Cys-loop Ligand-gated Ion Channels Research Articles

Species specific RNA A-to-I editing of mosquito RDL modulates GABA potency and influences agonistic, potentiating and antagonistic actions of ivermectin

The insect GABA receptor, RDL, is the target of several classes of pesticides. The peptide sequences of RDL are generally highly conserved between diverse insects. However, RNA A-to-I editing can effectively alter amino acid residues of RDL in a species specific manner, which can affect the potency of GABA and possibly insecticides. We report here that RNA A-to-I editing alters the gene products of Rdl in three mosquito disease vectors, recoding five amino acid residues in RDL of Aedes aegypti and six residues in RDLs of Anopheles gambiae and Culex pipiens, which is the highest extent of editing in RDL observed to date. Analysis of An. gambiae Rdl cDNA sequences identified 24 editing isoforms demonstrating a considerable increase in gene product diversity. RNA editing influenced the potency of the neurotransmitter, GABA, on An. gambiae RDL editing isoforms expressed in Xenopus laevis oocytes, as demonstrated by EC50s ranging from 5 ± 1 to 246 ± 41 μM. Fipronil showed similar potency on different editing isoforms, with IC50s ranging from 0.18 ± 0.08 to 0.43 ± 0.09 μM. In contrast, editing of An. gambiae RDL affected the activating, potentiating and inhibiting actions of ivermectin. For example, ivermectin potentiated currents induced by GABA at the EC20 concentration in the unedited isoform but not in the fully edited variant. Editing of a residue in the first transmembrane domain or the cys-loop influenced this potentiation, highlighting residues involved in the allosteric mechanisms of cys-loop ligand-gated ion channels. Understanding the interactions of ivermectin with molecular targets may have relevance to mosquito control in areas where people are administered with ivermectin to treat parasitic diseases.

RDL mutations predict multiple insecticide resistance in Anopheles sinensis in Guangxi, China

BackgroundAnopheles sinensis is a major vector of malaria in China. The gamma-aminobutyric acid (GABA)-gated chloride channel, encoded by the RDL (Resistant to dieldrin) gene, is the important target for insecticides of widely varied structures. The use of various insecticides in agriculture and vector control has inevitably led to the development of insecticide resistance, which may reduce the control effectiveness. Therefore, it is important to investigate the presence and distribution frequency of the resistance related mutation(s) in An. sinensis RDL to predict resistance to both the withdrawn cyclodienes (e.g. dieldrin) and currently used insecticides, such as fipronil.MethodsTwo hundred and forty adults of An. sinensis collected from nine locations across Guangxi Zhuang Autonomous Region were used. Two fragments of An. sinensis RDL (AsRDL) gene, covering the putative insecticide resistance related sites, were sequenced respectively. The haplotypes of each individual were reconstructed by the PHASE2.1 software, and confirmed by clone sequencing. The phylogenetic tree was built using maximum-likelihood and Bayesian inference methods. Genealogical relations among different haplotypes were also analysed using Network 5.0.ResultsThe coding region of AsRDL gene was 1674 bp long, encoding a protein of 557 amino acids. AsRDL had 98.0% amino acid identity to that from Anopheles funestus, and shared common structural features of Cys-loop ligand-gated ion channels. Three resistance-related amino acid substitutions (A296S, V327I and T345S) were detected in all the nine populations of An. sinensis in Guangxi, with the 296S mutation being the most abundant (77–100%), followed by 345S (22–47%) and 327I (8–60%). 38 AsRDL haplotypes were identified from 240 individuals at frequencies ranging from 0.2 to 34.8%. Genealogical analysis suggested multiple origins of the 345S mutation in AsRDL.ConclusionsThe near fixation of the 296S mutation and the occurrence of the 327I and 345S mutations in addition to 296S, in all the nine tested An. sinensis populations in Guangxi, strongly indicate a risk of multiple insecticide resistance. The haplotype diversity plus genetic heterogeneities in the geographical distribution, and multiple origins of AsRDL alleles call for a location-customized strategy for monitoring and management of insecticide resistance.

Analyzing the Effects of Lipid Type on the α-Hemolysin Nanopore and 5HT3 Receptor Structure and Gating using Molecular Dynamics Simulations

Alpha-hemolysin (α-HL) is a transmembrane protein used to measure the size and charge of macromolecules. In this process, α-HL is inserted into a tethered lipid bilayer composed of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (PHPC) lipids. However, all previous molecular dynamics (MD) simulations attempting to model this system have used a more common lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), to represent the PHPC-tethered bilayer. PHPC possesses highly methylated hydrophobic chains which creates a more rigid structure. The result is that simulations using POPC lipids yield channel currents at least 20% less than experimental values. MD simulations conducted with both PHPC and POPC lipids for both the full and a reduced form of the protein containing just the β-barrel indicate that PHPC lipids could more accurately describe the experimentally observed parameters. The 5-hydroxytryptamine receptor (5HT3) is the principle receptor responsible for modulating a wide range of neurological and digestive functions. Here we aim to better understand the response of the 5HT3 receptor to 5HT (serotonin) in an environment that mimics the native lipid composition. This work supports the experimental design of a field effect transistor (FET) device we are developing to measure the presence of ligands such as neurotransmitters, neuropeptides and drug molecules. To this end, MD simulations were conducted on a system composed of the Cys-loop ligand-gated ion channel protein receptor 5HT3, a 7:7:6 ratio POPC/1-stearoyl-2-docosahexaenoyl-sn-glyerco-3-phosphocholine (SDPC)/cholesterol membrane, and 5HT to determine if ligand binding causes experimentally observed protein reassembly and channel opening. This work is unique in that these simulations possess a more realistic membrane composition and that they compare the conformation of the glycosylated protein to the non-glycosylated form providing structural details of the effects of glycosylation on the binding pocket and channel pore radius.

Disruption of a putative intersubunit electrostatic bond enhances agonist efficacy at the human α1 glycine receptor

Partial agonists have lower efficacies than compounds considered ‘full agonists’, eliciting submaximal responses even at saturating concentrations. Taurine is a partial agonist at the glycine receptor (GlyR), a member of the cys-loop ligand-gated ion channel superfamily. The molecular mechanisms responsible for agonism are not fully understood but evidence suggests that efficacy at these receptors is determined by conformational changes that occur early in the process of receptor activation. We previously identified a residue located near the human α1 glycine binding site (aspartate-97; D97) that, when mutated to arginine (D97R), results in GlyR channels opening spontaneously with a high open probability, mimicking the effects of saturating glycine concentrations on wildtype GlyR. This D97 residue is hypothesized to form an electrostatic interaction with arginine-119 on an adjacent subunit, stabilizing the channel in a shut state. Here we demonstrate that the disruption of this putative bond increases the efficacy of partial agonists including taurine, as well as two other β-amino acid partial agonists, β-aminobutyric acid (β-ABA) and β-aminoisobutyric acid (β-AIBA). Even the subtle charge-conserving mutation of D97 to glutamate (D97E) markedly affects partial agonist efficacy. Mutation to the neutral alanine residue in the D97A mutant mimics the effects seen with D97R, indicating that charge repulsion does not significantly affect these findings. Our findings suggest that the determination of efficacy following ligand binding to the glycine receptor may involve the disruption of an intersubunit electrostatic interaction occurring near the agonist binding site.

Instant Integrated Ultradeep Quantitative-structural Membrane Proteomics Discovered Post-translational Modification Signatures for Human Cys-loop Receptor Subunit Bias

Neurotransmitter ligand-gated ion channels (LGICs) are widespread and pivotal in brain functions. Unveiling their structure-function mechanisms is crucial to drive drug discovery, and demands robust proteomic quantitation of expression, post-translational modifications (PTMs) and dynamic structures. Yet unbiased digestion of these modified transmembrane proteins-at high efficiency and peptide reproducibility-poses the obstacle. Targeting both enzyme-substrate contacts and PTMs for peptide formation and detection, we devised flow-and-detergent-facilitated protease and de-PTM digestions for deep sequencing (FDD) method that combined omni-compatible detergent, tandem immobilized protease/PNGase columns, and Cys-selective reduction/alkylation, to achieve streamlined ultradeep peptide preparation within minutes not days, at high peptide reproducibility and low abundance-bias. FDD transformed enzyme-protein contacts into equal catalytic travel paths through enzyme-excessive columns regardless of protein abundance, removed products instantly preventing inhibition, tackled intricate structures via sequential multiple micro-digestions along the flow, and precisely controlled peptide formation by flow rate. Peptide-stage reactions reduced steric bias; low contamination deepened MS/MS scan; distinguishing disulfide from M oxidation and avoiding gain/loss artifacts unmasked protein-endogenous oxidation states. Using a recent interactome of 285-kDa human GABA type A receptor, this pilot study validated FDD platform's applicability to deep sequencing (up to 99% coverage), H/D-exchange and TMT-based structural mapping. FDD discovered novel subunit-specific PTM signatures, including unusual nontop-surface N-glycosylations, that may drive subunit biases in human Cys-loop LGIC assembly and pharmacology, by redefining subunit/ligand interfaces and connecting function domains.

Novel mechanism of modulation at a ligand-gated ion channel; action of 5-Cl-indole at the 5-HT3 A receptor.

The 5-HT3 receptor is a prototypical member of the Cys-loop ligand-gated ion channel (LGIC) superfamily and an established therapeutic target. In addition to activation via the orthosteric site, receptor function can be modulated by allosteric ligands. We have investigated the pharmacological action of Cl-indole upon the 5-HT3 A receptor and identified that this positive allosteric modulator possesses a novel mechanism of action for LGICs. The impact of Cl-indole upon the 5-HT3 receptor was assessed using single cell electrophysiological recordings and [3 H]-granisetron binding in HEK293 cells stably expressing the 5-HT3 receptor. Cl-indole failed to evoke 5-HT3 A receptor-mediated responses (up to 30μM) or display affinity for the [3 H]-granisetron binding site. However, in the presence of Cl-indole, termination of 5-HT application revealed tail currents mediated via the 5-HT3 A receptor that were independent of the preceding 5-HT concentration but were antagonized by the 5-HT3 receptor antagonist, ondansetron. These tail currents were absent in the 5-HT3 AB receptor. Furthermore, the presence of 5-HT revealed a concentration-dependent increase in the affinity of Cl-indole for the orthosteric binding site of the human 5-HT3 A receptor. Cl-indole acts as both an orthosteric agonist and an allosteric modulator, but the presence of an orthosteric agonist (e.g. 5-HT) is a prerequisite to reveal both actions. Precedent for ago-allosteric action is available, yet the essential additional presence of an orthosteric agonist is now reported for the first time. This widening of the pharmacological mechanisms to modulate LGICs may offer further therapeutic opportunities.

Molecular characterization and expression pattern of three GABA receptor-like subunits in the small brown planthopper Laodelphax striatellus (Hemiptera: Delphacidae)

Ionotropic γ-aminobutyric acid (GABA)-gated chloride channel receptors mediate rapid inhibitory neurotransmission in vertebrates and invertebrates. GABA receptors are well known to be the molecular targets of synthetic insecticides or parasiticides. Three GABA receptor-like subunits, LsLCCH3, LsGRD and LS8916, of the small brown planthopper, Laodelphax striatellus (Fallén), a major insect pest of crop systems in East Asia, had been identified and characterized in this study. All three genes were cloned using the reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). They shared common structural features with known Cys-loop ligand-gated ion channels (LGICs): the well-conserved dicysteine-loop structures, an extracellular N-terminal domain containing six distinct regions (loops A–F) that form the ligand binding sites and four transmembrane regions (TM1–4). Additionally, temporal and spatial transcriptional profiling analysis indicated that Lslcch3 was significantly higher than the other two genes. All of them were expressed at higher levels in fifth-instar nymph and adults than in eggs and from first- to fourth-instar nymph. They were predominantly expressed in the heads of 2-d old female adults. These findings enhanced our understanding of cys-loop LGIC functional characterization in Hemiptera and provided a useful basis for the development of improved insecticides that targeting this important agricultural pest.

α9α10 Acetylcholine receptors: structure and functions

The α9 and α10 nicotinic acetylcholine receptor (nAChR) subunits assemble as pentameric complexes with a central ligand-gated cation-selective pore. However, these subunits do not assemble with other nAChR subunits. The general topology of each subunit is similar to that of other Cys-loop ligand-gated ion channels. Although the sequence and genomic organization of the α9 and α10 subunits determined their classification as nicotinic subunits, α9α10 nAChRs exhibit peculiar pharmacological properties that are not common to other nicotinic or muscarinic receptors. α9α10 nAChRs are expressed in diverse tissues, including auditory sensory hair cells, afferent neurons in the vestibular sensory organs, neurons of the dorsal root ganglion, skin, and bronchial epithelia. Furthermore, it has become apparent that nAChRs containing α9 and α10 subunits play essential roles in various physiological processes. For example, α9 is required for the normal development of the efferent synapses in the inner ear, and after the onset of hearing α9α10 nAChRs serve as the unique source of extracellular calcium for Ca 2+ -activated potassium channels in mature auditory hair cells. The development of drugs that selectively target α9/α10 receptors promises to be of therapeutic benefit since these receptors have distinct physiologic functions. They hold promise as pharmacological targets to treat complex auditory and vestibular pathologies, neuropathic pain, and pathologies of the skin and bronchia.

Molecular characterization and expression profiles of nicotinic acetylcholine receptors in the rice striped stem borer, Chilo suppressalis (Lepidoptera: Crambidae).

Nicotinic acetylcholine receptors (nAChRs) are members of the cys-loop ligand-gated ion channel (cysLGIC) superfamily, mediating fast synaptic cholinergic transmission in the central nervous system in insects. Insect nAChRs are the molecular targets of economically important insecticides, such as neonicotinoids and spinosad. Identification and characterization of the nAChR gene family in the rice striped stem borer, Chilo suppressalis, could provide beneficial information about this important receptor gene family and contribute to the investigation of the molecular modes of insecticide action and resistance for current and future chemical control strategies. We searched our C. suppressalis transcriptome database using Bombyx mori nAChR sequences in local BLAST searches and obtained the putative nAChR subunit complementary DNAs (cDNAs) via reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends methods. Similar to B. mori, C. suppressalis possesses 12 nAChR subunits, including nine α-type and three β-type subunits. Quantitative RT-PCR analysis revealed the expression profiles of the nAChR subunits in various tissues, including the brain, subesophageal ganglion, thoracic ganglion, abdominal ganglion, hemocytes, fat body, foregut, midgut, hindgut and Malpighian tubules. Developmental expression analyses showed clear differential expression of nAChR subunits throughout the C. suppressalis life cycle. The identification of nAChR subunits in this study will provide a foundation for investigating the diverse roles played by nAChRs in C. suppressalis and for exploring specific target sites for chemicals that control agricultural pests while sparing beneficial species.

Dipicrylamine Modulates GABAρ1 Receptors through Interactions with Residues in the TM4 and Cys-Loop Domains.

Dipicrylamine (DPA) is a commonly used acceptor agent in Förster resonance energy transfer experiments that allows the study of high-frequency neuronal activity in the optical monitoring of voltage in living cells. However, DPA potently antagonizes GABAA receptors that contain α1 and β2 subunits by a mechanism which is not clearly understood. In this work, we aimed to determine whether DPA modulation is a general phenomenon of Cys-loop ligand-gated ion channels (LGICs), and whether this modulation depends on particular amino acid residues. For this, we studied the effects of DPA on human homomeric GABAρ1, α7 nicotinic, and 5-HT3A serotonin receptors expressed in Xenopus oocytes. Our results indicate that DPA is an allosteric modulator of GABAρ1 receptors with an IC50 of 1.6 µM, an enhancer of α7 nicotinic receptors at relatively high concentrations of DPA, and has little, if any, effect on 5-HT3A receptors. DPA antagonism of GABAρ1 was strongly enhanced by preincubation, was slightly voltage-dependent, and its washout was accelerated by bovine serum albumin. These results indicate that DPA modulation is not a general phenomenon of LGICs, and structural differences between receptors may account for disparities in DPA effects. In silico modeling of DPA docking to GABAρ1, α7 nicotinic, and 5-HT3A receptors suggests that a hydrophobic pocket within the Cys-loop and the M4 segment in GABAρ1, located at the extracellular/membrane interface, facilitates the interaction with DPA that leads to inhibition of the receptor. Functional examinations of mutant receptors support the involvement of the M4 segment in the allosteric modulation of GABAρ1 by DPA.

Characterizing Nanopore-Polymer Interactions and Cys-loop Protein Receptor Gating

The first objective is to better understand how the size and charge of an analyte affect the flow of ions through a transmembrane nanopore to support the development of a bio-nanosensor that can measure the mass and identify of complex macromolecules such as DNA. To this end, molecular dynamics (MD) simulations were conducted on a transmembrane nanopore system composed of the β-barrel region of the transmembrane protein α-hemolysin (α-HL), a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer membrane, and an aqueous solution of potassium chloride (KCl), in which the β-barrel forms a channel through the membrane. After the system was allowed to equilibrate, a single molecule of polyethylene glycol (PEG) was added into the channel and a transmembrane potential was applied to one side of the membrane. The ionic current through the channel was measured, and compared to the measured current without a PEG molecule present. This work concluded that a system composed of only the β-barrel region of α-HL (not including the head region) can accurately depict the flow of ions through the nanopore in response to an applied transmembrane potential with greater computational ease than with the entire protein.The second objective is to better understand the response of the 5HT3 receptor to the ligand 5HT (serotonin) in an environment that mimics the surface of a proposed microelectromechanical system (MEMS) device designed to measure the presence of ligands such as neurotransmitters, neuropeptides and drug molecules. To this end, MD simulations were conducted on a system composed of the Cys-loop ligand-gated ion channel protein receptor 5HT3, a 7:7:6 ratio POPC/1-stearoyl-2-docosahexaenoyl-sn-glyerco-3-phosphocholine (SDPC)/cholesterol membrane, and 5HT to determine if binding of the ligand causes protein reassembly and channel opening.

Transcriptome Analysis of the Central and Peripheral Nervous Systems of the Spider Cupiennius salei Reveals Multiple Putative Cys-Loop Ligand Gated Ion Channel Subunits and an Acetylcholine Binding Protein.

Invertebrates possess a diverse collection of pentameric Cys-loop ligand gated ion channel (LGIC) receptors whose molecular structures, evolution and relationships to mammalian counterparts have been intensely investigated in several clinically and agriculturally important species. These receptors are targets for a variety of control agents that may also harm beneficial species. However, little is known about Cys-loop receptors in spiders, which are important natural predators of insects. We assembled de novo transcriptomes from the central and peripheral nervous systems of the Central American wandering spider Cupiennius salei, a model species for neurophysiological, behavioral and developmental studies. We found 15 Cys-loop receptor subunits that are expected to form anion or cation permeable channels, plus a putative acetylcholine binding protein (AChBP) that has only previously been reported in molluscs and one annelid. We used phylogenetic and sequence analysis to compare the spider subunits to homologous receptors in other species and predicted the 3D structures of each protein using the I-Tasser server. The quality of homology models improved with increasing sequence identity to the available high-resolution templates. We found that C. salei has orthologous γ-aminobutyric acid (GABA), GluCl, pHCl, HisCl and nAChα LGIC subunits to other arthropods, but some subgroups are specific to arachnids, or only to spiders. C. salei sequences were phylogenetically closest to gene fragments from the social spider, Stegodyphus mimosarum, indicating high conservation within the Araneomorphae suborder of spiders. C. salei sequences had similar ligand binding and transmembrane regions to other invertebrate and vertebrate LGICs. They also had motifs associated with high sensitivity to insecticides and antiparasitic agents such as fipronil, dieldrin and ivermectin. Development of truly selective control agents for pest species will require information about the molecular structure and pharmacology of Cys-loop receptors in beneficial species.

Computational analysis of a cys-loop ligand gated ion channel from the green alga Chlamydomonas reinhardtii

Computational analysis of a cys-loop ligand gated ion channel from the green alga Chlamydomonas reinhardtii

Isolation, characterization, and tissue-specific expression of GABA A receptor α1 subunit gene of Carassius auratus gibelio after avermectin treatment.

Carassius auratus gibelio has been widely cultivated in fish farms in China, with avermectin (AVM) being used to prevent parasite infection. Recently, AVM was found to pass through the Carassius auratus gibelio blood-brain barrier (BBB). Although AVM acts mainly through a GABA receptor and specifically the α1 subunit gene, the most common isoform of the GABA A receptor, which is widely expressed in brain neurons and has been studied in other fish, Carassius auratus gibelio GABA A receptor α1 subunit gene cloning, and whether AVM passes through the BBB to induce Carassius auratus gibelio GABA A receptor α1 subunit gene expression have not been studied. The aim of this study was to clone, sequence, and phylogenetically analyze the GABA A receptor α1 subunit gene and to investigate the correlation of its expression with neurotoxicity in brain, liver, and kidney after AVM treatment by quantitative real-time reverse transcription polymerase chain reaction. The α1 subunit gene was 1550 bp in length with an open reading frame of 1380 bp encoding a predicted protein with 459 amino acid residues. The gene contained 128 bp of 5' terminal untranslated region (URT) and 72 bp of 3' terminal UTR. The α1 subunit structural features conformed to the Cys-loop ligand-gated ion channels family, which includes a signal peptide, an extracellular domain at the N-terminal, and four transmembrane domains. The established phylogenetic tree indicated that the α1 subunits of Carassius auratus gibelio and Danio rerio were the most closely related to each other. The α1 subunit was found to be highly expressed in brain and ovary, and the α1 mRNA transcription level increased significantly in brain. Moreover, the higher the concentration of AVM was, the higher the GABA A receptor expression was, indicating that AVM can induce significant neurotoxicity to Carassius auratus gibelio. Therefore, the α1 subunit mRNA expression was positively correlated with the neurotoxicity of AVM in Carassius auratus gibelio. Our findings suggest that AVM should be used carefully in Carassius auratus gibelio farming, and other alternate antibiotics with lower toxicity should be investigated with respect to toxicity via the induction of GABA A receptor expression for fish farming.

Putative nicotinic acetylcholine receptor subunits express differentially through the life cycle of codling moth, Cydia pomonella (Lepidoptera: Tortricidae).

Nicotinic acetylcholine receptors (nAChRs) are the targets of neonicotinoids and spinosads, two insecticides used in orchards to effectively control codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae). Orchardists in Washington State are concerned about the possibility of codling moth field populations developing resistance to these two insecticides. In an effort to help mitigate this issue, we initiated a project to identify and characterize codling moth nAChR subunits expressed in heads. This study had two main goals; (i) identify transcripts from a codling moth head transcriptome that encode for nAChR subunits, and (ii) determine nAChR subunit expression profiles in various life stages of codling moth. From a codling moth head transcriptome, 24 transcripts encoding for 12 putative nAChR subunit classes were identified and verified by PCR amplification, cloning, and sequence determination. Characterization of the deduced protein sequences encoded by putative nAChR transcripts revealed that they share the distinguishing features of the cys-loop ligand-gated ion channel superfamily with 9 α-type subunits and 3 β-type subunits identified. Phylogenetic analysis comparing these protein sequences to those of other insect nAChR subunits supports the identification of these proteins as nAChR subunits. Stage expression studies determined that there is clear differential expression of many of these subunits throughout the codling moth life cycle. The information from this study will be used in the future to monitor for potential target-site resistance mechanisms to neonicotinoids and spinosads in tolerant codling moth populations.

Cys-loop ligand-gated ion channel gene discovery in the Locusta migratoria manilensis through the neuron transcriptome

As an ideal model, Locusta migratoria manilensis (Meyen) has been widely used in the study of endocrinological and neurobiological processes. Here we created a large transcriptome of the locust neurons, which enriched ion channels whose potential for functional genetic experiments is currently limited. With high-throughput Illumina sequencing technology, we obtained more than 50 million raw reads, which were assembled into 61,056 unique sequences with average size of 737bp. Among the unigenes, a total 24,884 sequences had significant similarities with proteins in the five public databases (NR, SwissProt, GO, COG and KEGG) with a cut-off E-value of 10−5 using BLASTx. Moreover, the number of potential genes of the cys-loop ligand-gated ion channels (LGICs) was manually curated, including 39 putative nicotinic acetylcholine receptors (nAChRs), 6 putative γ-aminobutyric acid (GABA) gated anion channels, 21 putative glutamate-gated chloride channels (GluCls) and 1 histamine-gated chloride channels (HisCls). In addition, the full-length of 11 nAChRs subunits (9 alpha and 2 beta) were obtained by RACE technique that would be helpful to further studies on nAChR neurochemistry and pharmacological aspects. To our knowledge, this is the first study to characterize the locust neuron transcriptome, which will provide a useful resource especially for future studies on the neuro-function and behavior of the locust.

Phenylalanine in the pore of the Erwinia ligand-gated ion channel modulates picrotoxinin potency but not receptor function.

The Erwinia ligand-gated ion channel (ELIC) is a bacterial homologue of eukaryotic Cys-loop ligand-gated ion channels. This protein has the potential to be a useful model for Cys-loop receptors but is unusual in that it has an aromatic residue (Phe) facing into the pore, leading to some predictions that this protein is incapable of ion flux. Subsequent studies have shown this is not the case, so here we probe the role of this residue by examining the function of the ELIC in cases in which the Phe has been substituted with a range of alternative amino acids, expressed in Xenopus oocytes and functionally examined. Most of the mutations have little effect on the GABA EC50, but the potency of the weak pore-blocking antagonist picrotoxinin at F16′A-, F16′D-, F16′S-, and F16′T-containing receptors was increased to levels comparable with those of Cys-loop receptors, suggesting that this antagonist can enter the pore only when residue 16′ is small. T6′S has no effect on picrotoxinin potency when expressed alone but abolishes the increased potency when combined with F16′S, indicating that the inhibitor binds at position 6′, as in Cys-loop receptors, if it can enter the pore. Overall, the data support the proposal that the ELIC pore is a good model for Cys-loop receptor pores if the role of F16′ is taken into consideration.

Differential involvement of glutamate-gated chloride channel splice variants in the olfactory memory processes of the honeybee Apis mellifera

Glutamate-gated chloride channels (GluCl) belong to the cys-loop ligand-gated ion channel superfamily and their expression had been described in several invertebrate nervous systems. In the honeybee, a unique gene amel_glucl encodes two alternatively spliced subunits, Amel_GluCl A and Amel_GluCl B. The expression and differential localization of those variants in the honeybee brain had been previously reported. Here we characterized the involvement of each variant in olfactory learning and memory processes, using specific small-interfering RNA (siRNA) targeting each variant. Firstly, the efficacy of the two siRNAs to decrease their targets' expression was tested, both at mRNA and protein levels. The two proteins showed a decrease of their respective expression 24h after injection. Secondly, each siRNA was injected into the brain to test whether or not it affected olfactory memory by using a classical paradigm of conditioning the proboscis extension reflex (PER). Amel_GluCl A was found to be involved only in retrieval of 1-nonanol, whereas Amel_GluCl B was involved in the PER response to 2-hexanol used as a conditioned stimulus or as new odorant. Here for the first time, a differential behavioral involvement of two highly similar GluCl subunits has been characterized in an invertebrate species.

Natural compounds boldine and menthol are antagonists of human 5‐HT3 receptors: implications for treating gastrointestinal disorders

Impaired 5-HT3 receptor function is likely involved in the pathogenesis of functional gastrointestinal disorders (FGID) and 5-HT3 receptor antagonists are effective treatments for chemotherapy-induced nausea and vomiting (CINV) and irritable bowel syndrome (IBS). The monoterpene alcohol menthol and the aporphine alkaloid boldine combat symptoms of gastrointestinal diseases; both interact with other members of the Cys-loop ligand-gated ion channel family and may therefore also act on 5-HT3 receptors. The impact of boldine and menthol on human recombinant homomeric 5-HT3 A- and heteromeric 5-HT3 AB receptors in HEK293 cells was determined by radioligand binding, a luminescence-based Ca(2+) assay, and a membrane potential assay. 5-HT3 protein and mRNA expression was assessed in human colon tissue. Boldine and menthol inhibited the 5-HT-induced activation of 5-HT3 receptors in the low and middle micromolar range, respectively. Boldine was a competitive antagonist of both receptors being 6.5- to 10-fold more potent at 5-HT3 A- vs 5-HT3 AB receptors. Menthol non-competitively and stereoselectively inhibited both receptors: In contrast to (+)-menthol, (-)-menthol was significantly more potent toward 5-HT3 A- vs 5-HT3 AB receptors. We show co-expression of 5-HT3A and 5-HT3B subunits in the human gut epithelium, the lamina propria, the myenteric plexus, and the muscular cell layer. The demonstrated 5-HT3 inhibitory effects may be relevant for boldine's and menthol's alleviating properties on FGID and may encourage clinical studies with the compounds or the plant extracts for CINV and IBS treatment. The found receptor-discriminative properties make boldine and (-)-menthol to potentially useful tools for analyzing structural differences between these receptor subtypes.

The locust genome provides insight into swarm formation and long-distance flight

Locusts are one of the world’s most destructive agricultural pests and represent a useful model system in entomology. Here we present a draft 6.5 Gb genome sequence of Locusta migratoria, which is the largest animal genome sequenced so far. Our findings indicate that the large genome size of L. migratoria is likely to be because of transposable element proliferation combined with slow rates of loss for these elements. Methylome and transcriptome analyses reveal complex regulatory mechanisms involved in microtubule dynamic-mediated synapse plasticity during phase change. We find significant expansion of gene families associated with energy consumption and detoxification, consistent with long-distance flight capacity and phytophagy. We report hundreds of potential insecticide target genes, including cys-loop ligand-gated ion channels, G-protein-coupled receptors and lethal genes. The L. migratoria genome sequence offers new insights into the biology and sustainable management of this pest species, and will promote its wide use as a model system.