Cloning and Functional Characterisation of Two Novel Nicotinic Acetylcholine Receptor α Subunits from the Insect Pest Myzus persicae

Abstract: Nicotinic acetylcholine receptors play a major role in excitatory neurotransmission in insect CNSs and constitute an important target for insecticides. Here, we report the isolation and functional characterisation of two cDNAs encoding nicotinic acetylcholine receptor α subunits from a major insect pest, the peach-potato aphid Myzus persicae . These two subunits, termed Mpα1 and Mpα2, are respective structural homologues of the Drosophila Dα2/ Schistocerca gregaria αL1 α-subunit pair and the Drosophila ALS α subunit. Xenopus oocyte expression confirmed that each Myzus subunit can form functional acetylcholine- or nicotine-gated channels. However, some electrophysiological and pharmacological properties of the Myzus subunits were distinct from those encoded by the corresponding Drosophila subunits. Coexpression of the Myzus subunits with the chick β2 subunit revealed other differences from the Drosophila system, as only very limited potentiation of agonist-induced currents was observed with Mpα2 and none with Mpα1. Available data therefore indicate that structurally homologous insect nicotinic acetylcholine receptor α subunits from different species can exhibit distinctive physiological and pharmacological characteristics.     

Cloning of a Putative Neuronal Nicotinic Acetylcholine Receptor Subunit

A cDNA clone was isolated from a rat superior cervical ganglion cDNA library with an oligonucleotide that hybridized to muscle-like nicotinic acetylcholine receptor (nAChR) subunit cDNA. The deduced amino acid sequence possesses characteristics expected of a nAChR subunit that does not bind acetylcholine, in addition to distinctive features such as unique cysteine residues and N-linked glycosylation sites.     

Host Cell-Specific Folding and Assembly of the Neuronal Nicotinic Acetylcholine Receptor α7 Subunit

Abstract: Expression of the cloned neuronal nicotinic acetylcholine receptor (nAChR) α7 subunit in several cultured mammalian cell lines has revealed that the folding, assembly, and subcellular localization of this protein are critically dependent upon the nature of the host cell. In all cell lines that were examined, high levels of α7 protein were detected by metabolic labelling and immunoprecipitation after transfection with the cloned α7 cDNA. In contrast, elevated levels of α-bungarotoxin binding could be detected in only two of the nine cell lines. Both of these "α7-permissive" cell lines [rat phaeochromocytoma (PC12) and human neuroblastoma (SH-SY5Y)] express an endogenous α7 subunit. However, by expression of an epitope-tagged α7 subunit, it has been possible to show that the elevation in surface α-bungarotoxin binding in these two cell lines is due to expression of cDNA-encoded α7. The cell-specific misfolding of the neuronal nAChR α7 subunit is a phenomenon that is not shared by either the hetero-oligomeric muscle nAChR or the homo-oligomeric serotonin receptor 5-HT₃ subunit. Our data also indicate that the cell-specific misfolding cannot be explained by a requirement for the coassembly with other known nAChR subunits and cannot be alleviated by treatments that have been reported to affect the assembly efficiency of other neurotransmitter-gated ion channels.     

Dα3, a New Functional α Subunit of Nicotinic Acetylcholine Receptors from Drosophila

Abstract: Nicotinic acetylcholine (ACh) receptors (nAChRs) are important excitatory neurotransmitter receptors in the insect CNS. We have isolated and characterized the gene and the cDNA of a new nAChR subunit from Drosophila . The predicted mature nAChR protein consists of 773 amino acid residues and has the structural features of an ACh-binding α subunit. It was therefore named Dα3, for D rosophila α -subunit 3 . The dα3 gene maps to the X chromosome at position 7E. The properties of the Dα3 protein were assessed by expression in Xenopus oocytes. Dα3 did not form functional receptors on its own or in combination with any Drosophila β-type nAChR subunit. Nondesensitizing ACh-evoked inward currents were observed when Dα3 was coexpressed with the chick β2 subunit. Half-maximal responses were at ∼0.15 µ M ACh with a Hill coefficient of ∼1.5. The snake venom component α-bungarotoxin (100 n M ) efficiently but reversibly blocked Dα3/β2 receptors, suggesting that Dα3 may be a component of one of the previously described two classes of toxin binding sites in the Drosophila CNS.     

Mapping of Exposed Surfaces of the Nicotinic Acetylcholine Receptor by Identification of Iodinated Tyrosine Residues

Here we report on the use of iodination of the membrane-bound nicotinic acetylcholine receptor (nAChR) from Torpedo californica electric tissue in order to define surface-exposed portions of the receptor molecule. Membrane-bound nAChR was ¹²⁵I-iodinated using the oxidation agent Iodo-Gen. The iodinated subunits were separated by preparative gel electrophoresis, desalted, and cleaved with trypsin. The resulting peptides were separated by reverse-phase HPLC and the radioactive peptides were identified by mass spectrometry and protein sequencing. For the -subunit, we identified five iodinated peptides containing the tyrosine residues Tyr17, Tyr74, Tyr365, Tyr372, and Tyr428. The surface exposition of these amino acids is in agreement with the four-transmembrane-segment model (4TM model) of the nAChR, but the assignment to the intra- or extracellular surface is doubtful. According to this model, the N-terminal portion of the receptor subunits including the iodinated residues Tyr17 and Tyr74 is extracellular and Tyr372 as a site of tyrosine phosphorylation is located on the cytoplasmic side. But since this latter residue is among the first to be iodinated using an immobilized iodination agent, its true position with respect to the membrane bilayer is not clear.     

Affinity to the Nicotinic Acetylcholine Receptor and Insecticidal Activity of Chiral Imidacloprid Derivatives with a Methylated Imidazolidine Ring

Four imidacloprid derivatives with an asymmetrically methylated imidazolidine ring were synthesized. Their affinity to the nicotinic acetylcholine receptor of housefly Musca domestica and insecticidal activity against the housefly were measured. The compound with a 5R-methylated imidazolidine ring demonstrated intrinsic activity comparable to that of the unsubstituted compound. Most of the compounds were synergized by oxygenase inhibitors.     

Production of Polyclonal Antisera that Recognize and Distinguish Between the Extracellular Domains of Neuronal Nicotinic Acetylcholine Receptor Subunits

Abstract: Ligand-gated ion channels are oligomeric transmembrane proteins that usually contain more than one kind of monomer. The variety of monomers available to participate in oligomer formation and the apparent latitude in acceptable monomer combinations allows considerable diversity. Mechanisms for identifying the monomers comprising specific receptors are needed. We have generated affinity-purified polyclonal antisera that recognize the extracellular domain of nine neuronal nicotinic acetylcholine receptor (nAChR) subunits and distinguish between them. We prepared these antisera by immunizing rabbits with bacterially expressed recombinant protein representing the N-terminal extracellular domain of each neuronal nAChR subunit followed by affinity purification of antibodies against synthetic peptides corresponding to residues 68–81 of the α1 subunit. We demonstrate subunit specificity of each affinity-purified antisera by western blots of the bacterially expressed protein and immunoblot against peptide. We further used these antibodies to demonstrate expression of neuronal nAChR subunits on the surface of transiently transfected simian kidney (COS-7) cells.     

Effects of Substance P on the Binding of Agonists to the Nicotinic Acetylcholine Receptor of Torpedo Electroplaque

Abstract: Abstract: The effect of the neuropeptide substance P on the binding of the cholinergic ligands to the nicotinic acetylcholine receptor of Torpedo electroplaque membranes was examined at a physiological concentration of NaCl (150 m M ). Substance P had no effect on the initial rate of ¹²⁵I-α-bungarotoxin binding at concentrations of <100 μ M . The peptide did not bind to the high-affinity local anesthetic site but allosterically modulated [³H]phencyclidine binding, positively in the absence of agonist and negatively in the presence of agonist. Substance P increased the apparent affinity of the cholinergic agonists carbamylcholine and acetylcholine at equilibrium. The effect of substance P on the equilibrium binding of [³H]acetylcholine was examined directly, and the peptide appeared to increase the affinity of the binding of the second molecule of agonist, with no effect on the binding of the first. This indicates that substance P can affect the cooperative interactions between agonist binding sites. Substance P appeared to increase the rate of carbamylcholine-induced desensitization; however, the data are also consistent with an allosteric mechanism that does not involve the desensitized state. To attempt to differentiate between these mechanisms, the rates of recovery were determined after exposure to peptide and/or agonist. The kinetics of recovery are consistent with stabilization of the desensitized state by substance P if the peptide remains bound long enough to allow rapid recovery to the low-affinity state. However, an allosteric modulation of agonist binding that does not involve the desensitized state cannot be ruled out.     

Target-Dependent Regulation of Retinal Nicotinic Acetylcholine Receptor and Tubulin RNAs During Optic Nerve Regeneration in Goldfish

A fundamental issue in central nervous system development regards the effect of target tissue on the differentiation of innervating neurons. We address this issue by characterizing the role the retinal ganglion cell target, i.e., the optic tectum, plays in regulating expression of tubulin and nicotinic acetylcholine receptor genes in regenerating retinal ganglion cells. Tubulins are involved in axonal growth, whereas nicotinic acetylcholine receptors mediate communication across synapses. Retinal ganglion cell axons were induced to regenerate by crushing the optic nerve. Following crush, there was a rapid increase in alpha-tubulin RNAs (3 days), which preceded the increase in nicotinic acetylcholine receptor RNAs (10-15 days). Both classes of RNAs approached control levels by the time retinotectal synapses and functional recovery were restored (4-6 weeks). If the optic nerve was repeatedly crushed or its target ablated, tubulin RNAs remained elevated, and the increase in receptor RNAs that would otherwise be seen 2 weeks after a single nerve crush did not occur. The interaction of retinal ganglion cell axons with their targets in the optic tectum appears, then, to exert a suppressive effect on the RNA encoding a cytoskeletal protein, tubulin, and an inductive effect on RNAs encoding nicotinic acetylcholine receptors involved in synaptic communication.     

Tetrapeptides on N- and C-Terminal Regions of Mastoparan Inhibit Catecholamine Release from Chromaffin Cells by Blocking Nicotinic Acetylcholine Receptor

Mastoparan (MP), a tetradecapeptide in wasp venom, has been reported to evoke catecholamine release, but also reported to inhibit secretory response upon nicotinic stimulation in adrenal chromaffin cells. To elucidate the inhibitory mechanism of MP, we examined the effect of two MP fragments (INLK-NH2 and KKIL-NH2) on catecholamine release in bovine adrenal chromaffin cells. These MP fragments inhibited catecholamine release induced by nicotinic stimulation in a noncompetitive manner. These fragments did not affect catecholamine release evoked by high [K+] or by other secretagogues, neither caused catecholamine release by themselves. Replacement by hydrophobic and basic amino acids of the MP fragments enhanced the inhibitory effects on ACh-evoked catecholamine release. Among 23 analogs of the MP fragments, (Nle)3-R-NH2 showed the most potent inhibition with IC50 = 541 microM. These results suggested that the MP fragments selectively inhibit the secretory response to nicotinic stimulation by attacking nAChR on the site(s) made up of hydrophobic and acidic amino acids but other than ACh-binding sites. This mechanism may explain the inhibitory action of MP on nicotine-evoked catecholamine release.     

Neonicotinoid Binding,Toxicity and Expression of Nicotinic Acetylcholine Receptor Subunits in the Aphid Acyrthosiphon pisum

Neonicotinoid insecticides act on nicotinic acetylcholine receptor and are particularly effective against sucking pests. They are widely used in crops protection to fight against aphids, which cause severe damage. In the present study we evaluated the susceptibility of the pea aphid Acyrthosiphon pisum to the commonly used neonicotinoid insecticides imidacloprid (IMI), thiamethoxam (TMX) and clothianidin (CLT). Binding studies on aphid membrane preparations revealed the existence of high and low-affinity binding sites for [³H]-IMI (Kd of 0.16±0.04 nM and 41.7±5.9 nM) and for the nicotinic antagonist [¹²⁵I]-α-bungarotoxin (Kd of 0.008±0.002 nM and 1.135±0.213 nM). Competitive binding experiments demonstrated that TMX displayed a higher affinity than IMI for [¹²⁵I]-α-bungarotoxin binding sites while CLT affinity was similar for both [¹²⁵I]-α-bungarotoxin and [³H]-IMI binding sites. Interestingly, toxicological studies revealed that at 48 h, IMI (LC₅₀ = 0.038 µg/ml) and TMX (LC₅₀ = 0.034 µg/ml) were more toxic than CLT (LC₅₀ = 0.118 µg/ml). The effect of TMX could be associated to its metabolite CLT as demonstrated by HPLC/MS analysis. In addition, we found that aphid larvae treated either with IMI, TMX or CLT showed a strong variation of nAChR subunit expression. Using semi-quantitative PCR experiments, we detected for all insecticides an increase of Apisumα10 and Apisumβ1 expressions levels, whereas Apisumβ2 expression decreased. Moreover, some other receptor subunits seemed to be differently regulated according to the insecticide used. Finally, we also demonstrated that nAChR subunit expression differed during pea aphid development. Altogether these results highlight species specificity that should be taken into account in pest management strategies.     

Use-Dependent Suppression of the Nicotinic Acetylcholine Receptor Response by the Proadrenomedullin N-Terminal 20-Amino Acid Peptide in Rat Locus Coeruleus Neurons

Abstract: The effects of the proadrenomedullin N-terminal 20-amino acid peptide (PAMP) on the nicotinic acetylcholine (ACh) receptor (nAChR)-mediated inward current were investigated in neurons acutely dissociated from the rat locus coeruleus using whole-cell recording under voltage clamp. Nicotine and cytidine mimicked the ACh response, whereas the maximal response to dimethyl-phenylpiperazinium was lower in amplitude compared with that to ACh. Nicotine-induced current ( I _nic) was suppressed more effectively by mecamylamine than by hexamethonium. In addition, neither atropine nor α-bungarotoxin affected the I _nic. PAMP reversibly and noncompetitively suppressed the peak amplitude of 10⁻⁴ M I _nic. PAMP concentrations for the threshold, half-maximal inhibition, and maximal inhibition of 10⁻⁴ M I _nic were 10⁻⁸, 2.6 × 10⁻⁷, and 10⁻⁵ M , respectively. The peak amplitudes of 10⁻⁴ M I _nic elicited at 2-min intervals showed a gradual decline in the presence of 10⁻⁷ M PAMP. This decline in the I _nic was independent of the period of PAMP pretreatment. The suppression of I _nic by PAMP did not show any voltage dependency at a holding potential ( V _H) of <0 mV, although the inhibitory effect was masked by the marked inward rectification of I _nic at a V _H of 0 mV. These results suggest that PAMP could thus be a unique endogenous peptide that antagonizes the nAChR in the CNS.     

Identification of Two Corticotropin-Releasing Factor Receptors from Xenopus laevis with High Ligand Selectivity: Unusual Pharmacology of the Type 1 Receptor

Abstract: Two cDNA clones encoding distinct members of the corticotropin-releasing factor (CRF) receptor family have been isolated from Xenopus laevis with PCR-based approaches. The first full-length cDNA amplified from Xenopus brain encoded a 415-amino acid protein with ∼80% identity to mammalian CRF receptor type 1 (CRF-R1). The second full-length cDNA isolated from Xenopus brain and heart encoded a 413-amino acid protein with ∼81% identity to the α-variant of mammalian CRF receptor, type 2 (CRF-R2). No evidence could be obtained that the β-variant of CRF-R2 existed in Xenopus laevis . Binding studies using human embryonic kidney 293 (HEK 293) cells stably transfected with xenopus CRF-R2 showed that the CRF analogues urotensin I, urocortin, and sauvagine were bound with higher affinities than human/rat CRF, xenopus CRF, and ovine CRF. In contrast to human CRF-R1, xenopus CRF-R1 (xCRF-R1) was very selective for different CRF ligands. Urotensin I, urocortin, human/rat CRF, and xenopus CRF were bound with significantly (10–22-fold) higher affinities than ovine CRF ( K _D = 31.7 n M ) and sauvagine ( K _D = 51.4 n M ). In agreement with these binding data, EC₅₀ values of 39.7 and 1.1 n M were found for sauvagine and for human/rat CRF or xenopus CRF, respectively, when the cyclic AMP production in HEK 293 cells stably transfected with xCRF-R1 was determined.     

α4 but Not α3 and α7 Nicotinic Acetylcholine Receptor Subunits Are Lost from the Temporal Cortex in Alzheimer's Disease

Neuronal nicotinic acetylcholine receptors labelled with tritiated agonists are reduced in the cerebral cortex in Alzheimer's disease (AD), but to date it has not been demonstrated which nicotinic receptor subunits contribute to this deficit. In the present study, autopsy tissue from the temporal cortex of 14 AD cases and 15 age-matched control subjects was compared using immunoblotting with antibodies against recombinant peptides specific for alpha3, alpha4, and alpha7 subunits, in conjunction with [3H]epibatidine binding. Antibodies to alpha3, alpha4, and alpha7 produced one major band on western blots at 59, 51, and 57 kDa, respectively. [3H]Epibatidine binding and alpha4-like immunoreactivity (using antibodies against the extracellular domain and cytoplasmic loop of the alpha4 subunit) were reduced in AD cases compared with control subjects (p < 0.02) and with a subgroup of control subjects (n = 9) who did not smoke prior to death (p < 0.05) for the former two parameters. [3H]Epibatidine binding and cytoplasmic alpha4-like immunoreactivity were significantly elevated in a subgroup of control subjects (n = 4) known to have smoked prior to death (p < 0.05). There were no significant changes in alpha3- or alpha7-like immunoreactivity associated with AD or tobacco use. The selective involvement of alpha4 has implications for understanding the role of nicotinic receptors in AD and potential therapeutic targets.     

Characterization and Localization of Adrenal Nicotinic Acetylcholine Receptors: Evidence that mAb35-Nicotinic Receptors Are the Principal Receptors Mediating Adrenal Catecholamine Secretion

Abstract: Adrenal chromaffin cells contain at least two subtypes of nicotinic acetylcholine receptors (nAChRs). These studies were designed to identify and characterize the subtype of nAChR mediating adrenal catecholamine release using the monoclonal antibody mAb35, which recognizes the α-subunit of muscle nAChRs and cross-reacts with some neuronal nAChRs. Immunocytochemical studies demonstrated that mAb35 interacts with specific sites on cultured chromaffin cells. Pretreatment with mAb35 reduced nAChR-stimulated catecholamine release (IC₅₀ of ∼10 n M ). mAb35 had no effects on release stimulated through non-nAChR mechanisms. Unlike agonist-induced nAChR desensitization, the mAb35-induced reduction in nAChR-mediated secretion developed slowly. Although not immediately reversible, nAChR-stimulated release recovered after mAb35 removal. However, unlike recovery from agonist pretreatment, recovery from mAb35 pretreatment was relatively slow and was partially blocked by vinblastine. Hybridization of adrenal chromaffin RNA with a rat α3 cDNA revealed two strong bands and two fainter bands: two higher-molecular-weight bands, 6.9 and 8.5 kb; a strong band of 3.2 kb; and a lower amount of a 2.3-kb RNA. With recovery of nAChR function after agonist or mAb35 treatment, no significant effects on α3 subunit mRNA levels were seen. In summary, these studies demonstrate the presence of mAb35-nAChRs on adrenal chromaffin cells and provide evidence that these receptors represent the major population that regulates secretory events in adrenal chromaffin cells.     

Ion Channel Topography of the Neuronal Nicotinic Acetylcholine Receptor: Pharmacochemical Approaches

Forty-three bisammonium ganglionic blockers were synthesized to study the structure of the ion channel of nicotinic acetylcholine receptor. The conformational parameters of these blockers were studied, and their effects toward the ganglionic transmission in situ on the sympathetic feline superior cervical ganglia and in vitro on the parasympathetic guinea-pig small intestine ganglia were determined. A model of the binding site for the bisammonium ganglionic blockers in the neuronal ion channel was proposed.     

Molecular Cloning, Expression, and Pharmacological Characterization of humEAA1, a Human Kainate Receptor Subunit

Abstract: Kainate is a potent neuroexcitatory agent; its neurotoxicity is thought to be mediated by an ionotropic receptor with a nanomolar affinity for kainate. In this report, we describe the cloning of a cDNA encoding a human glutamate ionotropic receptor subunit protein from a human hippocampal library. This cDNA, termed humEAA1, is most closely related to rat and human cDNAs for kainate receptor proteins and, when expressed in COS or Chinese hamster ovary cells, is associated with high-affinity kainate receptor binding. We have successfully established cell lines stably expressing humEAA1. This is the first report of establishment of stable cell lines expressing a glutamate receptor subunit. The relative potency of compounds for displacing [³H] kainate binding of humEAA1 receptors expressed in these stable cell lines was kainate > quisqualate > domoate > L-glutamate > ( RS )-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid > dihydro-kainate > 6, 7-dinitroquinoxaline-2, 3-dione > 6-cyano-7-nitroquinoxaline-2, 3-dione. Homooligomeric expression of humEAA1 does not appear to elicit ligand-gated ion channel activity. Nevertheless, the molecular structure and pharmacological characterization of high-affinity kainate binding of the humEAA1 expressed in the stable cell line (ppEAA1–16) suggest that the humEAA1 is a subunit protein of a human kainate receptor complex.