The use of specific ligands for the vertebrate acetylcholine receptor in the characterization of invertebrate acetylcholine receptors

1. The interaction of two specific ligands for the vertebrate nicotinic acetylcholine receptor were investigated on the solubilized form of a proposed acetylcholine receptor from the invertebrate Limulus polyphemus. 2. The affinity agent 4-(N-maleimodo)benzyltrimethylammonium iodide exhibited no effect on the binding of alpha-bungarotoxin to the Limulus receptor protein. 3. Torpedo acetylcholine receptor antibody neither inhibited alpha-bungarotoxin binding nor produced any alteration in the sedimentation profile of the Limulus receptor. 4. The lack of interaction of 4-(N-maleimido)benzyltrimethylammonium iodide and Torpedo acetylcholine receptor antibody with the Limulus acetylcholine receptor was interpreted to reflect significant difference between the molecular structures of this invertebrate receptor and the acetylcholine receptor of vertebrate.     

The identification of an invertebrate acetylcholine receptor

The results of a series of experimental studies have culminated in the identification of an acetylcholine receptor from the invertebrate $\text{Limulus polyphemus}$. The binding ligand α-bungarotoxin was used to identify a specific protein in the central nervous system tissue of this organism. The specific interaction of α-bungarotoxin with an acetylcholine receptor has been confirmed by physiological, competitive binding, subcellular fractionation and autoradiographic techniques. The toxin binding protein was solubilized and exhibited properties consistent with the nature of a nicotinic cholinergic receptor. Therefore, the identified protein is proposed as an acetylcholine receptor protein from the central nervous system of this invertebrate species.     

A Dalpha6 knockout strain of Drosophila melanogaster confers a high level of resistance to spinosad

A null mutation of the nicotinic acetylcholine receptor (nAChR) subunit Dalpha6, in Drosophila melanogaster, confers 1181-fold resistance to a new and increasingly important biopesticide, spinosad. This study's molecular characterisation of a spinosad resistance mechanism identifies Dalpha6 as a major spinosad target in D. melanogaster. Although D. melanogaster is not a major field pest, target site resistances found in this species are often conserved in pest species. This, combined with the high degree of evolutionary conservation of nAChR subunits, suggests that mutations in Dalpha6 orthologues may underpin the spinosad resistance identified in several economically important field pests.     

Role of a key cysteine residue in the gating of the acetylcholine receptor

We have examined changes in single-channel behavior that result from conservative amino acid substitutions at the Cys230 residue in the putative first transmembrane region (M1) of the murine nicotinic acetylcholine receptor. Mutations made in the gamma subunit altered the energy barrier for a single closing rate constant in proportion to the size of the substituted side chain. One of these substitutions, when made in the alpha subunits, had no effect on gating. No mutations altered permeation. We conclude that the region surrounding the M1 Cys is involved in the gating of the nicotinic acetylcholine receptor and that the gamma subunit contributes significantly to the control of channel closure.     

Mutations in the extracellular domain and in the membrane-spanning domains interfere with nicotinic acetylcholine receptor maturation

The deg-3(u662) mutation is a degeneration-causing mutation in a Caenorhabditis elegans nicotinic acetylcholine receptor. In a large screen for mutations that suppress the deleterious effects of this mutation we identified 32 mutations in the deg-3 gene. Among these, 11 are missense mutations, affecting seven residues within the extracellular domain or the membrane-spanning domains. All of these mutations greatly reduce the degeneration-causing activity of deg-3(u662). All but one of these mutations cause defective localization of the DEG-3 protein, as seen in immunohistochemical analysis. Thus our screen identifies multiple residues within the nicotinic acetylcholine receptor needed for normal folding, assembly, or trafficking of this receptor. Interestingly, these mutations lead to distinct localization defects suggesting differences in their effect on DEG-3's maturation process. Specifically, mutations in the extracellular domain lead to a phenotype more severe than mutations in the membrane-spanning domains. Differences in the effects of the mutations are also predicted by homology-based modeling, showing that some mutations in the extracellular domain are likely to disrupt the native fold of the protein, while others are likely to disrupt trafficking.     

Nicotinic alpha5 subunit deletion locally reduces high-affinity agonist activation without altering nicotinic receptor numbers

Neuronal nicotinic acetylcholine receptor subunit alpha5 mRNA is widely expressed in the CNS. An alpha5 gene polymorphism has been implicated in behavioral differences between mouse strains, and alpha5-null mutation induces profound changes in mouse acute responses to nicotine. In this study, we have examined the distribution and prevalence of alpha5* nicotinic acetylcholine receptor in mouse brain, and quantified the effects of alpha5-null mutation on pre-synaptic nicotinic acetylcholine receptor function (measured using synaptosomal (86)Rb(+) efflux) and overall [(125)I]epibatidine binding site expression. alpha5* nicotinic acetylcholine receptor expression was found in nine of fifteen regions examined, although < 20% of the total nicotinic acetylcholine receptor population in any region contained alpha5. Deletion of the alpha5 subunit gene resulted in localized loss of function (thalamus, striatum), which was itself confined to the DHbetaE-sensitive receptor population. No changes in receptor expression were seen. Consequently, functional changes must occur as a result of altered function per unit of receptor. The selective depletion of high agonist activation affinity sites results in overall nicotinic function being reduced, and increases the overall agonist activation affinity. Together, these results describe the receptor-level changes underlying altered behavioral responses to nicotine in nicotinic acetylcholine receptor alpha5 subunit-null mutants.     

Nicotinic acetylcholine receptor is involved in acetylcholine regulating stomatal movement

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Phosphorylation and cytoskeletal anchoring of the acetylcholine receptor by Src class protein-tyrosine kinases. Activation by rapsyn.

Src class protein-tyrosine kinases bind to and phosphorylate the nicotinic acetylcholine receptor of skeletal muscle. This study provided evidence for the functional importance of Src kinases in regulating the nicotinic acetylcholine receptor at the neuromuscular junction. Three Src class kinases, Fyn, Fyk, and Src, each formed a complex with the endplate-specific cytoskeletal protein rapsyn. In addition, cellular phosphorylation by each kinase was stimulated by rapsyn in heterologous transfected cells. Several lines of evidence supported rapsyn as a substrate for Src kinases. Most importantly, rapsyn regulation of Fyn, Fyk, and Src resulted in phosphorylation of the nicotinic acetylcholine receptor beta and delta subunits and anchoring of the receptor to the cytoskeleton. Both nicotinic acetylcholine receptor phosphorylation and cytoskeletal anchoring were blocked by the Src kinase-selective inhibitor herbimycin A. Rapsyn alone also induced a modest increase in nicotinic acetylcholine receptor phosphorylation and cytoskeletal translocation. However, inhibition by herbimycin A and a catalytically inactive dominant negative Src demonstrated that the effects of rapsyn were mediated by endogenous Src kinases. These data support the importance of Src class kinases for stabilization of the nicotinic acetylcholine receptor at the endplate during synaptic differentiation at the neuromuscular junction.     

Ubiquilin-1 regulates nicotine-induced up-regulation of neuronal nicotinic acetylcholine receptors

Chronic exposure to nicotine, as in tobacco smoking, up-regulates nicotinic acetylcholine receptor surface expression in neurons. This up-regulation has been proposed to play a role in nicotine addiction and withdrawal. The regulatory mechanisms behind nicotine-induced up-regulation of surface nicotinic acetylcholine receptors remain to be determined. It has recently been suggested that nicotine stimulation acts through increased assembly and maturation of receptor subunits into functional pentameric receptors. Studies of muscle nicotinic acetylcholine receptors suggest that the availability of unassembled subunits in the endoplasmic reticulum can be regulated by the ubiquitin-proteosome pathway, resulting in altered surface expression. Here, we describe a role for ubiquilin-1, a ubiquitin-like protein with the capacity to interact with both the proteosome and ubiquitin ligases, in regulating nicotine-induced up-regulation of neuronal nicotinic acetylcholine receptors. Ubiquilin-1 interacts with unassembled alpha3 and alpha4 subunits when coexpressed in heterologous cells and interacts with endogenous nicotinic acetylcholine receptors in neurons. Coexpression of ubiquilin-1 and neuronal nicotinic acetylcholine receptors in heterologous cells dramatically reduces the expression of the receptors on the cell surface. In cultured superior cervical ganglion neurons, expression of ubiquilin-1 abolishes nicotine-induced up-regulation of nicotinic acetylcholine receptors but has no effect on the basal level of surface receptors. Coimmunostaining shows that the interaction of ubiquilin-1 with the alpha3 subunit draws the receptor subunit and proteosome into a complex. These data suggest that ubiquilin-1 limits the availability of unassembled nicotinic acetylcholine receptor subunits in neurons by drawing them to the proteosome, thus regulating nicotine-induced up-regulation.     

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.     

CHARACTERIZATION OF NICOTINE ACETYLCHOLINE RECEPTOR SUBUNITS IN THE COCKROACH Periplaneta americana MUSHROOM BODIES REVEALS A STRONG EXPRESSION OF β1 SUBUNIT: INVOLVEMENT IN NICOTINE‐INDUCED CURRENTS

Nicotinic acetylcholine receptors are ligand‐gated ion channels expressed in many insect structures, such as mushroom bodies, in which they play a central role. We have recently demonstrated using electrophysiological recordings that different native nicotinic receptors are expressed in cockroach mushroom bodies Kenyon cells. In the present study, we demonstrated that eight genes coding for cockroach nicotinic acetylcholine receptor subunits are expressed in the mushroom bodies. Quantitative real‐time polymerase chain reaction (PCR) experiments demonstrated that β1 subunit was the most expressed in the mushroom bodies. Moreover, antisense oligonucleotides performed against β1 subunit revealed that inhibition of β1 expression strongly decreases nicotine‐induced currents amplitudes. Moreover, co‐application with 0.5 μM α‐bungarotoxin completely inhibited nicotine currents whereas 10 μM d‐tubocurarine had a partial effect demonstrating that β1‐containing neuronal nicotinic acetylcholine receptor subtypes could be sensitive to the nicotinic acetylcholine receptor antagonist α‐bungarotoxin.     

Increased sensitivity of the neuronal nicotinic receptor alpha 2 subunit causes familial epilepsy with nocturnal wandering and ictal fear

Sleep has traditionally been recognized as a precipitating factor for some forms of epilepsy, although differential diagnosis between some seizure types and parasomnias may be difficult. Autosomal dominant frontal lobe epilepsy is characterized by nocturnal seizures with hyperkinetic automatisms and poorly organized stereotyped movements and has been associated with mutations of the alpha 4 and beta 2 subunits of the neuronal nicotinic acetylcholine receptor. We performed a clinical and molecular genetic study of a large pedigree segregating sleep-related epilepsy in which seizures are associated with fear sensation, tongue movements, and nocturnal wandering, closely resembling nightmares and sleep walking. We identified a new genetic locus for familial sleep-related focal epilepsy on chromosome 8p12.3-8q12.3. By sequencing the positional candidate neuronal cholinergic receptor alpha 2 subunit gene (CHRNA2), we detected a heterozygous missense mutation, I279N, in the first transmembrane domain that is crucial for receptor function. Whole-cell recordings of transiently transfected HEK293 cells expressing either the mutant or the wild-type receptor showed that the new CHRNA2 mutation markedly increases the receptor sensitivity to acetylcholine, therefore indicating that the nicotinic alpha 2 subunit alteration is the underlying cause. CHRNA2 is the third neuronal cholinergic receptor gene to be associated with familial sleep-related epilepsies. Compared with the CHRNA4 and CHRNB2 mutations reported elsewhere, CHRNA2 mutations cause a more complex and finalized ictal behavior.     

Purification and characterization of a nicotinic acetylcholine receptor from chick brain

Immunohistochemical studies have previously shown that both the chick brain and chick ciliary ganglion neurons contain a component which shares antigenic determinants with the main immunogenic region of the nicotinic acetylcholine receptor from electric organ and skeletal muscle. Here we describe the purification and initial characterization of this putative neuronal acetylcholine receptor. The component was purified by monoclonal antibody affinity chromatography. The solubilized component sediments on sucrose gradients as a species slightly larger than Torpedo acetylcholine receptor monomers. It was affinity labeled with bromo[3H]acetylcholine. Labeling was prevented by carbachol, but not by alpha-bungarotoxin. Two subunits could be detected in the affinity-purified component, apparent molecular weights 48 000 and 59 000. The 48 000 molecular weight subunit was bound both by a monoclonal antibody directed against the main immunogenic region of electric organ and skeletal muscle acetylcholine receptor and by antisera raised against the alpha subunit of Torpedo receptor. Evidence suggests that there are two alpha subunits in the brain component. Antisera from rats immunized with the purified brain component exhibited little or no cross-reactivity with Torpedo electric organ or chick muscle acetylcholine receptor. One antiserum did, however, specifically bind to all four subunits of Torpedo receptor. Experiments to be described elsewhere (J. Stollberg et al., unpublished results) show that antisera to the purified brain component specifically inhibit the electrophysiological function of acetylcholine receptors in chick ciliary ganglion neurons without inhibiting the function of acetylcholine receptors in chick muscle cells. All of these properties suggest that this component is a neuronal nicotinic acetylcholine receptor with limited structural homology to muscle nicotinic acetylcholine receptor.     

Nicotinic receptors in circuit excitability and epilepsy

Neuronal nicotinic acetylcholine receptors belong to the family of excitatory ligand-gated channels and result from the assembly of five subunits. Functional heteromeric nictonic receptors are present in the hippocampus and neocortex, thalamus, mesolimbic dopamine system and brainstem motor nuclei, where they may play a role, respectively, in memory, sensory processing, addiction and motor control. Some forms of autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) have been found to be associated with mutations in the genes coding for the alpha 4 or beta2 subunits of the nicotinic receptor. Mutant receptors display an increased acetylcholine sensitivity with respect to normal receptors. Since the thalamus and the cortex are strongly innervated by cholinergic neurons projecting from the brainstem and basal forebrain, an unbalance between excitation and inhibition, brought about by the presence of mutant receptors, could generate seizures by facilitating and synchronizing spontaneous oscillations in thalamo-cortical circuits.     

Nicotinic Agonists Regulate α-Bungarotoxin Binding Sites of TE671 Human Medulloblastoma Cells

The TE671 human medulloblastoma cell line expresses a variety of characteristics of human neurons. Among these characteristics is the expression of membrane-bound high-affinity binding sites for alpha-bungarotoxin, which is a potent antagonist of functional nicotinic acetylcholine receptors on these cells. These toxin binding sites represent a class of nicotinic receptor isotypes present in mammalian brain. Treatment of TE671 cells during proliferative growth phase with nicotine or carbamylcholine, but not with muscarine or d-tubocurarine, induced up to a five-fold increase in the density of radiolabeled toxin binding sites in crude membrane fractions. This effect was blocked by co-incubation with the nicotinic antagonists d-tubocurarine and decamethonium, but not by mecamylamine or by muscarinic antagonists. Following a 10-13 h lag phase upon removal of agonist, recovery of the up-regulated sites to control values occurred within an additional 10-20 h. These studies indicate that the expression of functional nicotinic acetylcholine receptors on TE671 cells is subject to regulation by nicotinic agonists. Studies of the murine CNS have consistently indicated nicotine-induced up-regulation of nicotinic acetylcholine receptors, thereby supporting the identification of the toxin binding site on these cells as the functional nicotinic receptor. Although a mechanism for this effect is not apparent, nicotine-induced receptor blockade does not appear to be involved.     

Biochemical characterization of two nicotinic receptors from the optic lobe of the chick

We have studied putative nicotinic acetylcholine receptors in the optic lobe of the newborn chick, using 125I-labeled alpha-bungarotoxin, a specific blocker of acetylcholine receptors in the neuromuscular junction, and [3H]acetylcholine, a ligand which in the presence of atropine selectively labels binding sites of nicotinic character in rat brain cortex (Schwartz et al., 1982). [3H]Acetylcholine binds reversibly to a single class of high affinity binding sites (KD = 2.2 X 10(-8) M) which occur at a tissue concentration of 5.7 pmol/g. A large fraction (approximately 60%) of these binding sites is solubilized by Triton X-100, sodium cholate, or the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Solubilization increases the affinity for acetylcholine and several nicotinic drugs from 1.5- to 7-fold. The acetylcholine-binding macromolecule resembles the receptor for alpha-bungarotoxin present in the same tissue with respect to subcellular distribution, hydrodynamic properties, lectin binding, and agonist affinity rank order. It differs from the toxin receptor in affinity for nicotinic antagonists, sensitivity to thermal inactivation, and regional distribution. The solubilized [3H]acetylcholine binding activity is separated from the toxin receptor by incubation with agarose-linked acetylcholine, by affinity chromatography on immobilized Naja naja siamensis alpha-toxin, and by precipitation with a monoclonal antibody to chick optic lobe toxin receptor.     

The effect of amantadine on nicotinic acetylcholine receptor (nAchR) in reconstituted membranes

The antiviral drug amantadine is also a potent neuromuscular blocking agent. When the nicotinic receptor from a Torpedinidae species is reconstituted into soybean liposomes, the binding of α-bungarotoxin is not altered although the carbamylcholine induced radioactive cation influx is blocked.By studying cation fluxes in amantadine preincubated membranes previously exposed to different concentrations of carbamylcholine for different periods of time, we have shown that the drug accelerates the conversion of the nicotinic acetylcholine receptor from a state of low affinity to a state of high affinity for carbamyalcholine, a phenomenon correlated with receptor desensitization. The drug did not induce such a shift by itself.The present data and those by Earnest et al. (Biochemistry22, 5523–5535, 1984) show that the nicotinic acetylcholine receptor reconstituted into liposomes is a good model for studying the effects of noncompetitive blockers of nicotinic acetylcholine receptor function.     

Cholinergic agonists inhibit HMGB1 release and improve survival in experimental sepsis

Physiological anti-inflammatory mechanisms can potentially be exploited for the treatment of inflammatory disorders. Here we report that the neurotransmitter acetylcholine inhibits HMGB1 release from human macrophages by signaling through a nicotinic acetylcholine receptor. Nicotine, a selective cholinergic agonist, is more efficient than acetylcholine and inhibits HMGB1 release induced by either endotoxin or tumor necrosis factor-alpha (TNF-alpha). Nicotinic stimulation prevents activation of the NF-kappaB pathway and inhibits HMGB1 secretion through a specific 'nicotinic anti-inflammatory pathway' that requires the alpha7 nicotinic acetylcholine receptor (alpha7nAChR). In vivo, treatment with nicotine attenuates serum HMGB1 levels and improves survival in experimental models of sepsis, even when treatment is started after the onset of the disease. These results reveal acetylcholine as the first known physiological inhibitor of HMGB1 release from human macrophages and suggest that selective nicotinic agonists for the alpha7nAChR might have therapeutic potential for the treatment of sepsis.     

Super agonist actions of clothianidin and related compounds on the SAD beta 2 nicotinic acetylcholine receptor expressed in Xenopus laevis oocytes

To compare the actions of clothianidin, a neonicotinoid acting on insect nicotinic acetylcholine receptors, and related compounds with that of imidacloprid, the compounds were tested on the Drosophila SAD-chicken beta2 nicotinic acetylcholine receptor expressed in Xenopus laevis oocytes using two-electrode voltage-clamp electrophysiology. The maximum response of the SAD beta 2 nicotinic receptor to clothianidin was larger than that observed for acetylcholine. Ring breakage of the imidazolidine ring of imidacloprid resulting in the generation of a guanidine group was critical for this super agonist action.     

Administration of keratinocyte growth factor (KGF) modulates the pulmonary expression of nicotinic acetylcholine receptor subunits alpha7, alpha9 and alpha10

Administration of recombinant human keratinocyte growth factor (rHuKGF, Delta23N-KGF, palifermin) protects the lung against a variety of injurious stimuli. The exact mechanisms leading to lung protection are unknown. Alterations in the non-neuronal cholinergic system of the lung might be involved, as vital pulmonary functions are regulated by acetylcholine. Here, we investigated the effect of KGF on the expression of nicotinic acetylcholine receptor subunits alpha7, alpha9 and alpha10 in rat lungs. Adult rats were treated via intratracheal instillation with rHuKGF or with an equivalent volume of PBS. The expression of nicotinic acetylcholine receptor subunits was analyzed by real-time RT-PCR, immunoblotting and immunohistochemistry. Treatment with rHuKGF led to a decreased expression of nicotinic receptor subunit alpha7 in the total lung. In contrast, the expression of the receptor subunits alpha9 and alpha10 was up-regulated. In conclusion, nicotinic acetylcholine receptors are differentially regulated by KGF treatment in vivo, which might result in changes in the biological effects of acetylcholine.