetylcholine receptor (nAChR) has been a long standing goal in the design of selective drugs useful in implicated diseases for this prevalent receptor family. Acetylcholine-binding proteins have proven to be valuable surrogates with structural similarity and sequence identity to the extracellular domain of the nicotinic receptor, yet these soluble proteins have their unique features and do not serve as exact replicates of the nAChRs of interest. Here we systematically modify the sequence of these proteins toward the homomeric human α7 nAChR. These chimeric proteins exhibit a shift in affinities to reflect α7 binding characteristics yet maintain expression levels and stability conducive for crystallization. We also present a pentameric humanoid nAChR extracellular domain with the structural determination of the α7 nAChR glycosylation site.

To further the understanding of functional α6α5*-nicotinic acetylcholine receptors (nAChR; the asterisk (*) indicates known or possible presence of other subunits), we have heterologously expressed in oocytes different, mouse or human, nAChR subunit combinations. Coexpression with wild-type α5 subunits or chimeric α5/β3 subunits (in which the human α5 subunit N-terminal, extracellular domain is linked to the remaining domains of the human β3 subunit) almost completely abolishes the very small amount of function seen for α6β4*-nAChR and does not induce function of α6β2*-nAChR. Coexpression with human α5(V9)'(S) subunits bearing a valine 290 to serine mutation in the 9' position of the second transmembrane domain does not rescue the function of α6β4*-nAChR or induce function of α6β2*-nAChR. However, coexpression with mutant chimeric α5/β3(V9)'(S) subunits has a gain-of-function effect (higher functional expression and agonist sensitivity and spontaneous opening inhibited by mecamylamine) on α6β4*-nAChR. Moreover, N143D + M145V mutations in the α6 subunit N-terminal domain enable α5/β3(V9)'(S) subunits to have a gain-of-function effect on α6β2*-nAChR. nAChR containing chimeric α6/α3 subunits plus either β2 or β4 subunits have some function that is modulated in the presence of α5 or α5/β3 subunits. Coexpression with α5/β3(V9)'(S) subunits has a gain-of-function effect more pronounced than that in the presence of α5(V9)'(S) subunits. Gain-of-function effects are dependent, sometimes subtly, on the nature and apparently the extracellular, cytoplasmic, and/or transmembrane domain topology of partner subunits. These studies yield insight into assembly of functional α6α5*-nAChR and provide tools for development of α6*-nAChR-selective ligands that could be important in the treatment of nicotine dependence, and perhaps other neurological diseases.

We investigated assembly and function of nicotinic acetylcholine receptors (nAChRs) composed of α7 and β2 subunits. We measured optical and electrophysiological properties of wild-type and mutant subunits expressed in cell lines and Xenopus laevis oocytes. Laser scanning confocal microscopy indicated that fluorescently tagged α7 and β2 subunits colocalize. Förster resonance energy transfer between fluorescently tagged subunits strongly suggested that α7 and β2 subunits coassemble. Total internal reflection fluorescence microscopy revealed that assemblies localized to filopodia-like processes of SH-EP1 cells. Gain-of-function α7 and β2 subunits confirmed that these subunits coassemble within functional receptors. Moreover, α7β2 nAChRs composed of wild-type subunits or fluorescently tagged subunits had pharmacological properties similar to those of α7 nAChRs, although amplitudes of α7β2 nAChR-mediated, agonist-evoked currents were generally ∼2-fold lower than those for α7 nAChRs. It is noteworthy that α7β2 nAChRs displayed sensitivity to low concentrations of the antagonist dihydro-β-erythroidine that was not observed for α7 nAChRs at comparable concentrations. In addition, cysteine mutants revealed that the α7-β2 subunit interface does not bind ligand in a functionally productive manner, partly explaining lower α7β2 nAChR current amplitudes and challenges in identifying the function of native α7β2 nAChRs. On the basis of our findings, we have constructed a model predicting receptor function that is based on stoichiometry and position of β2 subunits within the α7β2 nAChRs.

Hierarchical in silico screening protocols against the agonist bound acetylcholine binding protein (AChBP) crystal structure were efficient in identifying novel chemotypes for AChBP and the human α7 receptor. Two hit structures were cocrystallized with AChBP revealing intermolecular cation-π interactions with loop C but lacking intermolecular hydrogen bonding. The compounds act as competitive α7 receptor antagonists and as non-competitive α4β2 receptor inhibitors. These results underline the usability of AChBP in structure-based in silico screening strategies in finding novel scaffolds for the α7 receptor, but also illustrates some limitations of using AChBP as bait to find competitive α4β2 receptor ligands and α7 receptor agonists. © 2011 Elsevier Ltd. All rights reserved.

Glycine receptors (GlyRs) are chloride channels that mediate fast inhibitory neurotransmission and are members of the pentameric ligand-gated ion channel (pLGIC) family. The interface between the ligand binding domain and the transmembrane domain of pLGICs has been proposed to be crucial for channel gating and is lined by a number of charged and aromatic side chains that are highly conserved among different pLGICs. However, little is known about specific interactions between these residues that are likely to be important for gating in α1 GlyRs. Here we use the introduction of cysteine pairs and the in vivo nonsense suppression method to incorporate unnatural amino acids to probe the electrostatic and hydrophobic contributions of five highly conserved side chains near the interface, Glu-53, Phe-145, Asp-148, Phe-187, and Arg-218. Our results suggest a salt bridge between Asp-148 in loop 7 and Arg-218 in the pre-M1 domain that is crucial for channel gating. We further propose that Phe-145 and Phe-187 play important roles in stabilizing this interaction by providing a hydrophobic environment. In contrast to the equivalent residues in loop 2 of other pLGICs, the negative charge at Glu-53 α1 GlyRs is not crucial for normal channel function. These findings help decipher the GlyR gating pathway and show that distinct residue interaction patterns exist in different pLGICs. Furthermore, a salt bridge between Asp-148 and Arg-218 would provide a possible mechanistic explanation for the pathophysiologically relevant hyperekplexia, or startle disease, mutant Arg-218 → Gln.

Prokaryotic members of the Cys-loop receptor ligand-gated ion channel superfamily were recently identified. Previously, Cys-loop receptors were only known from multicellular organisms (metazoans). Contrary to the metazoan Cys-loop receptors, the prokaryotic ones consist of an extracellular (ECD) and a transmembrane domain (TMD), lacking the large intracellular domain (ICD) present in metazoa (between transmembrane segments M3 and M4). Using a chimera approach, we added the 115-amino acid ICD from mammalian serotonin type 3A receptors (5-HT(3A)) to the prokaryotic proton-activated Gloeobacter violaceus ligand-gated ion channel (GLIC). We created 12 GLIC-5-HT(3A)-ICD chimeras by replacing a variable number of amino acids in the short GLIC M3M4 linker with the entire 5-HT(3A)-ICD. Two-electrode voltage clamp recordings after expression in Xenopus laevis oocytes showed that only two chimeras were functional and produced currents upon acidification. The pH(50) was comparable with wild-type GLIC. 5-HT(3A) receptor expression can be inhibited by the chaperone protein RIC-3. We have shown previously that the 5-HT(3A)-ICD is required for the attenuation of 5-HT-induced currents when RIC-3 is co-expressed with 5-HT(3A) receptors in X. laevis oocytes. Expression of both functional 5-HT(3A) chimeras was inhibited by RIC-3 co-expression, indicating appropriate folding of the 5-HT(3A)-ICD in the chimeras. Our results indicate that the ICD can be considered a separate domain that can be removed from or added to the ECD and TMD while maintaining the overall structure and function of the ECD and TMD.

Lynx1 expresses in the central nervous system and plays important role in a regulation of nicotinic acetylcholine receptors. Successful milligram-quantitive expression of ws-Lynx1 was achieved only in the case of its production in the form of cytoplasm inclusion bodies. Different conditions of ws-Lynx1 refolding for yield optimization were performed. The obtained recombinant protein was characterized by means of mass spectrometry and CD spectroscopy. The binding experiments on the nAChRs from Torpedo californica membranes revealed that ws-Lynxl is biologically active and blocks muscle nAChR with IC50-20-30 microM.

Non-technical summary Nerve signals are transmitted across cell membranes by receptors that can consist of multiple different subunits. The 5-HT3 receptor is a pentamer which can function with A subunits alone, or with a mixture of A and B subunits. As 5-HT activates the receptor by binding at the interface of adjacent subunits, it is important to know which subunits are adjacent. Here we show that in both A-only and A+B receptors there is at least one A–A interface, without which the receptor cannot function. This knowledge is important for understanding the receptor mechanism, and also will allow the design of more specific drugs that act at the 5-HT binding site.

The molecular chaperone prefoldin (PFD) is a complex comprised of six different subunits, PFD1-PFD6, and delivers newly synthesized unfolded proteins to cytosolic chaperonin TRiC/CCT to facilitate the folding of proteins. PFD subunits also have functions different from the function of the PFD complex. We previously identified MM-1α/PFD5 as a novel c-Myc-binding protein and found that MM-1α suppresses transformation activity of c-Myc. However, it remains unclear how cells regulate protein levels of individual subunits and what mechanisms alter the ratio of their activities between subunits and their complex. In this study, we found that knockdown of one subunit decreased protein levels of other subunits and that transfection of five subunits other than MM-1α into cells increased the level of endogenous MM-1α. We also found that treatment of cells with MG132, a proteasome inhibitor, increased the level of transfected/overexpressed MM-1α but not that of endogenous MM-1α, indicating that overexpressed MM-1α, but not endogenous MM-1α, was degraded by the ubiquitin proteasome system (UPS). Experiments using other PFD subunits showed that the UPS degraded a monomer of PFD subunits, though extents of degradation varied among subunits. Furthermore, the level of one subunit was increased after co-transfection with the respective subunit, indicating that there are specific combinations between subunits to be stabilized. These results suggest mutual regulation of protein levels among PFD subunits and show how individual subunits form the PFD complex without degradation.

Single-channel conductance in Cys-loop channels is controlled by the nature of the amino acids in the narrowest parts of the ion conduction pathway, namely the second transmembrane domain (M2) and the intracellular helix. In cationic channels, such as Torpedo ACh nicotinic receptors, conductance is increased by negatively charged residues exposed to the extracellular vestibule. We now show that positively charged residues at the same loop 5 position boost also the conductance of anionic Cys-loop channels, such as glycine (α1 and α1β) and GABA(A) (α1β2γ2) receptors. Charge reversal mutations here produce a greater decrease on outward conductance, but their effect strongly depends on which subunit carries the mutation. In the glycine α1β receptor, replacing Lys with Glu in α1 reduces single-channel conductance by 41%, but has no effect in the β subunit. By expressing concatameric receptors with constrained stoichiometry, we show that this asymmetry is not explained by the subunit copy number. A similar pattern is observed in the α1β2γ2 GABA(A) receptor, where only mutations in α1 or β2 decreased conductance (to different extents). In both glycine and GABA receptors, the effect of mutations in different subunits does not sum linearly: mutations that had no detectable effect in isolation did enhance the effect of mutations carried by other subunits. As in the nicotinic receptor, charged residues in the extracellular vestibule of anionic Cys-loop channels influence elementary conductance. The size of this effect strongly depends on the direction of the ion flow and, unexpectedly, on the nature of the subunit that carries the residue.

The Cys-loop receptor super-family of neurotransmitter-gated ion channels mediates fast synaptic transmission throughout the human nervous system. These receptors exhibit widely varying pharmacologies, yet their structural characterization has relied heavily on their homology with the naturally abundant muscle-type Torpedo nicotinic acetylcholine receptor. Here we examine for the first time the structure of a human α4β2 neuronal nicotinic acetylcholine receptor. We show that human α4β2 nicotinic receptors adopt a secondary/tertiary fold similar to that of the Torpedo nicotinic receptor with a large proportion of both α-helix and β-sheet, but exhibit a substantially increased thermal stability. Both receptors bind agonist, but with different patterns of agonist recognition - particularly in the nature of the interactions between aromatic residues and the agonist quaternary amine functional group. By comparing α4β2 and Torpedo receptors, we begin to delineate their structural similarities and differences.