Insect nicotinic acetylcholine receptor gene families: from genetic model organism to vector,pest and beneficial species

Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic transmission in the insect nervous system and are targets of a major group of insecticides, the neonicotinoids. Analyses of genome sequences have shown that nAChR gene families remain compact in diverse insect species, when compared to their mammalian counterparts. Thus, Drosophila melanogaster and Anopheles gambiae each possess 10 nAChR genes while Apis mellifera has 11. Although these are among the smallest nAChR gene families known, receptor diversity can be considerably increased by alternative splicing and mRNA A-to-I editing, thereby generating species-specific subunit isoforms. In addition, each insect possesses at least one highly divergent nAChR subunit. Species-specific subunit diversification may offer promising targets for future rational design of insecticides that act on particular pests while sparing beneficial insects. Electrophysiological studies on cultured Drosophila cholinergic neurons show partial agonist actions of the neonicotinoid imidacloprid and super-agonist actions of another neonicotinoid, clothianidin, on native nAChRs. Recombinant hybrid heteromeric nAChRs comprising Drosophila Dα2 and a vertebrate β2 subunit have been instructive in mimicking such actions of imidacloprid and clothianidin. Unitary conductance measurements on native nAChRs indicate that more frequent openings of the largest conductance state may offer an explanation for the superagonist actions of clothianidin.     

Dbeta3, an atypical nicotinic acetylcholine receptor subunit from Drosophila : molecular cloning, heterologous expression and coassembly

Insect nicotinic acetylcholine receptors (nAChRs) play a central role in mediating neuronal synaptic transmission and are the target sites for the increasingly important group of neonicotinoid insecticides. Six nicotinic acetylcholine receptor (nAChR) subunits (four alpha-type and two beta-type) have been cloned previously from the model insect species Drosophila melanogaster. Despite extensive efforts, it has not been possible to generate functional recombinant nAChRs by heterologous expression of any combination of these six subunits. It has, however, been possible to express functional hybrid receptors when Drosophila alpha subunits are co-expressed with vertebrate beta subunits. This has led to the assumption that successful heterologous expression might require an, as yet, uncloned beta-type insect subunit. Examination of the recently completed Drosophila genomic sequence data has identified a novel putative nAChR beta-type subunit. Here we report the molecular cloning, heterologous expression and characterization of this putative Drosophila nAChR subunit (Dbeta3). Phylogenetic comparisons with other ligand-gated ion channel subunit sequences support its classification as a nAChR subunit but show it to be a distantly related member of this neurotransmitter receptor subunit family. Evidence that the Dbeta3 subunit is able to coassemble with other Drosophila nAChR subunits and contribute to recombinant nAChRs has been obtained by both radioligand binding and coimmunoprecipitation studies in transfected Drosophila S2 cells.     

The nicotinic acetylcholine receptor gene family of the pufferfish,Fugu rubripes

Nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission at nerve-muscle junctions and in the brain. However, the complete gene family of nAChRs has not so far been reported for any vertebrate organism. We have identified the complete nAChR gene family from the reference genome of the pufferfish, Fugu rubripes. It consists of 16 alpha and 12 non-alpha candidate subunits, making it the largest vertebrate nAChR gene family known to date. The gene family includes an unusual set of muscle-like nAChR subunits comprising two alpha1s, two beta1s, one delta, one epsilon, and one gamma. One of the beta1 subunits possesses an aspartate residue and N-glycosylation sites hitherto shown to be necessary for delta-subunit function. Potential Fugu orthologs of neuronal nAChR subunits alpha2-4, alpha6, and beta2-4 have been identified. Interestingly, the Fugu alpha5 counterpart appears to be a non-alpha subunit. Fugu possesses an expanded set of alpha7-9-like subunits and no alpha10 ortholog has been found. Two new candidate beta subtypes, designated beta5 and beta6, may represent subunits yet to be found in the human genome. The Fugu nAChR gene structures are considerably more diverse than those of higher vertebrates, with evidence of "intron gain" in many cases. We show, using RT-PCR, that the Fugu nAChR subunits are expressed in a variety of tissues.     

Functional genomics of the nicotinic acetylcholine receptor gene family of the nematode, Caenorhabditis elegans

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that bring about a diversity of fast synaptic actions. Analysis of the Caenorhabditis elegans genome has revealed one of the most-extensive and diverse nAChR gene families known, consisting of at least 27 subunits. Striking variation with possible functional implications has been observed in normally conserved motifs at the acetylcholine-binding site and in the channel-lining region. Some nAChR subunits are particular to neurons whilst others are present in both neurons and muscles. The localization of subunits in non-synaptic regions suggests novel roles for nAChRs. Genetic and heterologous expression studies have identified a subset of nAChR subunits that are important drug targets while the study of mutants has identified genes functionally-linked to nAChRs. Future studies using C. elegans offer the prospect of increasing our understanding of the functional diversity of a complex nAChR gene family as well as addressing the role of nAChRs and associated proteins in human disorders.     

Temperature-Sensitive Expression of Drosophila Neuronal Nicotinic Acetylcholine Receptors

Abstract: Heterologous expression of cloned Drosophila nicotinic acetylcholine receptor (nAChR) subunits indicates that these proteins misfold when expressed in mammalian cell lines at 37°C. This misfolding can, however, be overcome either by growing transfected mammalian cells at lower temperatures or by the expression of Drosophila nAChR subunits in a Drosophila cell line. Whereas the Drosophila nAChR β subunit (SBD) cDNA, reported previously, lacked part of the SBD coding sequence, here we report the construction and expression of a full-length SBD cDNA. We have examined whether problems in expressing functional Drosophila nAChRs in either Xenopus oocytes or mammalian cell lines can be attributed to an inability of these expression systems to assemble correctly Drosophila nAChRs. Despite expression in what might be considered a more native cellular environment, we have been unable to detect functional nAChRs in a Drosophila cell line unless Drosophila nAChR subunit cDNAs are coexpressed with vertebrate nAChR subunits. Our results indicate that the folding of Drosophila nAChR subunits is temperature-sensitive and strongly suggest that the inability of these Drosophila nAChR subunits to generate functional channels in the absence of vertebrate subunits is due to a requirement for coassembly with as yet unidentified Drosophila nAChR subunits.     

Molecular Characterization and Imidacloprid Selectivity of Nicotinic Acetylcholine Receptor Subunits from the Peach-Potato Aphid Myzus persicae

The recent introduction of the chloronicotinyl insecticide imidacloprid, targeting insect nicotinic acetylcholine receptors (nAChRs), emphasises the importance of a detailed molecular characterisation of these receptors. We are investigating the molecular diversity of insect nAChR subunit genes in an important agricultural pest, the peach-potato aphid Myzus persicae. Two M. persicae alpha-subunit cDNAs, Mp alpha1 and Mp alpha2, have been cloned previously. Here we report the isolation of three novel alpha-subunit genes (Mp alpha3-5) with overall amino acid sequence identities between 43 and 76% to characterised insect nAChR subunits. Alignment of their amino acid sequences with other invertebrate and vertebrate nAChR subunits suggests that the insect alpha subunits evolved in parallel to the vertebrate neuronal nAChRs and that the insect non-alpha subunits are clearly different from vertebrate neuronal beta and muscle non-alpha subunits. The discovery of novel subtypes in M. persicae is a further indicator of the complexity of the insect nAChR gene family. Heterologous co-expression of M. persicae nAChR alpha-subunit cDNAs with the rat beta2 in Drosophila S2 cells resulted in high-affinity binding of nicotinic radioligands. The affinity of recombinant nAChRs for [3H]imidacloprid was influenced strongly by the alpha subtype. This is the first demonstration that imidacloprid selectively acts on Mp alpha2 and Mp alpha3 subunits, but not Mp alpha1, in M. persicae.     

Contributions from Caenorhabditis elegans functional genetics to antiparasitic drug target identification and validation: nicotinic acetylcholine receptors, a case study

Following the complete sequencing of the genome of the free-living nematode, Caenorhabditis elegans, in 1998, rapid advances have been made in assigning functions to many genes. Forward and reverse genetics have been used to identify novel components of synaptic transmission as well as determine the key components of antiparasitic drug targets. The nicotinic acetylcholine receptors (nAChRs) are prototypical ligand-gated ion channels. The functions of these transmembrane proteins and the roles of the different members of their extensive subunit families are increasingly well characterised. The simple nervous system of C. elegans possesses one of the largest nicotinic acetylcholine receptor gene families known for any organism and a combination of genetic, microarray, physiological and reporter gene expression studies have added greatly to our understanding of the components of nematode muscle and neuronal nAChR subtypes. Chemistry-to-gene screens have identified five subunits that are components of nAChRs sensitive to the antiparasitic drug, levamisole. A novel, validated target acting downstream of the levamisole-sensitive nAChR has also been identified in such screens. Physiology and molecular biology studies on nAChRs of parasitic nematodes have also identified levamisole-sensitive and insensitive subtypes and further subdivisions are under investigation.     

Assembly and trafficking of nicotinic acetylcholine receptors (Review)

Nicotinic acetylcholine receptors (nAChRs) are members of an extensive super-family of neurotransmitter-gated ion channels. In humans, nAChRs are expressed within the nervous system and at the neuromuscular junction and are important targets for pharmaceutical drug discovery. They are also the site of action for neuroactive pesticides in insects and other invertebrates. Nicotinic receptors are complex pentameric transmembrane proteins which are assembled from a large family of subunits; seventeen nAChR subunits (α1-α10, β1-β4, γ, δ and ε) have been identified in vertebrate species. This review will discuss nAChR subunit diversity and factors influencing receptor assembly and trafficking.     

Assembly and trafficking of nicotinic acetylcholine receptors (Review)

Nicotinic acetylcholine receptors (nAChRs) are members of an extensive super-family of neurotransmitter-gated ion channels. In humans, nAChRs are expressed within the nervous system and at the neuromuscular junction and are important targets for pharmaceutical drug discovery. They are also the site of action for neuroactive pesticides in insects and other invertebrates. Nicotinic receptors are complex pentameric transmembrane proteins which are assembled from a large family of subunits; seventeen nAChR subunits (alpha1-alpha10, beta1-beta4, gamma, delta and epsilon) have been identified in vertebrate species. This review will discuss nAChR subunit diversity and factors influencing receptor assembly and trafficking.     

Alternative splicing of the Anopheles gambiae nicotinic acetylcholine receptor, Agamαβ9, generates both alpha and beta subunits

Nicotinic acetylcholine receptors (nAChRs) are the members of the cys-loop ligand-gated ion channel superfamily and are formed by five subunits arranged around a central ion channel. Each subunit is encoded by a separate gene and is classified as either α or non-α depending on the presence or absence, respectively, of two adjacent cysteine residues which are important for acetylcholine binding. Here, we report for the first time a single nAChR gene encoding both α and non-α subunits. Specifically, alternative splicing of the Anopheles gambiae nAChR subunit, previously called Agamα9 and renamed here Agamαβ9, generates two variants, one possessing the two cysteines (denoted Agamαβ9^α) and the other lacking the cysteine doublet (Agamαβ9^β). Attempts to heterologously express functional nAChRs consisting of the Agamαβ9 splice variants in Xenopus laevis oocytes were unsuccessful. Our findings further characterise a potential target to control the malaria mosquito as well as provide insights into the diversification of nAChRs.     

Nicotinic acetylcholine receptors: from structure to brain function

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels and can be divided into two groups: muscle receptors, which are found at the skeletal neuromuscular junction where they mediate neuromuscular transmission, and neuronal receptors, which are found throughout the peripheral and central nervous system where they are involved in fast synaptic transmission. nAChRs are pentameric structures that are made up of combinations of individual subunits. Twelve neuronal nAChR subunits have been described, α2–α10 and β2–β4; these are differentially expressed throughout the nervous system and combine to form nAChRs with a wide range of physiological and pharmacological profiles. The nAChR has been proposed as a model of an allosteric protein in which effects arising from the binding of a ligand to a site on the protein can lead to changes in another part of the molecule. A great deal is known about the structure of the pentameric receptor. The extracellular domain contains binding sites for numerous ligands, which alter receptor behavior through allosteric mechanisms. Functional studies have revealed that nAChRs contribute to the control of resting membrane potential, modulation of synaptic transmission and mediation of fast excitatory transmission. To date, ten genes have been identified in the human genome coding for the nAChRs. nAChRs have been demonstrated to be involved in cognitive processes such as learning and memory and control of movement in normal subjects. Recent data from knockout animals has extended the understanding of nAChR function. Dysfunction of nAChR has been linked to a number of human diseases such as schizophrenia, Alzheimer's and Parkinson's diseases. nAChRs also play a significant role in nicotine addiction, which is a major public health concern. A genetically transmissible epilepsy, ADNFLE, has been associated with specific mutations in the gene coding for the α4 or β2 subunits, which leads to altered receptor properties. Electronic Publication     

The structures of the human neuronal nicotinic acetylcholine receptor β2- and α3-subunit genes (CHRNB2 and CHRNA3)

The α4-subunit gene (CHRNA4) of the neuronal nicotinic acetylcholine receptor (nAChR) subunit family has recently been identified in two families as the gene responsible for autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), a rare monogenic idiopathic epilepsy. As a result of this finding, other subunits of the neuronal nAChR gene family are being considered as candidate genes for ADNFLE in families not linked to CHRNA4 and for other idiopathic epilepsies. α4-subunitsoften assemble together with β2-subunits (gene symbol CHRNB2) to build heteromeric nAChRs. The gene encoding another abundant AChR subunit, the α3-subunit gene (CHRNA3), is present with those encoding two other subunits, CHRNB4 and CHRNA5, in a gene cluster whose functional role is still unclear. Here we provide the information on the genomic structures of both the CHRNB2 and the CHRNA3 genes that is necessary for comprehensive mutational analyses, and we refine the genomic assignment of CHRNB2 on chromosome 1. Received: 5 August 1998 / Accepted: 13 October 1998     

A motif present in the main cytoplasmic loop of nicotinic acetylcholine receptors and catalases

A motif containing five conserved amino acids (RXPXTH(X)14P) was detected in 111 proteins, including 82 nicotinic acetylcholine receptor (nAChR) subunits and 20 catalases. To explore possible functional roles of this motif in nAChRs two approaches were used: first, the motif sequences in nAChR subunits and catalases were analysed and compared; and, second, deletions in the rat alpha2 and beta4 nAChR subunits expressed in Xenopus oocytes were analysed. Compared to the three-dimensional structure of bovine hepatic catalase, structural coincidences were found in the motif of catalases and nAChRs. On the other hand, partial deletions of the motif in the alpha2 or beta4 subunits and injection of the mutants into oocytes was followed by a very weak expression of functional nAChRs; oocytes injected with alpha2 and beta4 subunits in which the entire motif had been deleted failed to elicit any acetylcholine currents. The results suggest that the motif may play a role in the activation of nAChRs.     

Neuronal nicotinic acetylcholine receptors of Drosophila melanogaster: the alpha-subunit dalpha3 and the beta-type subunit ARD co-assemble within the same receptor complex

Dalpha3 is a functional alpha-subunit of Drosophila melanogaster nicotinic acetylcholine receptors (nAChRs). Here, we produced Dalpha3-specific antibodies to study which other nAChR subunits can co-assemble with Dalpha3 in receptor complexes of the Drosophila nervous system. Immunohistochemical studies revealed that Dalpha3 is co-distributed with the beta-subunit ARD in synaptic neuropil regions of the optic lobe. Both subunits can be co-purified by alpha-bungarotoxin affinity chromatography. Dalpha3 antibodies co-immunoprecipitate Dalpha3 and ARD proteins and, vice versa, anti-ARD antibodies co-precipitate ARD and Dalpha3. These data demonstrate that one type of fly nAChRs includes these two subunits as integral components.