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Sample records for acetylcholine receptor alpha

  1. Acetylcholine receptors at neuromuscular synapses: phylogenetic differences detected by snake alpha-neurotoxins.

    PubMed Central

    Burden, S J; Hartzell, H C; Yoshikami, D

    1975-01-01

    Phylogenetic differences in acetylcholine receptors from skeletal neuromuscular synapses of various species of snakes and lizards have been investigated, using the snake venom alpha-neurotoxins alpha-atratoxin (cobrotoxin) and alpha-bungarotoxin. The acetylcholine receptors of the phylogenetically primitive lizards, like those from all other vertebrates previously tested, are blocked by these alpha-neurotoxins. In contrast, receptors from snakes and advanced lizards are insensitive to one or both of the toxins. It is suggested that toxin-resistant acetylcholine receptors appeared early in the evolution of Squamata and preceded the appearance of alpha-neurotoxins. Images PMID:1081230

  2. Functional characterization of mongoose nicotinic acetylcholine receptor alpha-subunit: resistance to alpha-bungarotoxin and high sensitivity to acetylcholine.

    PubMed

    Asher, O; Lupu-Meiri, M; Jensen, B S; Paperna, T; Fuchs, S; Oron, Y

    1998-07-24

    The mongoose is resistant to snake neurotoxins. The mongoose muscle nicotinic acetylcholine receptor (AChR) alpha-subunit contains a number of mutations in the ligand-binding domain and exhibits poor binding of alpha-bungarotoxin (alpha-BTX). We characterized the functional properties of a hybrid (alpha-mongoose/beta gamma delta-rat) AChR. Hybrid AChRs, expressed in Xenopus oocytes, respond to acetylcholine with depolarizing current, the mean maximal amplitude of which was greater than that mediated by the rat AChR. The IC50 of alpha-BTX to the hybrid AChR was 200-fold greater than that of the rat, suggesting much lower affinity for the toxin. Hybrid AChRs exhibited an apparent higher rate of desensitization and higher affinity for ACh (EC50 1.3 vs. 23.3 microM for the rat AChR). Hence, changes in the ligand-binding domain of AChR not only affect the binding properties of the receptor, but also result in marked changes in the characteristics of the current.

  3. Allosteric modulation of alpha4beta2 nicotinic acetylcholine receptors by HEPES.

    PubMed

    Weltzin, Maegan M; Huang, Yanzhou; Schulte, Marvin K

    2014-06-05

    A number of new positive allosteric modulators (PAMs) have been reported that enhance responses of neuronal alpha7 and alpha4beta2 nicotinic acetylcholine receptor subtypes to orthosteric ligands. PAMs represent promising new leads for the development of therapeutic agents for disorders involving alterations in nicotinic neurotransmission including Autism, Alzheimer's and Parkinson's disease. During our recent studies of alpha4beta2 PAMs, we identified a novel effect of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). The effects of HEPES were evaluated in a phosphate buffered recording solution using two-electrode voltage clamp techniques and alpha4beta2 and alpha7 nicotinic acetylcholine receptor subtypes expressed in Xenopus laevis oocytes. Acetylcholine induced responses of high-sensitivity alpha4beta2 receptors were potentiated 190% by co-exposure to HEPES. Responses were inhibited at higher concentrations (bell-shaped concentration/response curve). Coincidentally, at concentrations of HEPES typically used in oocyte recording (5-10mM), the potentiating effects of HEPES are matched by its inhibitory effects, thus producing no net effect. Mutagenesis results suggest HEPES potentiates the high-sensitivity stoichiometry of the alpha4beta2 receptors through action at the beta2+/beta2- interface and is dependent on residue beta2D218. HEPES did not potentiate low-sensitivity alpha4beta2 receptors and did not produce any observable effect on acetylcholine induced responses on alpha7 nicotinic acetylcholine receptors.

  4. Mapping of the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor.

    PubMed Central

    Neumann, D; Barchan, D; Safran, A; Gershoni, J M; Fuchs, S

    1986-01-01

    Synthetic peptides and their respective antibodies have been used in order to map the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor. By using antibodies to a synthetic peptide corresponding to residues 169-181 of the alpha subunit, we demonstrate that this sequence is included within the 18-kDa toxin binding fragment previously reported. Furthermore, the 18-kDa fragment was also found to bind a monoclonal antibody (5.5) directed against the cholinergic binding site. Sequential proteolysis of the acetylcholine receptor with trypsin, prior to Staphylococcus aureus V8 protease digestion, resulted in a 15-kDa toxin binding fragment that is included within the 18-kDa fragment but is shorter than it only at its carboxyl terminus. This 15-kDa fragment therefore initiates beyond Asp-152 and terminates in the region of Arg-313/Lys-314. In addition, experiments are reported that indicate that in the intact acetylcholine receptor, Cys-128 and/or Cys-142 are not crosslinked by disulfide bridges with any of the cysteines (at positions 192, 193, and 222) that reside in the 15-kDa toxin binding fragment. Finally, the synthetic dodecapeptide Lys-His-Trp-Val-Tyr-Tyr-Thr-Cys-Cys-Pro-Asp-Thr, which is present in the 15-kDa fragment (corresponding to residues 185-196 of the alpha subunit) was shown to bind alpha-bungarotoxin directly. This binding was completely inhibited by competition with d-tubocurarine. Images PMID:3458258

  5. Mapping of the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor.

    PubMed

    Neumann, D; Barchan, D; Safran, A; Gershoni, J M; Fuchs, S

    1986-05-01

    Synthetic peptides and their respective antibodies have been used in order to map the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor. By using antibodies to a synthetic peptide corresponding to residues 169-181 of the alpha subunit, we demonstrate that this sequence is included within the 18-kDa toxin binding fragment previously reported. Furthermore, the 18-kDa fragment was also found to bind a monoclonal antibody (5.5) directed against the cholinergic binding site. Sequential proteolysis of the acetylcholine receptor with trypsin, prior to Staphylococcus aureus V8 protease digestion, resulted in a 15-kDa toxin binding fragment that is included within the 18-kDa fragment but is shorter than it only at its carboxyl terminus. This 15-kDa fragment therefore initiates beyond Asp-152 and terminates in the region of Arg-313/Lys-314. In addition, experiments are reported that indicate that in the intact acetylcholine receptor, Cys-128 and/or Cys-142 are not crosslinked by disulfide bridges with any of the cysteines (at positions 192, 193, and 222) that reside in the 15-kDa toxin binding fragment. Finally, the synthetic dodecapeptide Lys-His-Trp-Val-Tyr-Tyr-Thr-Cys-Cys-Pro-Asp-Thr, which is present in the 15-kDa fragment (corresponding to residues 185-196 of the alpha subunit) was shown to bind alpha-bungarotoxin directly. This binding was completely inhibited by competition with d-tubocurarine.

  6. Rabies virus binding to an acetylcholine receptor alpha-subunit peptide.

    PubMed

    Lentz, T L

    1990-04-01

    The binding of 125I-labeled rabies virus to a synthetic peptide comprising residues 173-204 of the alpha 1-subunit of the nicotinic acetylcholine receptor was investigated. Binding of rabies virus to the receptor peptide was dependent on pH, could be competed with by unlabeled homologous virus particles, and was saturable. Synthetic peptides of snake venom, curaremimetic neurotoxins and of the structurally similar segment of the rabies virus glycoprotein, were effective in competing with labeled virus binding to the receptor peptide at micromolar concentrations. Similarly, synthetic peptides of the binding domain on the acetylcholine receptor competed for binding. These findings suggest that both rabies virus and neurotoxins bind to residues 173-204 of the alpha 1-subunit of the acetylcholine receptor. Competition studies with shorter alpha-subunit peptides within this region indicate that the highest affinity virus binding determinants are located within residues 179-192. A rat nerve alpha 3-subunit peptide, that does not bind alpha-bungarotoxin, inhibited binding of virus to the alpha 1 peptide, suggesting that rabies binds to neuronal nicotinic acetylcholine receptors. These studies indicate that synthetic peptides of the glycoprotein binding domain and of the receptor binding domain may represent useful antiviral agents by targeting the recognition event between the viral attachment protein and the host cell receptor, and inhibiting attachment of virus to the receptor.

  7. Solution structure of {alpha}-conotoxin PIA, a novel antagonist of {alpha}6 subunit containing nicotinic acetylcholine receptors

    SciTech Connect

    Chi, Seung-Wook; Lee, Si-Hyung; Kim, Do-Hyoung; Kim, Jae-Sung; Olivera, Baldomero M.; McIntosh, J. Michael; Han, Kyou-Hoon . E-mail: khhan600@kribb.re.kr

    2005-12-30

    {alpha}-Conotoxin PIA is a novel nicotinic acetylcholine receptor (nAChR) antagonist isolated from Conus purpurascens that targets nAChR subtypes containing {alpha}6 and {alpha}3 subunits. {alpha}-conotoxin PIA displays 75-fold higher affinity for rat {alpha}6/{alpha}3{beta}2{beta}3 nAChRs than for rat {alpha}3{beta}2 nAChRs. We have determined the three-dimensional structure of {alpha}-conotoxin PIA by nuclear magnetic resonance spectroscopy. The {alpha}-conotoxin PIA has an '{omega}-shaped' overall topology as other {alpha}4/7 subfamily conotoxins. Yet, unlike other neuronally targeted {alpha}4/7-conotoxins, its N-terminal tail Arg{sup 1}-Asp{sup 2}-Pro{sup 3} protrudes out of its main molecular body because Asp{sup 2}-Pro{sup 3}-Cys{sup 4}-Cys{sup 5} forms a stable type I {beta}-turn. In addition, a kink introduced by Pro{sup 15} in the second loop of this toxin provides a distinct steric and electrostatic environment from those in {alpha}-conotoxins MII and GIC. By comparing the structure of {alpha}-conotoxin PIA with other functionally related {alpha}-conotoxins we suggest structural features in {alpha}-conotoxin PIA that may be associated with its unique receptor recognition profile.

  8. Spinal alpha3beta2* nicotinic acetylcholine receptors tonically inhibit the transmission of nociceptive mechanical stimuli.

    PubMed

    Young, Tracey; Wittenauer, Shannon; McIntosh, J Michael; Vincler, Michelle

    2008-09-10

    The presence of non-alpha4beta2, non-alpha7 nicotinic acetylcholine receptors (nAChR) in the rat spinal cord has been suggested previously, but the identity of these nAChRs had not been shown. Intrathecal administration of the alpha3beta2*/alpha6beta2* selective alpha-conotoxin MII (alpha-CTX MII) dose- and time-dependently reduced paw withdrawal thresholds to mechanical pressure in normal rats. The pronociceptive effect of alpha-CTX MII was partially blocked by NMDA receptor antagonism and lost completely following ablation of C-fibers. The effect of spinal nerve ligation on alpha-CTX MII-induced mechanical hypersensitivity was also assessed. Sensitivity was lost in the hind paw ipsilateral to spinal nerve ligation, but maintained in the contralateral hind paw at control levels. Radioligand binding in spinal cord membranes revealed high and low affinity alpha-CTX MII binding sites. Spinal nerve ligation did not significantly alter alpha-CTX MII binding ipsilateral to ligation. Finally, no evidence for the presence of alpha6-containing nAChRs was identified. The results of these studies show the presence of 2 populations of alpha-CTX MII-sensitive nAChRs containing the alpha3 and beta2, but not the alpha6, subunits in the rat spinal cord that function to inhibit the transmission of nociceptive mechanical stimuli via inhibiting the release of glutamate from C-fibers. Spinal nerve ligation produces a unilateral loss of alpha-CTX MII-induced mechanical hypersensitivity without altering alpha-CTX MII binding sites. Our data support a peripheral injury-induced loss of a cholinergic inhibitory tone at spinal alpha3beta2* nAChRs, without the loss of the receptors themselves, which may contribute to mechanical hypersensitivity following spinal nerve ligation.

  9. Acetylcholine receptor-inducing factor from chicken brain increases the level of mRNA encoding the receptor. alpha. subunit

    SciTech Connect

    Harris, D.A.; Falls, D.L.; Dill-Devor, R.M.; Fischbach, G.D. )

    1988-03-01

    A 42-kDa glycoprotein isolated from chicken brain, referred to as acetylcholine receptor-inducing activity (ARIA), that stimulates the rate of incorporation of acetylcholine receptors into the surface of chicken myotubes may play a role in the nerve-induced accumulation of receptors at developing neuromuscular synapses. Using nuclease-protection assays, the authors have found that ARIA causes a 2- to 16-fold increase in the level of mRNA encoding the {alpha} subunit of the receptor, with little or no change in the levels of {gamma}- and {delta}-subunit messengers. ARIA also increases the amount of a putative nuclear precursor of {alpha}-subunit mRNA, consistent with an activation of gene transcription. These results suggest that the concentration of {alpha} subunit may limit the rate of biosynthesis of the acetylcholine receptors in chicken myotubes. They also indicate that neuronal factors can regulate the expression of receptor subunit genes in a selective manner. Tetrodotoxin, 8-bromo-cAMP, and forskolin also increase the amount of {alpha}-subunit mRNA, with little change in the amount of {gamma}- and {delta}-subunit mRNAs. Unlike ARIA, however, these agents have little effect on the concentration of the {alpha}-subunit nuclear precursor.

  10. Some properties of human neuronal alpha 7 nicotinic acetylcholine receptors fused to the green fluorescent protein.

    PubMed

    Palma, Eleonora; Mileo, Anna M; Martinez-Torres, Ataulfo; Eusebi, Fabrizio; Miledi, Ricardo

    2002-03-19

    The functional properties and cellular localization of the human neuronal alpha7 nicotinic acetylcholine (AcCho) receptor (alpha7 AcChoR) and its L248T mutated (mut) form were investigated by expressing them alone or as gene fusions with the enhanced version of the green fluorescent protein (GFP). Xenopus oocytes injected with wild-type (wt), mutalpha7, or the chimeric subunit cDNAs expressed receptors that gated membrane currents when exposed to AcCho. As already known, AcCho currents generated by wtalpha7 receptors decay much faster than those elicited by the mutalpha7 receptors. Unexpectedly, the fusion of GFP to the wt and mutated alpha7 receptors led to opposite results: the AcCho-current decay of the wt receptors became slower, whereas that of the mutated receptors was accelerated. Furthermore, repetitive applications of AcCho led to a considerable "run-down" of the AcCho currents generated by mutalpha7-GFP receptors, whereas those of the wtalpha7-GFP receptors remained stable or increased in amplitude. The AcCho-current run-down of mutalpha7-GFP oocytes was accompanied by a marked decrease of alpha-bungarotoxin binding activity. Fluorescence, caused by the chimeric receptors expressed, was seen over the whole oocyte surface but was more intense and abundant in the animal hemisphere, whereas it was much weaker in the vegetal hemisphere. We conclude that fusion of GFP to wtalpha7 and mutalpha7 receptors provides powerful tools to study the distribution and function of alpha7 receptors. We also conclude that fused genes do not necessarily recapitulate all of the properties of the original receptors. This fact must be borne close in mind whenever reporter genes are attached to proteins.

  11. alpha7 Nicotinic acetylcholine receptor knockout selectively enhances ethanol-, but not beta-amyloid-induced neurotoxicity.

    PubMed

    de Fiebre, Nancyellen C; de Fiebre, Christopher M

    2005-01-03

    The alpha7 subtype of nicotinic acetylcholine receptor (nAChR) has been implicated as a potential site of action for two neurotoxins, ethanol and the Alzheimer's disease related peptide, beta-amyloid. Here, we utilized primary neuronal cultures of cerebral cortex from alpha7 nAChR null mutant mice to examine the role of this receptor in modulating the neurotoxic properties of subchronic, "binge" ethanol and beta-amyloid. Knockout of the alpha7 nAChR gene selectively enhanced ethanol-induced neurotoxicity in a gene dosage-related fashion. Susceptibility of cultures to beta-amyloid induced toxicity, however, was unaffected by alpha7 nAChR gene null mutation. Further, beta-amyloid did not inhibit the binding of the highly alpha7-selective radioligand, [(125)I]alpha-bungarotoxin. On the other hand, in studies in Xenopus oocytes ethanol efficaciously inhibited alpha7 nAChR function. These data suggest that alpha7 nAChRs modulate the neurotoxic effects of binge ethanol, but not the neurotoxicity produced by beta-amyloid. It is hypothesized that inhibition of alpha7 nAChRs by ethanol provides partial protection against the neurotoxic properties of subchronic ethanol.

  12. The neuronal nicotinic acetylcholine receptor {alpha}7 subunit gene: Cloning, mapping, structure, and targeting in mouse

    SciTech Connect

    Orr-Urtreger, A.; Baldini, A.; Beaudet, A.L.

    1994-09-01

    The neuronal nicotinic acetylcholine receptor {alpha}7 subunit is a member of a family of ligand-gated ion channels, and is the only subunit know to bind {alpha}-bungarotoxin in mammalian brain. {alpha}-Bungarotoxin binding sites are known to be more abundant in the hippocampus of mouse strains that are particularly sensitive to nicotine-induced seizures. The {alpha}7 receptor is highly permeable to calcium, which could suggest a role in synaptic plasticity in the nervous system. Auditory gating deficiency, an abnormal response to a second auditory stimulus, is characteristic of schizophrenia. Mouse strains that exhibit a similar gating deficit have reduced hippocampal expression of the {alpha}7 subunit. We have cloned and sequenced the full length cDNA for the mouse {alpha}7 gene (Acra-7) and characterized its gene structure. The murine {alpha}7 shares amino acid identity of 99% and 93% with the rat and human {alpha}7 subunits, respectively. Using an interspecies backcross panel, the murine gene was mapped to chromosome 7 near the p locus, a region syntenic with human chromosome 15; the human gene (CHRNA7) was confirmed to map to 15q13-q14 by FISH. To generate a mouse {alpha}7 mutant by homologous recombination, we have constructed a replacement vector which will delete transmembrane domains II-IV and the cytoplasmic domain from the gene product. Recombinant embryonic stem (ES) cell clones were selected and used to develop mouse chimeras that are currently being bred to obtain germline transmission.

  13. The alpha7 nicotinic acetylcholine receptor as a pharmacological target for inflammation

    PubMed Central

    de Jonge, W J; Ulloa, L

    2007-01-01

    The physiological regulation of the immune system encompasses comprehensive anti-inflammatory mechanisms that can be harnessed for the treatment of infectious and inflammatory disorders. Recent studies indicate that the vagal nerve, involved in control of heart rate, hormone secretion and gastrointestinal motility, is also an immunomodulator. In experimental models of inflammatory diseases, vagal nerve stimulation attenuates the production of proinflammatory cytokines and inhibits the inflammatory process. Acetylcholine, the principal neurotransmitter of the vagal nerve, controls immune cell functions via the alpha7 nicotinic acetylcholine receptor (alpha7nAChR). From a pharmacological perspective, nicotinic agonists are more efficient than acetylcholine at inhibiting the inflammatory signaling and the production of proinflammatory cytokines. This ‘nicotinic anti-inflammatory pathway' may have clinical implications as treatment with nicotinic agonists can modulate the production of proinflammatory cytokines from immune cells. Nicotine has been tested in clinical trials as a treatment for inflammatory diseases such as ulcerative colitis, but the therapeutic potential of this mechanism is limited by the collateral toxicity of nicotine. Here, we review the recent advances that support the design of more specific receptor-selective nicotinic agonists that have anti-inflammatory effects while eluding its collateral toxicity. PMID:17502850

  14. Sequence and functional expression of a single alpha subunit of an insect nicotinic acetylcholine receptor.

    PubMed Central

    Marshall, J; Buckingham, S D; Shingai, R; Lunt, G G; Goosey, M W; Darlison, M G; Sattelle, D B; Barnard, E A

    1990-01-01

    We report the isolation and sequence of a cDNA clone that encodes a locust (Schistocerca gregaria) nervous system nicotinic acetylcholine receptor (AChR) subunit (alpha L1). The calculated molecular weight of the unglycosylated polypeptide, which contains in the proposed extracellular domain two adjacent cysteine residues which are characteristic of alpha (ligand binding) subunits, is 60,641 daltons. Injection into Xenopus oocytes, of RNA synthesized from this clone in vitro, results in expression of functional nicotinic receptors in the oocyte membrane. In these, nicotine opens a cation channel; the receptors are blocked by both alpha-bungarotoxin (alpha-Bgt) and kappa-bungarotoxin (kappa-Bgt). Reversible block of the expressed insect AChR by mecamylamine, d-tubocurarine, tetraethylammonium, bicuculline and strychnine has also been observed. These data are entirely consistent with previously reported electrophysiological studies on in vivo insect nicotinic receptors and also with biochemical studies on an alpha-Bgt affinity purified locust AChR. Thus, a functional receptor exhibiting the characteristic pharmacology of an in vivo insect nicotinic AChR can be expressed in Xenopus oocytes by injection with a single subunit RNA. PMID:1702381

  15. Potentiation of alpha7 nicotinic acetylcholine receptors via an allosteric transmembrane site.

    PubMed

    Young, Gareth T; Zwart, Ruud; Walker, Alison S; Sher, Emanuele; Millar, Neil S

    2008-09-23

    Positive allosteric modulators of alpha7 nicotinic acetylcholine receptors (nAChRs) have attracted considerable interest as potential tools for the treatment of neurological and psychiatric disorders such as Alzheimer's disease and schizophrenia. However, despite the potential therapeutic usefulness of these compounds, little is known about their mechanism of action. Here, we have examined two allosteric potentiators of alpha7 nAChRs (PNU-120596 and LY-2087101). From studies with a series of subunit chimeras, we have identified the transmembrane regions of alpha7 as being critical in facilitating potentiation of agonist-evoked responses. Furthermore, we have identified five transmembrane amino acids that, when mutated, significantly reduce potentiation of alpha7 nAChRs. The amino acids we have identified are located within the alpha-helical transmembrane domains TM1 (S222 and A225), TM2 (M253), and TM4 (F455 and C459). Mutation of either A225 or M253 individually have particularly profound effects, reducing potentiation of EC(20) concentrations of acetylcholine to a tenth of the level seen with wild-type alpha7. Reference to homology models of the alpha7 nAChR, based on the 4A structure of the Torpedo nAChR, indicates that the side chains of all five amino acids point toward an intrasubunit cavity located between the four alpha-helical transmembrane domains. Computer docking simulations predict that the allosteric compounds such as PNU-120596 and LY-2087101 may bind within this intrasubunit cavity, much as neurosteroids and volatile anesthetics are thought to interact with GABA(A) and glycine receptors. Our findings suggest that this is a conserved modulatory allosteric site within neurotransmitter-gated ion channels.

  16. Characterization of alpha-conotoxin interactions with the nicotinic acetylcholine receptor and monoclonal antibodies.

    PubMed Central

    Ashcom, J D; Stiles, B G

    1997-01-01

    The venoms of predatory marine cone snails, Conus species, contain numerous peptides and proteins with remarkably diverse pharmacological properties. One group of peptides are the alpha-conotoxins, which consist of 13-19 amino acids constrained by two disulphide bonds. A biologically active fluorescein derivative of Conus geographus alpha-conotoxin GI (FGI) was used in novel solution-phase-binding assays with purified Torpedo californica nicotinic acetylcholine receptor (nAchR) and monoclonal antibodies developed against the toxin. The binding of FGI to nAchR or antibody had apparent dissociation constants of 10-100 nM. Structure-function studies with alpha-conotoxin GI analogues composed of a single disulphide loop revealed that different conformational restraints are necessary for effective toxin interactions with nAchR or antibodies. PMID:9359860

  17. Heterogeneity of Drosophila nicotinic acetylcholine receptors: SAD, a novel developmentally regulated alpha-subunit.

    PubMed Central

    Sawruk, E; Schloss, P; Betz, H; Schmitt, B

    1990-01-01

    Two genes, ard and als, are known to encode subunits of the nicotinic acetylcholine receptor (nAChR) in Drosophila. Here we describe the isolation of cDNA clones encoding a novel member (SAD, or alpha 2) of this receptor protein family. The deduced amino acid sequence displays high homology to the ALS protein and shares structural features with ligand binding nAChR alpha-subunits. Sad transcripts accumulate during major periods of neuronal differentiation and, in embryos, are localized in the central nervous system. Expression of SAD cRNA in Xenopus oocytes generates cation channels that are gated by nicotine. These data indicate heterogeneity of nAChRs in Drosophila. Images Fig. 3. Fig. 4. PMID:1697262

  18. alpha7 nicotinic acetylcholine receptors and modulation of gabaergic synaptic transmission in the hippocampus.

    PubMed

    Alkondon, M; Braga, M F; Pereira, E F; Maelicke, A; Albuquerque, E X

    2000-03-30

    The present report provides new findings regarding modulation of gamma-aminobutyric acid (GABA) transmission by alpha7 nicotinic receptor activity in CA1 interneurons of rat hippocampal slices. Recordings were obtained from tight-seal cell-attached patches of the CA1 interneurons, and agonists were delivered to the neurons via a modified U-tube. Application for 6 s of the alpha7 nicotinic receptor-selective agonist choline (> or =1 mM) to all CA1 interneurons tested triggered action potentials that were detected as fast current transients. The activity triggered by choline terminated well before the end of the agonist pulse, was blocked by the alpha7 nicotinic receptor antagonist methyllycaconitine (50 nM) and was concentration dependent; the higher the concentration of choline the higher the frequency of events and the shorter the delay for detection of the first event. In 40% of the neurons tested, choline-triggered action potentials decreased in amplitude progressively until no more events could be detected despite the presence of the agonist. Primarily, this finding could be explained by Na(+)-channel inactivation associated with membrane depolarization induced by alpha7 nicotinic receptor activation. In 60% of the neurons, the amplitude of choline-induced action potentials was sustained at the intial level, but again the activity did not last as long as the agonist pulse, in this case apparently because of agonist-induced receptor desensitization. These results altogether demonstrate that agonists interacting with alpha7 nicotinic receptors, including the natural transmitter acetylcholine and its metabolite choline, influence GABAergic transmission, not only by activating these receptors, but also by controlling the rate of Na(+)-channel inactivation and/or by inducing receptor desensitization.

  19. Solution conformation of a neuronal nicotinic acetylcholine receptor antagonist {alpha}-conotoxin OmIA that discriminates {alpha}3 vs. {alpha}6 nAChR subtypes

    SciTech Connect

    Chi, Seung-Wook; Kim, Do-Hyoung; Olivera, Baldomero M.; McIntosh, J. Michael; Han, Kyou-Hoon . E-mail: khhan600@kribb.re.kr

    2006-06-23

    {alpha}-Conotoxin OmIA from Conus omaria is the only {alpha}-conotoxin that shows a {approx}20-fold higher affinity to the {alpha}3{beta}2 over the {alpha}6{beta}2 subtype of nicotinic acetylcholine receptor. We have determined a three-dimensional structure of {alpha}-conotoxin OmIA by nuclear magnetic resonance spectroscopy. {alpha}-Conotoxin OmIA has an '{omega}-shaped' overall topology with His{sup 5}-Asn{sup 12} forming an {alpha}-helix. Structural features of {alpha}-conotoxin OmIA responsible for its selectivity are suggested by comparing its surface characteristics with other functionally related {alpha}4/7 subfamily conotoxins. Reduced size of the hydrophilic area in {alpha}-conotoxin OmIA seems to be associated with the reduced affinity towards the {alpha}6{beta}2 nAChR subtype.

  20. BDNF up-regulates alpha7 nicotinic acetylcholine receptor levels on subpopulations of hippocampal interneurons.

    PubMed

    Massey, Kerri A; Zago, Wagner M; Berg, Darwin K

    2006-12-01

    In the hippocampus, brain-derived neurotrophic factor (BDNF) regulates a number of synaptic components. Among these are nicotinic acetylcholine receptors containing alpha7 subunits (alpha7-nAChRs), which are interesting because of their relative abundance in the hippocampus and their high relative calcium permeability. We show here that BDNF elevates surface and intracellular pools of alpha7-nAChRs on cultured hippocampal neurons and that glutamatergic activity is both necessary and sufficient for the effect. Blocking transmission through NMDA receptors with APV blocked the BDNF effect; increasing spontaneous excitatory activity with the GABA(A) receptor antagonist bicuculline replicated the BDNF effect. BDNF antibodies blocked the BDNF-mediated increase but not the bicuculline one, consistent with enhanced glutamatergic activity acting downstream from BDNF. Increased alpha7-nAChR clusters were most prominent on interneuron subtypes known to directly innervate excitatory neurons. The results suggest that BDNF, acting through glutamatergic transmission, can modulate hippocampal output in part by controlling alpha7-nAChR levels.

  1. Antipsychotic clozapine inhibits the function of alpha7-nicotinic acetylcholine receptors.

    PubMed

    Singhal, Sachin K; Zhang, Li; Morales, Marisela; Oz, Murat

    2007-02-01

    The effects of the antipsychotic clozapine on the function of the cloned alpha(7) subunit of the nicotinic acetylcholine (nACh) receptor expressed in Xenopus oocytes was investigated by using the two-electrode voltage-clamp technique. Clozapine reversibly inhibited nicotine (10 microM)-induced currents in a concentration-dependent manner (300 nM to 90 microM), with an IC(50) value of 3.2+/-0.4 microM. The effect of clozapine was not dependent on the membrane potential. Clozapine did not affect the activity of endogenous Ca(2+)-dependent Cl(-) channels since the inhibition by clozapine was unaltered by the intracellularly injected Ca(2+) chelator BAPTA and perfusion with Ca(2+)-free bathing solution containing 2mM Ba(2+). Clozapine decreased the maximal nicotine-induced responses without significantly affecting its potency, indicating that it acts as a noncompetitive antagonist on alpha(7)-nACh receptors. In hippocampal slices, the whole-cell recordings from CA1 pyramidal neurons indicated that the increases in the frequency and amplitudes of the GABA-mediated spontaneous inhibitory postsynaptic currents induced by bath application of 2 mM choline, a specific agonist for alpha(7)-nACh receptors, were abolished after 10 min application of 5 microM clozapine. In conclusion, these results demonstrate that clozapine inhibits the function of alpha(7)-nACh receptors expressed in Xenopus oocytes and in hippocampal neurons.

  2. Topological dispositions of lysine. alpha. 380 and lysine. gamma. 486 in the acetylcholine receptor from Torpedo californica

    SciTech Connect

    Dwyer, B.P. )

    1991-04-23

    The locations have been determined, with respect to the plasma membrane, of lysine {alpha}380 and lysine {gamma}486 in the {alpha} subunit and the {gamma} subunit, respectively, of the nicotinic acetylcholine receptor from Torpedo californica. Immunoadsorbents were constructed that recognize the carboxy terminus of the peptide GVKYIAE released by proteolytic digestion from positions 378-384 in the amino acid sequence of the {alpha} subunit of the acetylcholine receptor and the carboxy terminus of the peptide KYVP released by proteolytic digestion from positions 486-489 in the amino acid sequence of the {gamma} subunit. They were used to isolate these peptides from proteolytic digests of polypeptides from the acetylcholine receptor. Sealed vesicles containing the native acetylcholine receptor were labeled with pyridoxal phosphate and sodium ({sup 3}H)-borohydride. The effect of saponin on the incorporation of pyridoxamine phosphate into lysine {alpha}380 and lysine {gamma}486 from the acetylcholine receptor in these vesicles was assessed with the immunoadsorbents. The conclusions that follow from these results are that lysine {alpha}380 is on the inside surface of a vesicle and lysine {gamma}486 is on the outside surface. Because a majority (85%) of the total binding sites for {alpha}-bungarotoxin bind the toxin in the absence of saponin, the majority of the vesicles are right side out with the inside of the vesicle corresponding to the cytoplasmic surface and the outside of the vesicle corresponding to the extracytoplasmic, synaptic surface. Because lysine {alpha}380 and lysine {gamma}486 lie on opposite sides of the membrane, a membrane-spanning segment must be located between the two positions occupied by these two amino acids in the common sequence of a polypeptide of the acetylcholine receptor.

  3. The neuronal nicotinic acetylcholine receptors alpha 4* and alpha 6* differentially modulate dopamine release in mouse striatal slices.

    PubMed

    Meyer, Erin L; Yoshikami, Doju; McIntosh, J Michael

    2008-06-01

    Striatal dopamine (DA) plays a major role in the regulation of motor coordination and in the processing of salient information. We used voltammetry to monitor DA-release evoked by electrical stimulation in striatal slices, where interneurons continuously release acetylcholine. Use of the alpha6-selective antagonist alpha-conotoxin MII[E11A] and alpha4 knockout mice enabled identification of two populations of DA-ergic fibers. The first population had a low action potential threshold, and action potential-evoked DA-release from these fibers was modulated by alpha6. The second population had a higher action potential threshold, and only alpha4(non-alpha6) modulated action potential-evoked DA-release. Striatal DA-ergic neurons fire in both tonic and phasic patterns. When stimuli were applied in a train to mimic phasic firing, more DA-release was observed in alpha4 knockout versus wild-type mice. Furthermore, block of alpha4(non-alpha6), but not of alpha6, increased DA release evoked by a train. These results indicate that there are different classes of striatal DA-ergic fibers that express different subtypes of nicotinic receptors.

  4. Cognitive improvement by activation of alpha7 nicotinic acetylcholine receptors: from animal models to human pathophysiology.

    PubMed

    Thomsen, Morten S; Hansen, Henrik H; Timmerman, Daniel B; Mikkelsen, Jens D

    2010-01-01

    Agonists and positive allosteric modulators of the alpha(7) nicotinic acetylcholine receptor (nAChR) are currently being developed for the treatment of cognitive disturbances in patients with schizophrenia or Alzheimer's disease. This review describes the neurobiological properties of the alpha nAChR and the cognitive effects of alpha(7) nAChR activation, focusing on the translational aspects in the development of these drugs. The functional properties and anatomical localization of the alpha(7) nAChR makes it well suited to modulate cognitive function. Accordingly, systemic administration of alpha(7) nAChR agonists improves learning, memory, and attentional function in variety of animal models, and pro-cognitive effects of alpha(7) nAChR agonists have recently been demonstrated in patients with schizophrenia or Alzheimer's disease. The alpha(7) nAChR desensitizes rapidly in vitro, and this has been a major concern in the development of alpha(7) nAChR agonists as putative drugs. Our review of the existing literature shows that development of tolerance to the behavioral effects of alpha(7) nAChR agonists does not occur in animal models or humans. However, the long-term memory-enhancing effects seen in animal models are not mimicked in healthy humans and schizophrenic patients, where attentional improvement predominates. This discrepancy may result from inherent differences in testing methods or from species differences in the level of expression of alpha(7) nAChRs in limbic brain regions, and may hamper preclinical evaluation of alpha(7) nAChR activation. It is therefore important to consider the translational power of the animal models used before entering into a clinical evaluation of the pro-cognitive effects of alpha(7) nAChR activation.

  5. The binding site of the nicotinic acetylcholine receptor in animal species resistant to alpha-bungarotoxin.

    PubMed

    Barchan, D; Ovadia, M; Kochva, E; Fuchs, S

    1995-07-18

    The ligand binding site of the nicotinic acetylcholine receptor (AChR) is located in the alpha-subunit, within a small fragment containing the tandem cysteines at positions 192 and 193. We have been analyzing the binding site domain of AChRs from several animal species exhibiting various degrees of resistance to alpha-bungarotoxin (alpha-BTX). Our earlier work on the snake and mongoose AChR, both of which do not bind alpha-BTX, suggested that amino acid substitutions at positions 187, 189, and 194 of the AChR alpha-subunit are important in determining the resistance of these AChRs to alpha-BTX. In the present study, we have examined the correlation between alpha-BTX binding and the structure of the binding site domain of AChR from the hedgehog, shrew, cat, and human. Fragments of the AChR alpha-subunit corresponding to residues 122-205 from these species were cloned, sequenced, and expressed in Escherichia coli. The hedgehog fragment does not bind alpha-BTX, in common with the snake and mongoose AChR, and the human fragment is a partial binder. The shrew and cat fragments bind alpha-BTX to a similar extent as the mouse fragment. The hedgehog and human AChRs have nonaromatic amino acid residues at positions 187 and 189 of the alpha-subunit, as is seen with the "toxin resistant" snake and mongoose, and in contrast with the "toxin binders", which have aromatic residues at these two positions.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Analysis of ligand binding to the synthetic dodecapeptide 185-196 of the acetylcholine receptor alpha subunit.

    PubMed Central

    Neumann, D; Barchan, D; Fridkin, M; Fuchs, S

    1986-01-01

    A synthetic dodecapeptide corresponding to residues 185-196 of the Torpedo acetylcholine receptor alpha subunit, which contains the adjacent cysteine residues at positions 192 and 193, was recently shown by us to contain the essential elements for alpha-bungarotoxin binding. In the present study, we have used Sepharose-linked peptides for quantitative analysis of the cholinergic binding properties of this and other synthetic peptides. Sepharose-linked peptides corresponding to residues 1-20, 126-143, 143-158, 169-181, 185-196, 193-210, and 394-409 of the alpha subunit of Torpedo acetylcholine receptor, as well as a peptide corresponding to residues 185-196 of the alpha subunit of human acetylcholine receptor, were tested for their toxin-binding capacity. Of these immobilized peptides, only peptide 185-196 of the Torpedo acetylcholine receptor bound toxin significantly, thus verifying that this synthetic peptide contains essential components of the receptor toxin-binding site. Analysis of toxin binding to the peptide yielded a dissociation constant of 3.5 X 10(-5) M. This binding was inhibited by various cholinergic ligands. The inhibition potency obtained was alpha-bungarotoxin greater than Naja naja siamensis toxin greater than d-tubocurarine greater than decamethonium greater than acetylcholine greater than carbamoylcholine. This pharmacological profile resembles that of the nicotinic acetylcholine receptor and therefore suggests that the synthetic dodecapeptide also includes the neurotransmitter binding site. Reduction and carboxymethylation of the cysteine residues on peptide 185-196 inhibit its capacity to bind toxin, demonstrating that an intact disulfide is required for toxin binding. A decrease in toxin binding was also obtained following chemical modification of the tryptophan residue at position 187, thus implying its possible involvement in toxin binding. The failure to detect binding of toxin to the corresponding human sequence 185-196, in which the

  7. Analysis of ligand binding to the synthetic dodecapeptide 185-196 of the acetylcholine receptor alpha subunit.

    PubMed

    Neumann, D; Barchan, D; Fridkin, M; Fuchs, S

    1986-12-01

    A synthetic dodecapeptide corresponding to residues 185-196 of the Torpedo acetylcholine receptor alpha subunit, which contains the adjacent cysteine residues at positions 192 and 193, was recently shown by us to contain the essential elements for alpha-bungarotoxin binding. In the present study, we have used Sepharose-linked peptides for quantitative analysis of the cholinergic binding properties of this and other synthetic peptides. Sepharose-linked peptides corresponding to residues 1-20, 126-143, 143-158, 169-181, 185-196, 193-210, and 394-409 of the alpha subunit of Torpedo acetylcholine receptor, as well as a peptide corresponding to residues 185-196 of the alpha subunit of human acetylcholine receptor, were tested for their toxin-binding capacity. Of these immobilized peptides, only peptide 185-196 of the Torpedo acetylcholine receptor bound toxin significantly, thus verifying that this synthetic peptide contains essential components of the receptor toxin-binding site. Analysis of toxin binding to the peptide yielded a dissociation constant of 3.5 X 10(-5) M. This binding was inhibited by various cholinergic ligands. The inhibition potency obtained was alpha-bungarotoxin greater than Naja naja siamensis toxin greater than d-tubocurarine greater than decamethonium greater than acetylcholine greater than carbamoylcholine. This pharmacological profile resembles that of the nicotinic acetylcholine receptor and therefore suggests that the synthetic dodecapeptide also includes the neurotransmitter binding site. Reduction and carboxymethylation of the cysteine residues on peptide 185-196 inhibit its capacity to bind toxin, demonstrating that an intact disulfide is required for toxin binding. A decrease in toxin binding was also obtained following chemical modification of the tryptophan residue at position 187, thus implying its possible involvement in toxin binding. The failure to detect binding of toxin to the corresponding human sequence 185-196, in which the

  8. The Protective Effect of Alpha 7 Nicotinic Acetylcholine Receptor Activation on Critical Illness and Its Mechanism

    PubMed Central

    REN, Chao; TONG, Ya-lin; LI, Jun-cong; LU, Zhong-qiu; YAO, Yong-ming

    2017-01-01

    Critical illnesses and injuries are recognized as major threats to human health, and they are usually accompanied by uncontrolled inflammation and dysfunction of immune response. The alpha 7 nicotinic acetylcholine receptor (α7nAchR), which is a primary receptor of cholinergic anti-inflammatory pathway (CAP), exhibits great benefits for critical ill conditions. It is composed of 5 identical α7 subunits that form a central pore with high permeability for calcium. This putative structure is closely associated with its functional states. Activated α7nAChR exhibits extensive anti-inflammatory and immune modulatory reactions, including lowered pro-inflammatory cytokines levels, decreased expressions of chemokines as well as adhesion molecules, and altered differentiation and activation of immune cells, which are important in maintaining immune homeostasis. Well understanding of the effects and mechanisms of α7nAChR will be of great value in exploring effective targets for treating critical diseases. PMID:28123345

  9. The endogenous cannabinoid anandamide inhibits alpha7 nicotinic acetylcholine receptor-mediated responses in Xenopus oocytes.

    PubMed

    Oz, Murat; Ravindran, Arippa; Diaz-Ruiz, Oscar; Zhang, Li; Morales, Marisela

    2003-09-01

    The effect of the endogenous cannabinoid ligand anandamide on the function of the cloned alpha7 subunit of the nicotinic acetylcholine (ACh) receptor expressed in Xenopus oocytes was investigated by using the two-electrode voltage-clamp technique. Anandamide reversibly inhibited nicotine (10 microM) induced-currents in a concentration-dependent manner (10 nM to 30 microM), with an IC50 value of 229.7 +/- 20.4 nM. The effect of anandamide was neither dependent on the membrane potential nor meditated by endogenous Ca2+ dependent Cl- channels since it was unaffected by intracellularly injected BAPTA and perfusion with Ca2+-free bathing solution containing 2 mM Ba2+. Anandamide decreased the maximal nicotine-induced responses without significantly affecting its potency, indicating that it acts as a noncompetitive antagonist on nicotinic acetylcholine (nACh) alpha7 receptors. This effect was not mediated by CB1 or CB2 receptors, as neither the selective CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR 141716A) nor CB2 receptor antagonist N-((1S)-endo-1,3,3-trimethyl-bicyclo-heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR 144528) reduced the inhibition by anandamide. In addition, inhibition of nicotinic responses by anandamide was not sensitive to either pertussis toxin treatment or to the membrane permeable cAMP analog 8-Br-cAMP (0.2 mM). Inhibitors of enzymes involved in anandamide metabolism including phenylmethylsulfonyl fluoride, superoxide dismutase, and indomethacin, or the anandamide transport inhibitor AM404 did not prevent anandamide inhibition of nicotinic responses, suggesting that anandamide itself acted on nicotinic receptors. In conclusion, these results demonstrate that the endogenous cannabinoid anandamide inhibits the function of nACh alpha7 receptors expressed in Xenopus oocytes in a cannabinoid receptor-independent and

  10. Activation and desensitization of nicotinic alpha7-type acetylcholine receptors by benzylidene anabaseines and nicotine.

    PubMed

    Papke, Roger L; Kem, William R; Soti, Ferenc; López-Hernández, Gretchen Y; Horenstein, Nicole A

    2009-05-01

    Nicotinic receptor activation is inextricably linked to desensitization. This duality affects our ability to develop useful therapeutics targeting nicotinic acetylcholine receptor (nAChR). Nicotine and some alpha7-selective experimental partial agonists produce a transient activation of alpha7 receptors followed by a period of prolonged residual inhibition or desensitization (RID). The object of the present study was to determine whether RID was primarily due to prolonged desensitization or due to channel block. To make this determination, we used agents that varied significantly in their production of RID and two alpha7-selective positive allosteric modulators (PAMs): 5-hydroxyindole (5HI), a type 1 PAM that does not prevent desensitization; and 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxanol-3-yl)-urea (PNU-120596), a type 2 PAM that reactivates desensitized receptors. The RID-producing compounds nicotine and 3-(2,4-dimethoxybenzylidene)anabaseine (diMeOBA) could obscure the potentiating effects of 5HI. However, through the use of nicotine, diMeOBA, and the RID-negative compound 3-(2,4-dihydroxybenzylidene)anabaseine (diOHBA) in combination with PNU-120596, we confirmed that diMeOBA produces short-lived channel block of alpha7 but that RID is because of the induction of a desensitized state that is stable in the absence of PNU-120596 and activated in the presence of PNU-120596. In contrast, diOHBA produced channel block but only readily reversible desensitization, whereas nicotine produced desensitization that could be converted into activation by PNU-120596 but no demonstrable channel block. Steady-state currents through receptors that would otherwise be desensitized could also be produced by the application of PNU-120596 in the presence of a physiologically relevant concentration of choline (60 microM), which may be significant for the therapeutic development of type 2 PAMs.

  11. Synthetic. cap alpha. subunit peptide 125-147 of human nicotinic acetylcholine receptor induces antibodies to native receptor

    SciTech Connect

    McCormick, D.J.; Griesmann, G.E.; Huang, Z.; Lennon, V.A.

    1986-03-05

    A synthetic peptide corresponding to residues 125-147 of the Torpedo acetylcholine receptor (AChR) ..cap alpha.. subunit proved to be a major antigenic region of the AChR. Rats inoculated with 50 ..mu..g of peptide (T ..cap alpha.. 125-147) developed T cell immunity and antibodies to native AChR and signs of experimental autoimmune myasthenia gravis. They report the synthesis and preliminary testing of a disulfide-looped peptide comprising residues 125-147 of the human AChR ..cap alpha.. subunit. Peptide H ..cap alpha.. 125-147 differs from T ..cap alpha.. 125-147 at residues 139 (Glu for Gln) and 143 (Ser for Thr). In immunoprecipitation assays, antibodies to Torpedo AChR bound /sup 125/I-labelled H..cap alpha.. 125-147 antibody bound H..cap alpha.. 125-147, but monoclonal antibodies to an immunodominant region of native AChR bound neither H..cap alpha.. 125-147 nor T ..cap alpha.. 125-147. Rats immunized with H ..cap alpha.. 125-147 produced anti-mammalian muscle AChR antibodies that induced modulation of AChRs from cultured human myotubes. Thus, region 125-147 of the human AChR ..cap alpha.. subunit is extracellular in muscle, and is both antigenic and immunogenic. It remains to be determined whether or not autoantibodies to this region may in part cause the weakness or myasthenia gravis in man.

  12. Cloning and mapping of the mouse {alpha}7-neuronal nicotinic acetylcholine receptor

    SciTech Connect

    Orr-Urtreger, A.; Baldini, A.; Beaudet, A.L.

    1995-03-20

    We report the isolation of cDNA clones for the mouse {alpha}7 neuronal nicotinic acetylcholine receptor subunit (gene symbol Acra7), the only nicotinic receptor subunit known to bind a-bungarotoxin in mammalian brain. This gene may have relevance to nicotine sensitivity and to some electrophysiologic findings in schizophrenia. The mouse {alpha}7 subunit gene encodes a protein of 502 amino acids with substantial identity to the rat (99.6%), human (92.8%), and chicken (87.5%) amino acid sequences. The {alpha}7 gene was mapped to mouse chromosome 7 near the p locus with the following gene order from proximal to distal: Myod1-3.5 {+-}1.7 cM-Gas2-0.9 cM {+-} 0.9 cM-D7Mit70-1.8 {+-} 1.2 cM- Acra7-4.4 {+-}1.0 cM-Hras1-ps11/Igf1r/Snrp2a. The human gene was confirmed to map to the homologous region of human chromosome 15q13-q14. 26 refs., 3 figs.

  13. Exon-intron structure of the human neuronal nicotinic acetylcholine receptor {alpha}4 subunit (CHRNA4)

    SciTech Connect

    Steinlein, O.; Weiland, S.; Stoodt, J.; Propping, P.

    1996-03-01

    The human neuronal nicotinic acetylcholine receptor {alpha}4 subunit gene (CHRNA4) is located in the candidate region for three different phenotypes: benign familial neonatal convulsions, autosomal dominant nocturnal frontal lobe epilepsy, and low-voltage EEG. Recently, a missense mutation in transmembrane domain 2 of CHRNA4 was found to be associated with autosomal dominant nocturnal frontal lobe epilepsy in one extended pedigree. We have determined the genomic organization of CHRNA4, which consists of six exons distributed over approximately 17 kb of genomic DNA. The nucleotide sequence obtained from the genomic regions adjacent to the exon boundaries enabled us to develop a set of primer pairs for PCR amplification of the complete coding region. The sequence analysis provides the basis for a comprehensive mutation screening of CHRNA4 in the above-mentioned phenotypes and possibly in other types of idopathic epilepsies. 29 refs., 3 figs., 1 tab.

  14. Snake acetylcholine receptor: cloning of the domain containing the four extracellular cysteines of the alpha subunit.

    PubMed Central

    Neumann, D; Barchan, D; Horowitz, M; Kochva, E; Fuchs, S

    1989-01-01

    The acetylcholine receptor (AcChoR) at the neuromuscular junction of elapid snakes binds cholinergic ligands but unlike other muscle AcChoRs does not bind alpha-bungarotoxin. Numerous studies indicate that the ligand-binding site of the AcChoR includes cysteine residues at positions 192 and 193 of the alpha subunit. We have previously shown that a synthetic dodecapeptide corresponding to residues 185-196 of the Torpedo AcChoR alpha subunit contains the essential elements of the ligand-binding site. In an attempt to elucidate the structural basis for the precise binding properties of snake AcChoR, we sequenced a portion of the snake AcChoR alpha subunit. First, a mouse AcChoR alpha-subunit cDNA probe was used to screen a size-selected snake (Natrix tessellata) genomic library. A genomic clone was isolated and was found to contain sequences homologous to the exon including the first two cysteines (Cys-128 and -142) of AcChoR alpha subunit. The domain of the alpha subunit from Natrix and cobra AcChoR (amino acid residues 119-222), which contains the four extracellular cysteines (128, 142, 192, and 193), was amplified by reverse transcription of mRNA and the polymerase chain reaction and then sequenced. The deduced amino acid sequence showed that the snake alpha subunit contains the two tandem cysteines at positions 192 and 193, resembling all other AcChoR alpha subunits. Sequence comparison revealed that the cloned region of the snake alpha subunit is highly homologous (75-80%) to other muscle AcChoRs and not to neuronal AcChoR, which also does not bind alpha-bungarotoxin. In the presumed ligand-binding site, in the vicinity of Cys-192 and Cys-193, four major substitutions occur in the snake sequence--at positions 184 (Trp----Phe), 185 (Lys----Trp), 187 (Trp----Ser), and 194 (Pro----Leu). In addition, Asn-189 is a putative N-glycosylation site, present only in the snake. These changes, or part of them, may explain the lack of alpha-bungarotoxin-binding to snake Ac

  15. Snake acetylcholine receptor: cloning of the domain containing the four extracellular cysteines of the alpha subunit.

    PubMed

    Neumann, D; Barchan, D; Horowitz, M; Kochva, E; Fuchs, S

    1989-09-01

    The acetylcholine receptor (AcChoR) at the neuromuscular junction of elapid snakes binds cholinergic ligands but unlike other muscle AcChoRs does not bind alpha-bungarotoxin. Numerous studies indicate that the ligand-binding site of the AcChoR includes cysteine residues at positions 192 and 193 of the alpha subunit. We have previously shown that a synthetic dodecapeptide corresponding to residues 185-196 of the Torpedo AcChoR alpha subunit contains the essential elements of the ligand-binding site. In an attempt to elucidate the structural basis for the precise binding properties of snake AcChoR, we sequenced a portion of the snake AcChoR alpha subunit. First, a mouse AcChoR alpha-subunit cDNA probe was used to screen a size-selected snake (Natrix tessellata) genomic library. A genomic clone was isolated and was found to contain sequences homologous to the exon including the first two cysteines (Cys-128 and -142) of AcChoR alpha subunit. The domain of the alpha subunit from Natrix and cobra AcChoR (amino acid residues 119-222), which contains the four extracellular cysteines (128, 142, 192, and 193), was amplified by reverse transcription of mRNA and the polymerase chain reaction and then sequenced. The deduced amino acid sequence showed that the snake alpha subunit contains the two tandem cysteines at positions 192 and 193, resembling all other AcChoR alpha subunits. Sequence comparison revealed that the cloned region of the snake alpha subunit is highly homologous (75-80%) to other muscle AcChoRs and not to neuronal AcChoR, which also does not bind alpha-bungarotoxin. In the presumed ligand-binding site, in the vicinity of Cys-192 and Cys-193, four major substitutions occur in the snake sequence--at positions 184 (Trp----Phe), 185 (Lys----Trp), 187 (Trp----Ser), and 194 (Pro----Leu). In addition, Asn-189 is a putative N-glycosylation site, present only in the snake. These changes, or part of them, may explain the lack of alpha-bungarotoxin-binding to snake AcChoR.

  16. Profile of the alpha-bungarotoxin-binding regions on the extracellular part of the alpha-chain of Torpedo californica acetylcholine receptor.

    PubMed Central

    Mulac-Jericevic, B; Atassi, M Z

    1987-01-01

    The continuous alpha-neurotoxin-binding regions on the extracellular part (residues 1-210) of the alpha-chain of Torpedo californica acetylcholine receptor were localized by reaction of 125I-labelled alpha-bungarotoxin with synthetic overlapping peptides spanning this entire part of the chain. The specificity of the binding was confirmed by inhibition with unlabelled toxin and, for appropriate peptides, with unlabelled anti-(acetylcholine receptor) antibodies. Five toxin-binding regions were localized within residues 1-10, 32-41, 100-115, 122-150 and 182-198. The third, fourth and fifth (and to a lesser extent the first and second) toxin-binding regions overlapped with regions recognized by anti-(acetylcholine receptor) antibodies. The five toxin-binding regions may be distinct sites or, alternatively, different 'faces' in one (or more) sites. PMID:3435488

  17. Characterization of the retina in the alpha7 nicotinic acetylcholine receptor knockout mouse

    NASA Astrophysics Data System (ADS)

    Smith, Marci L.

    Acetylcholine receptors (AChRs) are involved in visual processing and are expressed by inner retinal neurons in all species studied to date (Keyser et al., 2000; Dmitrieva et al., 2007; Liu et al., 2009), but their distribution in the mouse retina remains unknown. Reductions in alpha7 nicotinic AChRs (nAChRs) are thought to contribute to memory and visual deficits observed in Alzheimer's and schizophrenia (Coyle et al., 1983; Nordberg et al., 1999; Leonard et al., 2006). However, the alpha7 nAChR knockout (KO) mouse has a mild phenotype (Paylor et al., 1998; Fernandes et al., 2006; Young et al., 2007; Origlia et al., 2012). The purpose of this study was to determine the expression of AChRs in wildtype (WT) mouse retina and to assess whether up-regulation of other AChRs in the alpha7 nAChR KO retina may explain the minimal deficits described in the KO mouse. Reverse-transcriptase PCR (RT-PCR) showed that mRNA transcripts for alpha2-7, alpha 9, alpha10, beta2-4 nAChR subunits and m1-m5 muscarinic AChR (mAChR) subtypes were present in WT murine retina. Western blot analysis confirmed the presence of alpha3-5, alpha9, and m1-m5 AChR proteins and immunohistochemical analysis demonstrated nAChR and mAChR proteins expressed by subsets of bipolar, amacrine and ganglion cells. This is the first reported expression of alpha9 and alpha10 nAChR transcripts and alpha9 nAChR proteins in the retina of any species. Quantitative RT-PCR (qPCR) showed changes in AChR transcript expression in the alpha7 nAChR KO mouse retina relative to WT. Within whole retina alpha2, alpha9, alpha10, beta4, m1 and m4 AChR transcripts were up-regulated, while alpha5 nAChR transcripts were down-regulated. However, cell populations showed subtle differences; m4 mAChR transcripts were up-regulated in the ganglion cell layer and outer portion of the inner nuclear layer (oINL),while beta4 nAChR transcript up-regulation was limited to the oINL. Surprisingly, alpha2, alpha9, beta4, m2 and m4 transcripts were

  18. Inhibitory effects of tramadol on nicotinic acetylcholine receptors in adrenal chromaffin cells and in Xenopus oocytes expressing alpha 7 receptors.

    PubMed

    Shiraishi, Munehiro; Minami, Kouichiro; Uezono, Yasuhito; Yanagihara, Nobuyuki; Shigematsu, Akio; Shibuya, Izumi

    2002-05-01

    1. Tramadol has been used clinically as an analgesic; however, the mechanism of its analgesic effects is still unknown. 2. We used bovine adrenal chromaffin cells to investigate effects of tramadol on catecholamine secretion, nicotine-induced cytosolic Ca(2+) concentration ([Ca(2+)](i)) increases and membrane current changes. We also investigated effects of tramadol on alpha7 nicotinic acetylcholine receptors (AChRs) expressed in Xenopus oocytes. 3. Tramadol concentration-dependently suppressed carbachol-induced catecholamine secretion to 60% and 27% of the control at the concentration of 10 and 100 microM, respectively, whereas it had little effect on veratridine- or high K(+)-induced catecholamine secretion. 4. Tramadol also suppressed nicotine-induced ([Ca(2+)](i)) increases in a concentration-dependent manner. Tramadol inhibited nicotine-induced inward currents, and the inhibition was unaffected by the opioid receptor antagonist naloxone. 5. Tramadol inhibited nicotinic currents carried by alpha7 receptors expressed in Xenopus oocytes. 6. Tramadol inhibited both alpha-bungarotoxin-sensitive and -insensitive nicotinic currents in bovine adrenal chromaffin cells. 7. In conclusion, tramadol inhibits catecholamine secretion partly by inhibiting nicotinic AChR functions in a naloxone-insensitive manner and alpha7 receptors are one of those inhibited by tramadol.

  19. Acetylcholine receptor alpha-subunit and myogenin mRNAs in thymus and thymomas.

    PubMed Central

    Kornstein, M. J.; Asher, O.; Fuchs, S.

    1995-01-01

    Myasthenia gravis is an autoimmune disorder characterized in most cases by serological antibody against the acetylcholine receptor (AChR). Evidence for intrathymic localization of AChR suggests that the thymus has an important role in the pathogenesis of this disorder. Using reverse transcription followed by the polymerase chain reaction, we have demonstrated AChR alpha-subunit mRNA in thymuses and thymomas from patients with and without myasthenia gravis. We have also studied the expression of myogenin which is known to be involved in the regulation of AChR expression. By using the reverse transcription polymerase chain reaction, we found myogenin mRNAs in all of the thymuses and thymomas. Thus, both AChR alpha-subunit and myogenin mRNA are present in all of these specimens. By immunohistochemistry myoid cells (desmin and myoglobin positive) were present in all (four of four) thymuses studied and in two of five thymomas. Thus, in thymomas, nonmyoid cells might express both AChR and myogenin. These results indicate that cells within the thymus and thymoma express AChR and its regulatory protein myogenin and that such cells, under certain conditions, might play a role in the triggering of myasthenia gravis. Images Figure 2 Figure 3 PMID:7778671

  20. The mongoose acetylcholine receptor alpha-subunit: analysis of glycosylation and alpha-bungarotoxin binding.

    PubMed

    Asher, O; Jensen, B S; Lupu-Meiri, M; Oron, Y; Fuchs, S

    1998-04-17

    The mongoose AChR alpha-subunit has been cloned and shown to be highly homologous to other AChR alpha-subunits, with only six differences in amino acid residues at positions that are conserved in animal species that bind alpha-bungarotoxin (alpha-BTX). Four of these six substitutions cluster in the ligand binding site, and one of them, Asn-187, forms a consensus N-glycosylation site. The mongoose glycosylated alpha-subunit has a higher apparent molecular mass than that of the rat glycosylated alpha-subunit, probably resulting from the additional glycosylation at Asn-187 of the mongoose subunit. The in vitro translated mongoose alpha-subunit, in a glycosylated or non-glycosylated form, does not bind alpha-BTX, indicating that lack of alpha-BTX binding can be achieved also in the absence of glycosylation.

  1. The molecular cloning and characterisation of cDNA coding for the alpha subunit of the acetylcholine receptor.

    PubMed Central

    Sumikawa, K; Houghton, M; Smith, J C; Bell, L; Richards, B M; Barnard, E A

    1982-01-01

    A rare cDNA coding for most of the alpha subunit of the Torpedo nicotinic acetylcholine receptor has been cloned into bacteria. The use of a mismatched oligonucleotide primer of reverse transcriptase facilitated the design of an efficient, specific probe for recombinant bacteria. DNA sequence analysis has enabled the elucidation of a large part of the polypeptide primary sequence which is discussed in relation to its acetylcholine binding activity and the location of receptor within the plasma membrane. When used as a radioactive probe, the cloned cDNA binds specifically to a single Torpedo mRNA species of about 2350 nucleotides in length but fails to show significant cross-hybridisation with alpha subunit mRNA extracted from cat muscle. Images PMID:6183641

  2. Structural Characterization of the Putative Cholinergic Binding Region alpha(179-201) of the Nicotinic Acetylcholine Receptor. Part 1. Review and Experimental Design.

    DTIC Science & Technology

    1993-04-01

    alpha-subunit of the acetylcholine receptor. Proc. Natl. Acad. Sci USA, vol. 82, pp. 3490-3493, 1985 Neumann, D, Barchan , D., Safran, A., GershoniJ., and...pp. 3008-3011, 1986 MAY. Neumann, D., Barchan , D., Fuchs, S., Analysis of ligand binding to the synthetic dodeca-peptide 185-1% of the acetylcholine

  3. Neural regulation of muscle acetylcholine receptor epsilon- and alpha- subunit gene promoters in transgenic mice

    PubMed Central

    1993-01-01

    The effects of denervation were investigated in mice with transgenes containing promoter elements from the muscle acetylcholine receptor epsilon- and alpha-subunit genes. The promoter sequences were coupled to a nuclear localization signal-beta-galactosidase fusion gene (nlacZ) as a reporter. While many postsynaptic specializations form in the embryo, expression of the epsilon subunit is induced during the first two postnatal weeks. When muscles were denervated at birth, before the onset of epsilon expression, epsilon nlacZ still appeared at the former synaptic sites on schedule. This result suggests that the nerve leaves a localized "trace" in the muscle that can continue to regulate transcription. An additional finding was that epsilon nlacZ expression was much stronger in denervated than in intact muscles. This suggests that the epsilon promoter is similar to the other subunits in containing elements that are activated on cessation of neural activity. However, even after denervation, epsilon nlacZ expression was always confined to the synaptic region whereas alpha nlacZ expression increased in nuclei along the entire length of the fiber. This suggests that while the epsilon gene is similar in its activity dependence to other subunit genes, it is unique in that local nerve-derived signals are essential for its expression. Consequently, inactivity enhances epsilon expression only in synaptic nuclei where such signals are present, but enhances expression throughout the muscle fiber. Truncations and an internal deletion of the epsilon promoter indicate that cis-elements essential for the response to synaptic signals are contained within 280 bp of the transcription start site. In contrast to these results in young animals, denervation in older animals leads to an unexpected reduction in nlacZ activity. However, mRNA measurements indicated that transgene expression was increased in these animals. This discordance between nlacZ mRNA and enzyme activity, demonstrates a

  4. Expression of the alpha-bungarotoxin binding site of the nicotinic acetylcholine receptor by Escherichia coli transformants.

    PubMed Central

    Gershoni, J M

    1987-01-01

    Restriction fragments of DNA derived from a cDNA clone of the alpha subunit of the acetylcholine receptor were subcloned in Escherichia coli by using the trpE fusion vector, pATH2. Transformants expressing the amino acid sequences 166-315 or 166-200 are shown to produce a chimeric protein that bound alpha-bungarotoxin. Moreover, it is shown that sufficient amounts of toxin-binding proteins can be generated by individual colonies of bacteria. This provides a new approach for gene selection via functional expression--i.e., ligand overlays of colony blots. Images PMID:3295881

  5. Activity regulates the levels of acetylcholine receptor alpha-subunit mRNA in cultured chicken myotubes.

    PubMed Central

    Klarsfeld, A; Changeux, J P

    1985-01-01

    In vitro blocking the spontaneous activity of primary cultures of chicken embryo myotubes with tetrodotoxin increases approximately equal to 2-fold their content in surface acetylcholine receptor. To investigate this effect at the level of gene expression, chicken genomic DNA sequences coding for the acetylcholine receptor alpha subunit were isolated and characterized. They were shown to belong to a single-copy, polymorphic gene with at least two alleles in the chicken strain utilized. Probes derived from these genomic clones were used to quantitate levels of alpha-subunit mRNA. In culture, a 2-day exposure to tetrodotoxin increased these mRNA levels up to 13-fold, a value similar to that observed after denervation of chick leg muscle (approximately equal to 17-fold). Actin mRNA levels varied little in any of these experiments. These results support the notion that membrane electrical activity affects acetylcholine receptor expression by regulating the accumulation of the corresponding mRNAs. Images PMID:2989833

  6. Untranslated region-dependent exclusive expression of high-sensitivity subforms of alpha4beta2 and alpha3beta2 nicotinic acetylcholine receptors.

    PubMed

    Briggs, Clark A; Gubbins, Earl J; Marks, Michael J; Putman, C Brent; Thimmapaya, Rama; Meyer, Michael D; Surowy, Carol S

    2006-07-01

    alpha4beta2 nicotinic acetylcholine receptors (nAChRs) are recognized as the principal nicotine binding site in brain. Recombinant alpha4beta2 nAChR demonstrate biphasic concentration-response relationships with low- and high-EC50 components. This study shows that untranslated regions (UTR) can influence expression of high-sensitivity subforms of alpha4beta2 and alpha3beta2 nAChR. Oocytes injected with alpha4 and beta2 RNA lacking UTR expressed biphasic concentration-response relationships for acetylcholine with high-sensitivity EC50 values of 0.5 to 2.5 microM (14-24% of the population) and low-sensitivity EC50 values of 110 to 180 microM (76-86%). In contrast, message with UTR expressed exclusively the high-sensitivity alpha4beta2 nAChR subform with an acetylcholine EC50 value of 2.2 microM. Additional studies revealed pharmacological differences between high- and low-sensitivity alpha4beta2 subforms. Whereas the antagonists dihydro-beta-erythroidine (IC50 of 3-6 nM) and methyllycaconitine (IC50 of 40-135 nM) were not selective between high- and low-sensitivity alpha4beta2, chlorisondamine, mecamylamine, and d-tubocurarine were, respectively, 100-, 8-, and 5-fold selective for the alpha4beta2 subform with low sensitivity to acetylcholine. Conversely, agonists that selectively activated the high-sensitivity alpha4beta2 subform with respect to efficacy as well as potency were identified. Furthermore, two of these agonists were shown to activate mouse brain alpha4beta2 as well as the ferret high-sensitivity alpha4beta2 expressed in Xenopus laevis oocytes. With the use of UTR-containing RNA, exclusive expression of a novel high-sensitivity alpha3beta2 nAChR was also achieved. These studies 1) provide further evidence for the existence of multiple subforms of alpha4beta2 nAChR, 2) extend that to alpha3beta2 nAChR, 3) demonstrate UTR influence on beta2-containing nAChR properties, and 4) reveal compounds that interact with alpha4beta2 in a subform-selective manner.

  7. Role of the large cytoplasmic loop of the alpha 7 neuronal nicotinic acetylcholine receptor subunit in receptor expression and function.

    PubMed

    Valor, Luis M; Mulet, José; Sala, Francisco; Sala, Salvador; Ballesta, Juan J; Criado, Manuel

    2002-06-25

    The role of the large intracellular loop of the nicotinic acetylcholine receptor (nAChR) alpha7 subunit in the expression of functional channels was studied. For this purpose, systematic deletions and substitutions were made throughout the loop and the ability of the mutated alpha7 subunits to support expression of functional nAChRs at the Xenopus oocyte membrane was tested. Surface nAChR expression was abolished upon removal of sequences at two regions, a 29-amino acid segment close to the N-terminus of the loop (amino acids 297-325) and adjacent to the third transmembrane region and an 11-amino acid segment near the fourth transmembrane region. Some residues (amino acids 317-322) within the 29 amino acids N-terminal segment could be substituted by others but not deleted without loss of expression, suggesting that a certain structure, determined by the number of amino acids rather than by their identity, has to be maintained in this region. The contiguous sequence M323 K324 R325 did not tolerate deletions and substitutions. Removal of the rest of the cytoplasmic loop was not deleterious; even higher expression levels (2-4-fold) were obtained upon large deletions of the loop (Delta399-432 and Delta339-370). High expression levels were observed provided that a minimal sequence of three amino acids (E371, G372, and M373) was present. In addition, some electrophysiological properties of mutant nAChRs were modified. Substitution of the EGM sequence by other protein segments produced a variety of effects, but, in general, insertions were not well tolerated, suggesting the existence of tight structural restrictions in the large cytoplasmic region of the rat alpha7 subunit.

  8. An allosteric modulator of the alpha7 nicotinic acetylcholine receptor possessing cognition-enhancing properties in vivo.

    PubMed

    Timmermann, Daniel B; Grønlien, Jens Halvard; Kohlhaas, Kathy L; Nielsen, Elsebet Ø; Dam, Eva; Jørgensen, Tino D; Ahring, Philip K; Peters, Dan; Holst, Dorte; Christensen, Jeppe K; Chrsitensen, Jeppe K; Malysz, John; Briggs, Clark A; Gopalakrishnan, Murali; Olsen, Gunnar M

    2007-10-01

    Augmentation of nicotinic alpha7 receptor function is considered to be a potential therapeutic strategy aimed at ameliorating cognitive and mnemonic dysfunction in relation to debilitating pathological conditions, such as Alzheimer's disease and schizophrenia. In the present report, a novel positive allosteric modulator of the alpha7 nicotinic acetylcholine receptor (nAChR), 1-(5-chloro-2-hydroxy-phenyl)-3-(2-chloro-5-trifluoromethyl-phenyl)-urea (NS1738), is described. NS1738 was unable to displace or affect radioligand binding to the agonist binding site of nicotinic receptors, and it was devoid of effect when applied alone in electrophysiological paradigms. However, when applied in the presence of acetylcholine (ACh), NS1738 produced a marked increase in the current flowing through alpha7 nAChRs, as determined in both oocyte electrophysiology and patch-clamp recordings from mammalian cells. NS1738 acted by increasing the peak amplitude of ACh-evoked currents at all concentrations; thus, it increased the maximal efficacy of ACh. Oocyte experiments indicated an increase in ACh potency as well. NS1738 had only marginal effects on the desensitization kinetics of alpha7 nAChRs, as determined from patch-clamp studies of both transfected cells and cultured hippocampal neurons. NS1738 was modestly brain-penetrant, and it was demonstrated to counteract a (-)-scopolamine-induced deficit in acquisition of a water-maze learning task in rats. Moreover, NS1738 improved performance in the rat social recognition test to the same extent as (-)-nicotine, demonstrating that NS1738 is capable of producing cognitive enhancement in vivo. These data support the notion that alpha7 nAChR allosteric modulation may constitute a novel pharmacological principle for the treatment of cognitive dysfunction.

  9. Antipeptide monoclonal antibodies inhibit the binding of rabies virus glycoprotein and alpha-bungarotoxin to the nicotinic acetylcholine receptor.

    PubMed

    Bracci, L; Antoni, G; Cusi, M G; Lozzi, L; Niccolai, N; Petreni, S; Rustici, M; Santucci, A; Soldani, P; Valensin, P E

    1988-09-01

    It has been reported that binding to muscle nicotinic acetylcholine receptor at the post-synaptic membrane is an important event of the rabies virus neurotropism. The binding site can be located within the 190-203 region of the virus glycoprotein sharing a high degree of homology with the "toxic loop" of the curare-mimetic snake neurotoxins. We have synthesized a tetradecapeptide corresponding to this glycoprotein region and used it, following conjugation with an immunogenic carrier to raise MAbs. We found that some MAbs raised against the peptide were able to recognize both the virus glycoprotein and the snake neurotoxin alpha-bungarotoxin; moreover, they can inhibit the binding of rabies virus glycoprotein and alpha-bungarotoxin to the nicotinic acetylcholine receptor extracted from the electric organs of Torpedo marmorata. On the basis of this cross-reactivity, we suggest that rabies virus glycoprotein and curare-mimetic snake neurotoxins share three-dimensionally similar structures in order to bind to the nicotinic cholinergic receptor. The potential use of the immunogenic properties of the peptide for the rational design of a synthetic vaccine against rabies is proposed.

  10. Rabies virus binding to the nicotinic acetylcholine receptor alpha subunit demonstrated by virus overlay protein binding assay.

    PubMed

    Gastka, M; Horvath, J; Lentz, T L

    1996-10-01

    A virus overlay protein binding assay was used to study binding of 125I-labelled rabies virus to the acetylcholine receptor (AChR) from Torpedo californica electric organ membranes. After gel electrophoresis of electric organ membranes and transfer of proteins to nitrocellulose, 125I-labelled alpha-bungarotoxin, a curaremimetic neurotoxin, bound to a 40 kDa band and 125I-labelled rabies virus bound to 51 kDa and 40 kDa bands. Binding of rabies virus to the 40 kDa band was inhibited by unlabelled alpha-bungarotoxin. In blots of affinity-purified AChR, labelled virus bound to the 40 kDa alpha subunit and was competed by alpha-bungarotoxin. Based on binding of rabies virus to the alpha subunit and the ability of alpha-bungarotoxin to compete for binding, rabies virus appears to bind to the neurotoxin-binding site of the nicotinic AChR alpha subunit.

  11. Antibodies to synthetic peptides as probes for the binding site on the alpha subunit of the acetylcholine receptor.

    PubMed Central

    Neumann, D; Gershoni, J M; Fridkin, M; Fuchs, S

    1985-01-01

    Synthetic peptides and their respective antibodies were used in an attempt to localize and identify the ligand-binding site of the nicotinic acetylcholine receptor. Two peptides of the receptor alpha subunit were synthesized, the first corresponding to the NH2-terminal domain (positions 1-20) and the other, to a segment (residues 126-143) that contains the first two cysteine residues. Specific antipeptide antibodies were elicited in rabbits after immunization with the peptides conjugated to bovine serum albumin. The antipeptide antibodies thus obtained cross-reacted with the receptor and bound specifically to its alpha subunit. The antipeptide antibodies were used to test whether the peptide sequences corresponded to the alpha-bungarotoxin (alpha-BTX)-binding site. Staphylococcus aureus V8-protease digestion of the isolated receptor alpha subunit generated several fragments. Antipeptide (1-20) and antipeptide (126-143) both bound a 26-kDa fragment, whereas only antipeptide (126-143) bound a 17-kDa fragment. None of these fragments were found to bind alpha-BTX. On the other hand, alpha-BTX bound to an 18-kDa fragment that did not react with either of the antipeptide antibodies. Moreover, the 26-kDa and 17-kDa fragments were also found to contain the endoglycosidase H-susceptible oligosaccharide chain. Our results indicate that the toxin-binding site lies beyond the first possible V8 protease cleavage site after residues 126-143: i.e., Asp-152. This location is in agreement with the possibility that cysteine residues 192 and/or 193 are in close proximity to or contiguous with the ligand-binding site. Images PMID:2582416

  12. Wnt-7a induces presynaptic colocalization of alpha 7-nicotinic acetylcholine receptors and adenomatous polyposis coli in hippocampal neurons.

    PubMed

    Farías, Ginny G; Vallés, Ana S; Colombres, Marcela; Godoy, Juan A; Toledo, Enrique M; Lukas, Ronald J; Barrantes, Francisco J; Inestrosa, Nibaldo C

    2007-05-16

    Nicotinic acetylcholine receptors (nAChRs) contribute significantly to hippocampal function. Alpha7-nAChRs are present in presynaptic sites in hippocampal neurons and may influence transmitter release, but the factors that determine their presynaptic localization are unknown. We report here that Wnt-7a, a ligand active in the canonical Wnt signaling pathway, induces dissociation of the adenomatous polyposis coli (APC) protein from the beta-catenin cytoplasmic complex and the interaction of APC with alpha7-nAChRs in hippocampal neurons. Interestingly, Wnt-7a induces the relocalization of APC to membranes, clustering of APC in neurites, and coclustering of APC with different, presynaptic protein markers. Wnt-7a also increases the number and size of coclusters of alpha7-nAChRs and APC in presynaptic terminals. These short-term changes in alpha7-nAChRs occur in the few minutes after ligand exposure and involve translocation to the plasma membrane without affecting total receptor levels. Longer-term exposure to Wnt-7a increases nAChR alpha7 subunit levels in an APC-independent manner and increases clusters of alpha7-nAChRs in neurites via an APC-dependent process. Together, these results demonstrate that stimulation through the canonical Wnt pathway regulates the presynaptic localization of APC and alpha7-nAChRs with APC serving as an intermediary in the alpha7-nAChR relocalization process. Modulation by Wnt signaling may be essential for alpha7-nAChR expression and function in synapses.

  13. alpha-conotoxin AuIB selectively blocks alpha3 beta4 nicotinic acetylcholine receptors and nicotine-evoked norepinephrine release.

    PubMed

    Luo, S; Kulak, J M; Cartier, G E; Jacobsen, R B; Yoshikami, D; Olivera, B M; McIntosh, J M

    1998-11-01

    Neuronal nicotinic acetylcholine receptors (nAChRs) with putative alpha3 beta4-subunits have been implicated in the mediation of signaling in various systems, including ganglionic transmission peripherally and nicotine-evoked neurotransmitter release centrally. However, progress in the characterization of these receptors has been hampered by a lack of alpha3 beta4-selective ligands. In this report, we describe the purification and characterization of an alpha3 beta4 nAChR antagonist, alpha-conotoxin AuIB, from the venom of the "court cone," Conus aulicus. We also describe the total chemical synthesis of this and two related peptides that were also isolated from the venom. alpha-Conotoxin AuIB blocks alpha3 beta4 nAChRs expressed in Xenopus oocytes with an IC50 of 0.75 microM, a kon of 1.4 x 10(6) min-1 M-1, a koff of 0.48 min-1, and a Kd of 0.5 microM. Furthermore, alpha-conotoxin AuIB blocks the alpha3 beta4 receptor with >100-fold higher potency than other receptor subunit combinations, including alpha2 beta2, alpha2 beta4, alpha3 beta2, alpha4 beta2, alpha4 beta4, and alpha1 beta1 gamma delta. Thus, AuIB is a novel, selective probe for alpha3 beta4 nAChRs. AuIB (1-5 microM) blocks 20-35% of the nicotine-stimulated norepinephrine release from rat hippocampal synaptosomes, whereas nicotine-evoked dopamine release from striatal synaptosomes is not affected. Conversely, the alpha3 beta2-specific alpha-conotoxin MII (100 nM) blocks 33% of striatal dopamine release but not hippocampal norepinephrine release. This suggests that in the respective systems, alpha3 beta4-containing nAChRs mediate norepinephrine release, whereas alpha3 beta2-containing receptors mediate dopamine release.

  14. Alpha7-nicotinic acetylcholine receptors affect growth regulation of human mesothelioma cells: role of mitogen-activated protein kinase pathway.

    PubMed

    Trombino, Sonya; Cesario, Alfredo; Margaritora, Stefano; Granone, PierLuigi; Motta, Giovanni; Falugi, Carla; Russo, Patrizia

    2004-01-01

    This study presents data suggesting that both human mesothelioma (cell lines and human mesothelioma biopsies) and human normal mesothelial cells express receptors for acetylcholine and that stimulation of these receptors by nicotine prompted cell growth via activation of nicotinic cholinergic receptors. Thus, these data demonstrate that: (a) human mesothelioma cells and human biopsies of mesothelioma as well as of normal pleural mesothelial cells express functionally alpha-7 nicotinic acethlycholine receptors, evaluated by alpha-bungarotoxin-FITC binding, receptor binding assay, Western blot, and reverse transcription-PCR; (b) choline acetyltransferase immunostaining is present in mesothelioma cells; (c) mesothelioma cell growth is modulated by the cholinergic system in which agonists (i.e., nicotine) has a proliferative effect, and antagonists (i.e., curare) has an inhibitory effect, evaluated by cell cloning, DNA synthesis and cell cycle; (d) nicotine induces Ca(+2) influx, evaluated by [(45)Ca(2+)] uptake, and consequently activation of mitogen-activated protein kinase pathway (extracellular signal-regulated kinase and p90(RSK) phosphorylation), evaluated by Western blot; and (e) apoptosis mechanisms in mesothelioma cells are under the control of the cholinergic system (nicotine antiapoptotic via induction of nuclear factor-kappaB complexes and phosphorylation of Bad at Ser(112); curare proapoptotic via G(0)-G(1) arrest p21(waf-1) dependent but p53 independent). The involvement of the nonneuronal cholinergic system in mesothelioma appears reasonable and open up new therapeutic strategies.

  15. Postsynaptic action of brain-derived neurotrophic factor attenuates alpha7 nicotinic acetylcholine receptor-mediated responses in hippocampal interneurons.

    PubMed

    Fernandes, Catarina C; Pinto-Duarte, António; Ribeiro, Joaquim Alexandre; Sebastião, Ana M

    2008-05-21

    Nicotinic mechanisms acting on the hippocampus influence attention, learning, and memory and constitute a significant therapeutic target for many neurodegenerative, neurological, and psychiatric disorders. Here, we report that brain-derived neurotrophic factor (BDNF) (1-100 ng/ml), a member of the neurotrophin gene family, rapidly decreases alpha7 nicotinic acetylcholine receptor responses in interneurons of the hippocampal CA1 stratum radiatum. Such effect is dependent on the activation of the TrkB receptor and involves the actin cytoskeleton; noteworthy, it is compromised when the extracellular levels of the endogenous neuromodulator adenosine are reduced with adenosine deaminase (1 U/ml) or when adenosine A(2A) receptors are blocked with SCH 58261 (2-(2-furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine) (100 nm). The intracellular application of U73122 (1-[6[[(17beta)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione) (5 mum), a broad-spectrum inhibitor of phospholipase C, or GF 109203X (bisindolylmaleimide I) (2 mum), a general inhibitor of protein kinase C isoforms, blocks BDNF-induced inhibition of alpha7 nicotinic acetylcholine receptor function. Moreover, in conditions of simultaneous intracellular dialysis of the fast Ca(2+) chelator BAPTA (10 mm) and removal of extracellular Ca(2+) ions, the inhibitory action of BDNF is further prevented. The present findings disclose a novel target for rapid actions of BDNF that might play important roles on synaptic transmission and plasticity in the brain.

  16. Methanandamide allosterically inhibits in vivo the function of peripheral nicotinic acetylcholine receptors containing the alpha 7-subunit.

    PubMed

    Baranowska, Urszula; Göthert, Manfred; Rudz, Radoslaw; Malinowska, Barbara

    2008-09-01

    Methanandamide (MAEA), the stable analog of the endocannabinoid anandamide, has been proven in Xenopus oocytes to allosterically inhibit the function of the alpha7-nicotinic acetylcholine receptors (nAChRs) in a cannabinoid (CB) receptor-independent manner. The present study aimed at demonstrating that this mechanism can be activated in vivo. In anesthetized and vagotomized pithed rats treated with atropine, we determined the tachycardic response to electrical stimulation of preganglionic sympathetic nerves via the pithing rod or to i.v. nicotine (0.7 micromol/kg) activating nAChRs on the cardiac postganglionic sympathetic neurons. MAEA (3 and 10 micromol/kg) inhibited the electrically induced tachycardia (maximally by 15-20%; abolished by the CB(1) receptor antagonist AM 251 [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide]; 3 micromol/kg) in pentobarbitone-anesthetized pithed rats, but not in urethane-anesthetized pithed rats, which, thus, are suitable to study the CB(1) receptor-independent inhibition of nicotine-evoked tachycardia. The subunit-nonselective nAChR antagonist hexamethonium (100 micromol/kg) and the selective alpha7-subunit antagonist methyllycaconitine (MLA; 3 and 10 micromol/kg) decreased the nicotine-induced tachycardia by 100 and 40%, respectively (maximal effects), suggesting that nAChRs containing the alpha7-subunit account for 40% of the nicotine-induced tachycardia. MAEA (3 micromol/kg) produced an AM 251-insensitive inhibition (maximum again by 40%) of the nicotine-induced tachycardia. Simultaneous or sequential coadministration of MLA and MAEA inhibited the nicotine-induced tachycardia to the same extent (maximally by 40%) as each of the drugs alone. In conclusion, according to nonadditivity of the effects, MAEA mediates in vivo inhibition by the same receptors as MLA, namely alpha7-subunit-containing nAChRs, although at an allosteric instead of the orthosteric site.

  17. (-)-Spiro[1-azabicyclo[2.2.2]octane-3,5'-oxazolidin-2'-one], a conformationally restricted analogue of acetylcholine, is a highly selective full agonist at the alpha 7 nicotinic acetylcholine receptor.

    PubMed

    Mullen, G; Napier, J; Balestra, M; DeCory, T; Hale, G; Macor, J; Mack, R; Loch, J; Wu, E; Kover, A; Verhoest, P; Sampognaro, A; Phillips, E; Zhu, Y; Murray, R; Griffith, R; Blosser, J; Gurley, D; Machulskis, A; Zongrone, J; Rosen, A; Gordon, J

    2000-11-02

    Neuronal nicotinic acetylcholine receptors are members of the ligand-gated ion channel receptor superfamily and may play important roles in modulating neurotransmission, cognition, sensory gating, and anxiety. Because of its distribution and abundance in the CNS, the alpha 7 nicotinic receptor is a strong candidate to be involved in some of these functions. In this paper we describe the synthesis and in vitro profile of AR-R17779, (-)-spiro[1-azabicyclo[2.2. 2]octane-3,5'-oxazolidin-2'-one] (4a), a potent full agonist at the rat alpha 7 nicotinic receptor, which is highly selective for the rat alpha 7 nicotinic receptor over the alpha 4 beta 2 subtype. Preliminary SAR of AR-R17779 presented here indicate that there is little scope for modification of this rigid molecule as even minor changes result in significant loss of the alpha 7 nicotinic receptor affinity.

  18. Identification of regions involved in the binding of alpha-bungarotoxin to the human alpha7 neuronal nicotinic acetylcholine receptor using synthetic peptides.

    PubMed Central

    Marinou, Martha; Tzartos, Socrates J

    2003-01-01

    The neuronal alpha7 nicotinic acetylcholine receptor (AChR) binds the neurotoxin alpha-bungarotoxin (alpha-Bgt). Fine mapping of the alpha-Bgt-binding site on the human alpha7 AChR was performed using synthetic peptides covering the entire extracellular domain of the human alpha7 subunit (residues 1-206). Screening of these peptides for (125)I-alpha-Bgt binding resulted in the identification of at least two toxin-binding sites, one at residues 186-197, which exhibited the best (125)I-alpha-Bgt binding, and one at residues 159-165, with weak toxin-binding capacity; these correspond, respectively, to loops C and IV of the agonist-binding site. Toxin binding to the alpha7(186-197) peptide was almost completely inhibited by unlabelled alpha-Bgt or d -tubocurarine. Alanine substitutions within the sequence 186-198 revealed a predominant contribution of aromatic and negatively charged residues to the binding site. This sequence is homologous to the alpha-Bgt binding site of the alpha1 subunit (residues 188-200 in Torpedo AChR). In competition experiments, the soluble peptides alpha7(186-197) and Torpedo alpha1(184-200) inhibited the binding of (125)I-alpha-Bgt to the immobilized alpha7(186-197) peptide, to native Torpedo AChR, and to the extracellular domain of the human alpha1 subunit. These results suggest that the toxin-binding sites of the neuronal alpha7 and muscle-type AChRs bind to identical or overlapping sites on the alpha-Bgt molecule. In support of this, when synthetic alpha-Bgt peptides were tested for binding to the recombinant extracellular domains of the human alpha7 and alpha1 subunits, and to native Torpedo and alpha7 AChR, the results indicated that alpha-Bgt interacts with both neuronal and muscle-type AChRs through its central loop II and C-terminal tail. PMID:12614199

  19. Binding sites for. alpha. -bungarotoxin and the noncompetitive inhibitor phencyclidine on a synthetic peptide comprising residues 172-227 of the. alpha. -subunit of the nicotinic acetylcholine receptor

    SciTech Connect

    Donnelly-Roberts, D.L.; Lentz, T.L. )

    1991-07-30

    The binding of the competitive antagonist {alpha}-bungarotoxin ({alpha}-Btx) and the noncompetitive inhibitor phencyclidine (PCP) to a synthetic peptide comprising residues 172-227 of the {alpha}-subunit of the Torpedo acetylcholine receptor has been characterized. {sup 125}I-{alpha}-Btx bound to the 172-227 peptide in a solid-phase assay and was competed by {alpha}-Btx d-tubocurarine and NaCl. In the presence of 0.02% sodium dodecyl sulfate, {sup 125}I-{alpha}-Btx bound to the 56-residue peptide with a K{sub D} of 3.5 nM, as determined by equilibrium saturation binding studies. Because {alpha}Btx binds to a peptide comprising residues 173-204 with the same affinity and does not bind to a peptide comprising residues 205-227, the competitive antagonist and hence agonist binding site lies between residues 173 and 204. After photoaffinity labeling, ({sup 3}H)PCP was bound to the 172-227 peptide. ({sup 3}H)PCP binding was inhibited by chlorpromazine, tetracaine, and dibucaine. It is concluded that a high-affinity binding site for PCP is located between residues 205 and 227, which includes the first 18 residues of transmembrane segment M1, and that a low-affinity site is located in the competitive antagonist binding site between residues 173 and 204. These results show that a synthetic peptide comprising residues 172-227 of the {alpha} subunit contains three binding sites, one for {alpha}-Btx and two for PCP. Previous studies on the intact receptor indicate high-affinity PCP binding occurs in the receptor channel.

  20. A single gene codes for the nicotinic acetylcholine receptor alpha-subunit in Torpedo marmorata: structural and developmental implications.

    PubMed Central

    Klarsfeld, A; Devillers-Thiéry, A; Giraudat, J; Changeux, J P

    1984-01-01

    We have used Southern blot hybridization to analyze the genomic structure encoding the alpha-subunit of the acetylcholine receptor (AChR) in Torpedo marmorata, with cDNA probes isolated from the electric organ. Four different radiolabelled probes, corresponding to various parts of the alpha-subunit mRNA, hybridized to several genomic fragments of T. marmorata DNA generated by digestion with the restriction enzymes SstI, PvuII and PstI. The same hybridization pattern was observed after washing the blots under low- or high-stringency conditions. As a check for detection sensitivity of heterologous sequences, the same probes were hybridized to PvuII-digested chicken DNA, revealing bands at low stringency which disappeared at higher stringencies. Unambiguously, two of our probes (one of them entirely within the coding region) hybridized to a single genomic fragment from T. marmorata DNA. This feature, as well as the results of an extensive study of the whole hybridization pattern, points towards the uniqueness of alpha-subunit-specific sequences in the genome of T. marmorata. Since overall more bands were found than expected from the cDNA sequence, this alpha-subunit gene must be split by several introns (at least four, possibly more). The length of this gene is at least 20 kb. The existence of a single alpha-subunit gene is consistent with the absence of chemical heterogeneity in the NH2-terminal sequence of the purified alpha-chain, and supports the view that the two alpha-chains belonging to one AChR oligomer have an identical primary structure. It also suggests that localization and stabilization of the AChR in well-defined post-synaptic areas of T. marmorata electric organ basically relies, during development, on 'epigenetic' mechanisms. Images Fig. 2. Fig. 3. Fig. 4. PMID:6323168

  1. Potentiation of alpha7-containing nicotinic acetylcholine receptors by select albumins.

    PubMed

    Conroy, William G; Liu, Qing-Song; Nai, Qiang; Margiotta, Joseph F; Berg, Darwin K

    2003-02-01

    Nicotinic receptors containing alpha7 subunits are ligand-gated ion channels widely distributed in the nervous system; they influence a diverse array of events because of their high relative calcium permeability. We show here that nicotine-induced whole-cell responses generated by such receptors can be dramatically potentiated in a rapidly reversible manner by some but not all albumins. The potentiation involves increases both in potency and efficacy with no obvious differences in rise and fall times of the response. The potentiation is not reduced by removing absorbed components; it is abolished by proteolysis, suggesting that the albumin protein backbone is essential. The fact that some albumins are ineffective indicates that minor differences in amino acid sequence may be critical. Experiments with open channel blockers indicate that the potentiation involves increased responses from active receptors rather than recruitment of receptors from a previously silent pool. Single channel recordings reveal that the potentiation correlates with increased single channel opening probability, reflected in increased frequency of channel opening and increased mean channel open time. The potentiation can be exploited to overcome blockade by noncompetitive inhibitors such as beta-amyloid peptide. The results raise the possibility that endogenous compounds use the site to modulate receptor function in vivo, and suggest that the receptors may represent useful targets for therapeutic intervention in cases where they have been implicated in neuropathologies such as Alzheimer's disease.

  2. Alpha-9 Nicotinic Acetylcholine Receptor Immunoreactivity in the Rodent Vestibular Labyrinth

    PubMed Central

    Luebke, Anne E.; Maroni, Paul D.; Guth, Scott M.; Lysakowski, Anna

    2010-01-01

    Vestibular tissues (cristae ampullares, macular otolithic organs, and Scarpa’s ganglia) in chinchilla, rat, and guinea pig were examined for immunoreactivity to the α9 nicotinic acetylcholine receptor (nAChR) subunit. The α9 antibody was generated against a conserved peptide present in the intracellular loop of the predicted protein sequence of the guinea pig α9 nAChR subunit. In the vestibular periphery, staining was observed in calyces around type I hair cells, at the synaptic pole of type II hair cells, and in varying levels in Scarpa’s ganglion cells. Ganglion cells were also triply labeled to detect α9, calretinin, and peripherin. Calretinin labels calyx-only afferents. Peripherin labels bouton-only afferents. Dimorphic afferents, which have both calyx and bouton endings, are not labeled by calretinin or peripherin. In these experiments, α9 was expressed in both calyx and dimorphic afferents. A subpopulation of small ganglion cells did not contain the α9 nAChR but did stain for peripherin. We surmise that these are bouton-only afferents. Bouton (regularly discharging) afferents also show efferent responses, although they are qualitatively different from those in irregularly discharging (calyx and dimorphic) afferents, much slower and longer lasting. Thus, regular afferents are probably more affected via a muscarinic cholinergic or a peptidergic mechanism, with a much smaller superimposed fast nicotinic-type response. This latter response could be due to one of the other nicotinic receptors that have been described in studies from other laboratories. PMID:16217793

  3. Nicotine increases GABAergic input on rat dorsal raphe serotonergic neurons through alpha7 nicotinic acetylcholine receptor.

    PubMed

    Hernández-Vázquez, F; Chavarría, K; Garduño, J; Hernández-López, S; Mihailescu, S P

    2014-12-15

    The dorsal raphe nucleus (DRN) contains large populations of serotonergic (5-HT) neurons. This nucleus receives GABAergic inhibitory afferents from many brain areas and from DRN interneurons. Both GABAergic and 5-HT DRN neurons express functional nicotinic acetylcholine receptors (nAChRs). Previous studies have demonstrated that nicotine increases 5-HT release and 5-HT DRN neuron discharge rate by stimulating postsynaptic nAChRs and by increasing glutamate and norepinephrine release inside DRN. However, the influence of nicotine on the GABAergic input to 5-HT DRN neurons was poorly investigated. Therefore, the aim of this work was to determine the effect of nicotine on GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) of 5-HT DRN neurons and the subtype of nAChR(s) involved in this response. Experiments were performed in coronal slices obtained from young Wistar rats. GABAergic sIPSCs were recorded from post hoc-identified 5-HT DRN neurons with the whole cell voltage patch-clamp technique. Administration of nicotine (1 μM) increased sIPSC frequency in 72% of identified 5-HT DRN neurons. This effect was not reproduced by the α4β2 nAChR agonist RJR-2403 and was not influenced by TTX (1 μM). It was mimicked by the selective agonist for α7 nAChR, PNU-282987, and exacerbated by the positive allosteric modulator of the same receptor, PNU-120596. The nicotine-induced increase in sIPSC frequency was independent on voltage-gated calcium channels and dependent on Ca(2+)-induced Ca(2+) release (CICR). These results demonstrate that nicotine increases the GABAergic input to most 5-HT DRN neurons, by activating α7 nAChRs and producing CICR in DRN GABAergic terminals.

  4. Differential inhibition of nicotine- and acetylcholine-evoked currents through alpha4beta2 neuronal nicotinic receptors by tobacco cembranoids in Xenopus oocytes.

    PubMed

    Eaton, Misty J; Ospina, Claudia A; Rodríguez, Abimael D; Eterović, Vesna A

    2004-08-05

    In tobacco, there are two types of compounds that interact with neuronal nicotinic acetylcholine receptors (nnAChRs) in the brain. The first is the addictive component of tobacco and an agonist of these receptors, nicotine. The second are cyclic diterpenoids called cembranoids that non-competitively inhibit many types of nnAChRs. Nictotinic receptors composed of alpha4beta2 subunits are the predominant type of nicotinic receptors in the brain. These alpha4beta2 receptors are up-regulated upon chronic exposure to nicotine and have been implicated in nicotine addiction. The present study was designed to determine whether the inhibitory effects of two cembranoids from tobacco [(1S, 2E, 4R, 6R, 7E, 11E)-2,7,11-cembratriene-4,6-diol (4R) and its diastereoisomer (1S, 2E, 4S, 6R, 7E, 11E)-2,7,11-cembratriene-4,6-diol (4S)] were comparable on acetylcholine (ACh) and nicotine-evoked currents through alpha4beta2 nnAChRs. alpha4beta2 nnAChRs from rat brain were expressed in Xenopus oocytes and studied using the two-electrode voltage-clamp technique. The dose-response curves for acetylcholine and nicotine were hyperbolic and bell-shaped, respectively. Although there was no difference in the potency between cembranoids 4R and 4S, both of these cembranoids more potently inhibited nicotine-induced currents than acetylcholine-induced currents. Furthermore, both cembranoids were more potent inhibitors of this receptor when they were preincubated for 1 min prior to application of agonist. The finding that cembranoids preferentially inhibit nicotine-induced currents over those elicited by the natural neurotransmitter acetylcholine may have important implications when developing strategies to prevent nicotine addiction and tobacco use.

  5. [New approaches to the immunotherapy of Alzheimer's disease with the synthetic fragments of alpha7 subunit of the acetylcholine receptor].

    PubMed

    Vol'pina, O M; Volkova, T D; Titova, M A; Gershovich, Iu G; medvinskaia, N I; Samokhin, A N; Kamynina, A V; Shalgunov, V S; Koroev, D O; Filatova, M P; Oboznaia, M B; Bobkova, N V

    2008-01-01

    The effect of immunization with the synthetic fragments of the alpha7 subunit of the acetylcholine nicotine receptor on the spatial memory of mice subjected to olfactory bulbectomy, which causes the development of the neuro-degenetrative disease of Alzheimer's type, was studied. Mice of the NMRI line were immunized with the KLH conjugates of two peptide fragments of the N-terminal fragment of the alpha7 subunit extraxcellular fragment, subjected to olfactory bulbectomy to cause the development of the neurodegenetrative disease of Alzheimer's type, and then the state of the spartial memory was evaluated. It was shown that 20% of bulbectomized mice immunized with the N-terminal 1-23 fragment exhibited good spatial memory after training. Immunization with the peptide construct (159-167)-(179-188) consisting of two hydrophilic exposed regions of alpha7-subunit induced good spatial memory in 50% of bulbectomized mice, while in the control group, which received only KLH, none of the animals were educated. Thus, the development of immunotherapy with peptide (159-167)-(179-188) seems to be a promising approach to prophylaxis and treatment of Alzheimer's disease.

  6. Ca(2+)-sensitive inhibition by Pb(2+) of alpha7-containing nicotinic acetylcholine receptors in hippocampal neurons.

    PubMed

    Mike, A; Pereira, E F; Albuquerque, E X

    2000-08-04

    In the present study the patch-clamp technique was applied to cultured hippocampal neurons to determine the kinetics as well as the agonist concentration- and Ca(2+)-dependence of Pb(2+)-induced inhibition of alpha7 nicotinic receptors (nAChRs). Evidence is provided that more than two-thirds of the inhibition by Pb(2+) (3-30 microM) of alpha7 nAChR-mediated whole-cell currents (referred to as type IA currents) develops rapidly and is fully reversible upon washing. The estimated values for tau(onset) and tau(recovery) were 165 and 240 ms, respectively. The magnitude of the effect of Pb(2+) was the same regardless of whether acetylcholine or choline was the agonist. Pre-exposure of the neurons for 800 ms to Pb(2+) (30 microM) decreased the amplitude and accelerated the decay phase of currents evoked by moderate to high agonist concentrations. In contrast, only the amplitude of currents evoked by low agonist concentrations was reduced when the neurons were exposed simultaneously to Pb(2+) and the agonists. Taken together with the findings that Pb(2+) reduces the frequency of opening and the mean open time of alpha7 nAChR channels, these data suggest that Pb(2+) accelerates the rate of receptor desensitization. An additional reduction of type IA current amplitudes occurred after 2-min exposure of the neurons to Pb(2+). This effect was not reversible upon washing of the neurons and was most likely due to an intracellular action of Pb(2+). Pb(2+)-induced inhibition of alpha7 nAChRs, which was hindered by the enhancement of extracellular Ca(2+) concentrations, may contribute to the neurotoxicity of the heavy metal.

  7. Alpha7 nicotinic acetylcholine receptor agonists and PAMs as adjunctive treatment in schizophrenia. An experimental study.

    PubMed

    Marcus, Monica M; Björkholm, Carl; Malmerfelt, Anna; Möller, Annie; Påhlsson, Ninni; Konradsson-Geuken, Åsa; Feltmann, Kristin; Jardemark, Kent; Schilström, Björn; Svensson, Torgny H

    2016-09-01

    Nicotine has been found to improve cognition and reduce negative symptoms in schizophrenia and a genetic and pathophysiological link between the α7 nicotinic acetylcholine receptors (nAChRs) and schizophrenia has been demonstrated. Therefore, there has been a large interest in developing drugs affecting the α7 nAChRs for schizophrenia. In the present study we investigated, in rats, the effects of a selective α7 agonist (PNU282987) and a α7 positive allosteric modulator (PAM; NS1738) alone and in combination with the atypical antipsychotic drug risperidone for their utility as adjunct treatment in schizophrenia. Moreover we also investigated their utility as adjunct treatment in depression in combination with the SSRI citalopram. We found that NS1738 and to some extent also PNU282987, potentiated a subeffective dose of risperidone in the conditioned avoidance response test. Both drugs also potentiated the effect of a sub-effective concentration of risperidone on NMDA-induced currents in pyramidal cells of the medial prefrontal cortex. Moreover, NS1738 and PNU282987 enhanced recognition memory in the novel object recognition test, when given separately. Both drugs also potentiated accumbal but not prefrontal risperidone-induced dopamine release. Finally, PNU282987 reduced immobility in the forced swim test, indicating an antidepressant-like effect. Taken together, our data support the utility of drugs targeting the α7 nAChRs, perhaps especially α7 PAMs, to potentiate the effect of atypical antipsychotic drugs. Moreover, our data suggest that α7 agonists and PAMs can be used to ameliorate cognitive symptoms in schizophrenia and depression.

  8. First and second transmembrane segments of alpha3, alpha4, beta2, and beta4 nicotinic acetylcholine receptor subunits influence the efficacy and potency of nicotine.

    PubMed

    Rush, Ray; Kuryatov, Alexander; Nelson, Mark E; Lindstrom, Jon

    2002-06-01

    The first three transmembrane segments (M1-M3) of human nicotinic acetylcholine receptors (nAChRs) have been implicated in determining the efficacy of nicotine by studies of alpha3/alpha4 subunit chimeras. Nicotine has full efficacy on the alpha4beta2 nAChR and partial efficacy on the alpha3beta2 nAChR. Now, we have exchanged individually three amino acids between the alpha4 and the alpha3 subunits at positions 226(M1), 258(M2), and 262(M2). Also, similar exchanges were made in the beta2 and beta4 subunits at positions 224(M1), 226(M1), and 254(M2) (using alpha subunit numbering). Expression of these mutated nAChRs in Xenopus laevis oocytes showed that the mutated M1 amino acids were important in influencing the potency of ACh and nicotine. It is hypothesized that these M1 amino acids affect the stability between the resting and activated states of the nAChR. M2 amino acids altered the efficacy of nicotine, usually without altering its potency. When the residue located at position 258 in the M2 region of the alpha subunit was valine (as in the alpha3 subunit), the resulting nAChR exhibited partial efficacy for nicotine that was voltage-dependent. Therefore, we believe that these M2 amino acids contribute to the formation of a binding site for nicotine in the alpha3beta2 nAChR channel, which results in a low-affinity channel block, causing the lower efficacy of nicotine on this nAChR.

  9. Is the acetylcholine receptor a rabies virus receptor?

    PubMed

    Lentz, T L; Burrage, T G; Smith, A L; Crick, J; Tignor, G H

    1982-01-08

    Rabies virus was found on mouse diaphragms and on cultured chick myotubes in a distribution coinciding with that of the acetylcholine receptor. Treatment of the myotubes with alpha-bungarotoxin and d-tubocurarine before the addition of the virus reduced the number of myotubes that became infected with rabies virus. These findings together suggest that acetylcholine receptors may serve as receptors for rabies virus. The binding of virus to acetylcholine receptors, which are present in high density at the neuromuscular junction, would provide a mechanism whereby the virus could be locally concentrated at sites in proximity to peripheral nerves facilitating subsequent uptake and transfer to the central nervous system.

  10. Evaluation of alpha7 nicotinic acetylcholine receptor agonists and positive allosteric modulators using the parallel oocyte electrophysiology test station.

    PubMed

    Malysz, John; Grønlien, Jens H; Timmermann, Daniel B; Håkerud, Monika; Thorin-Hagene, Kirsten; Ween, Hilde; Trumbull, Jonathan D; Xiong, Yongli; Briggs, Clark A; Ahring, Philip K; Dyhring, Tino; Gopalakrishnan, Murali

    2009-08-01

    Neuronal acetylcholine receptors (nAChRs) of the alpha7 subtype are ligand-gated ion channels that are widely distributed throughout the central nervous system and considered as attractive targets for the treatment of various neuropsychiatric and neurodegenerative diseases. Both agonists and positive allosteric modulators (PAMs) are being developed as means to enhance the function of alpha7 nAChRs. The in vitro characterization of alpha7 ligands, including agonists and PAMs, relies on multiple technologies, but only electrophysiological measurements assess the channel activity directly. Traditional electrophysiological approaches utilizing two-electrode voltage clamp or patch clamp in isolated cells have very low throughput to significantly impact drug discovery. Abbott (Abbott Park, IL) has developed a two-electrode voltage clamp-based system, the Parallel Oocyte Electrophysiology Test Station (POETs()), that allows for the investigation of ligand-gated ion channels such as alpha7 nAChRs in a higher-throughput manner. We describe the utility of this technology in the discovery of selective alpha7 agonists and PAMs. With alpha7 agonists, POETs experiments involved both single- and multiple-point concentration-response testing revealing diverse activation profiles (zero efficacy desensitizing, partial, and full agonists). In the characterization of alpha7 PAMs, POETs testing has served as a reliable primary or secondary screen identifying compounds that fall into distinct functional types depending on the manner in which current potentiation occurred. Type I PAMs (eg, genistein, NS1738, and 5-hydroxyindole) increase predominantly the peak amplitude response, type II PAMs affect the peak current and current decay (eg, PNU-120,596 and 4-(naphthalen-1-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide), and anothertype slowing the current decay kinetics in the absence of increases in the peak current. In summary, POETs technology allows for significant

  11. [Sites of synthesis of acetylcholine receptors in denervated muscles].

    PubMed

    Giacobini Robecchi, M G; Garelli, M; Filogamo, G

    1980-09-01

    Muscle fibres binding with 125I alpha-bungarotoxine from Bungarus Multicinctus, after treatment with saponine, shows (in electron microscope autoradiography) intracellular binding sites identifying sites of acetylcholine receptor synthesis. In innervated muscle, the acetylcholine receptor is located only at the neuromuscular junction. In denervated muscle the receptor is distributed along the whole sarcolemma; in this situation the acetylcholine receptor is synthesized "ex novo" in the membrane system over the whole length of the muscle fibre.

  12. New ligands with affinity for the alpha4beta2 subtype of nicotinic acetylcholine receptors. Synthesis, receptor binding, and 3D-QSAR modeling.

    PubMed

    Audouze, Karine; Nielsen, Elsebet Østergaard; Olsen, Gunnar M; Ahring, Philip; Jørgensen, Tino Dyhring; Peters, Dan; Liljefors, Tommy; Balle, Thomas

    2006-06-01

    A new series of piperazines, diazepanes, diazocanes, diazabicyclononanes, and diazabicyclodecanes with affinity for the alpha4beta2 subtype of nicotinic acetylcholine receptors were synthesized on the basis of results from a previous computational study. A predictive 3D-QSAR model was developed using the GRID/GOLPE approach (R2 = 0.94, Q2 = 0.83, SDEP = 0.34). The SAR was interpreted in terms of contour maps of the PLS coefficients and in terms of a homology model of the alpha4beta2 subtype of the nicotinic acetylcholine receptors. The results reveal that hydrogen bonding from both hydrogens on the protonated amine and from the pyridine nitrogen to a water molecule as well as van der Waals interactions between the substituent bearing the protonated amine and the receptor is of importance for ligand affinity. The combination of 3D-QSAR and homology modeling proved successful for the interpretation of structure-affinity relationships as well as the validation of the individual modeling approaches.

  13. alpha4beta2 nicotinic receptors with high and low acetylcholine sensitivity: pharmacology, stoichiometry, and sensitivity to long-term exposure to nicotine.

    PubMed

    Moroni, Mirko; Zwart, Ruud; Sher, Emanuele; Cassels, Bruce K; Bermudez, Isabel

    2006-08-01

    alpha4 and beta2 nicotinic acetylcholine receptor (nAChR) subunits expressed heterologously assemble into receptors with high (HS) and low (LS) sensitivity to acetylcholine (ACh); their relative proportions depend on the alpha4to beta2 ratio. In this study, injection of oocytes with 1:10 alpha4/beta2 subunit cDNA ratios favored expression of HS alpha4beta2 nAChRs, as evidenced by monophasic ACh concentration-response curves, whereas injections with 10:1 cDNA ratios favored expression of LS alpha4beta2 receptors. The stoichiometry was inferred from the shifts in the ACh EC(50) values caused by Leu to Thr mutations at position 9' of the second transmembrane domain of alpha4 and beta2. The 1:10 injection ratio produced the (alpha4)(2)(beta2)(3) stoichiometry, whereas 10:1 injections produced the (alpha4)(3)(beta2)(2) stoichiometry. The agonists epibatidine, 3-[2(S)-azetidinylmethoxy]pyridine (A-85380), 5-ethoxy-metanicotine (TC-2559), cytisine, and 3-Br-cytisine and the antagonists dihydro-beta-erythroidine and d-tubocurarine were more potent at HS receptors. TC-2559 was more efficacious than ACh at HS receptors but was a partial agonist at LS receptors. Epibatidine was more efficacious than ACh at LS receptors and a partial agonist at HS receptors. Cytisine and 5-halogenated cytisines had moderate efficacy at LS receptors but had almost no efficacy at HS receptors. By exploiting the differential effects of ACh, TC-2559 and 5-I-cytisine we evaluated the effects of long-term exposure to nicotine on HS and LS receptors expressed in Xenopus laevis oocytes after cDNA injections or microtransplantation of alpha4beta2 receptors assembled in human embryonic kidney 293 cells. We conclude that nicotine up-regulates HS alpha4beta2 receptors, probably by influencing the assembly of receptors rather than by altering the functional state of LS alpha4beta2 nAChRs.

  14. Negative regulatory elements upstream of a novel exon of the neuronal nicotinic acetylcholine receptor alpha 2 subunit gene.

    PubMed Central

    Bessis, A; Savatier, N; Devillers-Thiéry, A; Bejanin, S; Changeux, J P

    1993-01-01

    The expression of the nicotinic acetylcholine receptor alpha 2 subunit gene is highly restricted to the Spiriform lateralis nucleus of the Chick diencephalon. As a first step toward understanding the molecular mechanism underlying this regulation, we have investigated the structural and regulatory properties of the 5' sequence of this gene. A strategy based on the ligation of an oligonucleotide to the first strand of the cDNA (SLIC) followed by PCR amplification was used. A new exon was found approximately 3kb upstream from the first coding exon, and multiple transcription start sites of the gene were mapped. Analysis of the flanking region shows many consensus sequences for the binding of nuclear proteins, suggesting that the 1 kb flanking region contains at least a portion of the promoter of the gene. We have analysed the negative regulatory elements present within this region and found that a silencer region located between nucleotide -144 and +76 is active in fibroblasts as well as in neurons. This silencer is composed of six tandem repeat Oct-like motifs (CCCCATGCAAT), but does not bind any member of the Oct family. Moreover these motifs were found to act as a silencer only when they were tandemly repeated. When two, four or five motifs were deleted, the silencer activity of the motifs unexpectedly became an enhancer activity in all cells we have tested. Images PMID:8502560

  15. Virtual screening studies of Chinese medicine Coptidis Rhizoma as alpha7 nicotinic acetylcholine receptor agonists for treatment of Alzheimer's disease

    NASA Astrophysics Data System (ADS)

    Xiang, Li; Xu, Youdong; Zhang, Yan; Meng, Xianli; Wang, Ping

    2015-04-01

    Alzheimer's disease (AD) is an age-related neurodegenerative disease. Extensive in vitro and in vivo experiments have proved that the decreased activity of the cholinergic neuron is responsible for the memory and cognition deterioration. The alpha7 nicotinic acetylcholine receptor (α7-nAChR) is proposed to a drug target of AD, and compounds which acting as α7-nAChR agonists are considered as candidates in AD treatment. Chinese medicine CoptidisRhizoma and its compounds are reported in various anti-AD effects. In this study, virtual screening, docking approaches and hydrogen bond analyses were applied to screen potential α7-nAChR agonists from CoptidisRhizome. The 3D structure of the protein was obtained from PDB database. 87 reported compounds were included in this research and their structures were accessed by NCBI Pubchem. Docking analysis of the compounds was performed using AutoDock 4.2 and AutoDock Vina. The images of the binding modes hydrogen bonds and the hydrophobic interaction were rendered with PyMOL1.5.0.4. and LigPlot+ respectively. Finally, N-tran-feruloyltyramine, isolariciresinol, flavanone, secoisolariciresinol, (+)-lariciresinol and dihydrochalcone, exhibited the lowest docking energy of protein-ligand complex. The results indicate these 6 compounds are potential α7 nAChR agonists, and expected to be effective in AD treatment.

  16. Pharmacological and kinetic properties of alpha 4 beta 2 neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes.

    PubMed Central

    Charnet, P; Labarca, C; Cohen, B N; Davidson, N; Lester, H A; Pilar, G

    1992-01-01

    1. Co-injection of RNA synthesized from cloned neuronal acetylcholine receptor (nAChR) subunits (alpha 4 and beta 2) in Xenopus oocytes produced functional receptors. In macroscopic voltage-clamp experiments, the agonist-induced current exhibited a strong inward rectification. 2. Voltage jumps from +50 mV to more negative potentials produced relaxations of the agonist-induced current with a single exponential time course. The relaxation rate constant was only weakly voltage dependent. 3. At the single-channel level, three conductances were recorded of 12, 22 and 34 pS. Their burst durations were similar and varied only weakly with voltage (e-fold for 120 to 370 mV), consistent with the poorly voltage-dependent relaxation rate constants. However, the burst durations were less than 10 ms, or less than 1/5 the value expected from voltage-jump relaxations. 4. Hexamethonium (Hex, 0.5 to 8 microM) inhibited the agonist-induced current and produced voltage-jump relaxations characterized by a rapid conductance increase and a slower conductance decrease. Analysis of these relaxations suggested that the Hex-receptor interaction is open-channel blockade characterized by a forward binding rate of 1 x 10(7) M-1 s-1 and a dissociation rate constant of about 25 s-1. 5. For the relaxations produced by QX222, the slowest phase was a conductance increase, suggesting that the dissociation rate constant for QX222 is 10-30-fold greater than for Hex. 6. Hex but not QX222 produced an additional use-dependent blockade that was manifest during repetitive hyperpolarizing pulses. 7. With mouse muscle ACh receptors expressed in oocytes, the blockade by Hex did not depend strongly on voltage. Neither Hex nor QX222 produced appreciable use-dependent block on muscle ACh receptors. 8. Of the four conditions studied (neuronal and muscle receptors, Hex and QX222), only the blockade of the neuronal AChR by Hex is characterized by a residence time longer than the normal open time. 9. It is concluded

  17. Acetylcholine receptors in the human retina

    SciTech Connect

    Hutchins, J.B.; Hollyfield, J.G.

    1985-11-01

    Evidence for a population of acetylcholine (ACh) receptors in the human retina is presented. The authors have used the irreversible ligand TH-propylbenzilylcholine mustard (TH-PrBCM) to label muscarinic receptors. TH- or SVI-alpha-bungarotoxin (alpha-BTx) was used to label putative nicotinic receptors. Muscarinic receptors are apparently present in the inner plexiform layer of the retina. Autoradiographic grain densities are reduced in the presence of saturating concentrations of atropine, quinuclidinyl benzilate or scopolamine; this indicates that TH-PrBCM binding is specific for a population of muscarinic receptors in the human retina. Binding sites for radiolabeled alpha-BTx are found predominantly in the inner plexiform layer of the retina. Grain densities are reduced in the presence of d-tubocurarine, indicating that alpha-BTx may bind to a pharmacologically relevant nicotinic ACh receptor. This study provides evidence for cholinergic neurotransmission in the human retina.

  18. Nicotinic acetylcholine receptor from chick optic lobe.

    PubMed Central

    Norman, R I; Mehraban, F; Barnard, E A; Dolly, J O

    1982-01-01

    An alpha-bungarotoxin-sensitive nicotinic cholinergic receptor from chick optic lobe has been completely purified. Its standard sedimentation coefficient is 9.1 S. The value near 12 S reported for the related component from other brain regions can be reproduced when the initial extraction is by Triton X-100 (rather than Lubrol PX), but other protein is then complexed with it. A single subunit of apparent molecular weight 54,000 is detected, and this subunit is specifically labeled by bromo-[3H]acetylcholine, but only after disulfide reduction. The same size subunit likewise is labeled in the protein (purified similarly) from the rest of the chick brain which can also bind alpha-bungarotoxin and nicotinic ligands. Immunological crossreactivity is demonstrated between both of these proteins with an antiserum to pure acetylcholine receptor from skeletal muscle. The acetylcholine receptor from chick optic lobe and the alpha-bungarotoxin-binding protein from the rest of the brain appear similar or identical by a series of criteria and are related to (but with differences from) peripheral acetylcholine receptors. Images PMID:6175967

  19. Molecular mimicry and myasthenia gravis. An autoantigenic site of the acetylcholine receptor alpha-subunit that has biologic activity and reacts immunochemically with herpes simplex virus.

    PubMed Central

    Schwimmbeck, P L; Dyrberg, T; Drachman, D B; Oldstone, M B

    1989-01-01

    The large majority of patients with the autoimmune disease myasthenia gravis characteristically have detectable antibodies against the acetylcholine receptor (AChR). We used synthetic peptides to identify antibodies in sera of myasthenia gravis patients reactive with the human acetylcholine receptor (HuAChR) alpha-subunit, residues 160-167. Affinity purification of these antibodies, using the HuAChR alpha-subunit 157-170 peptide immobilized on thiopropyl-Sepharose, yielded IgG antibodies that bound to the native AChR and inhibited the binding of alpha-bungarotoxin to the receptor. The HuAChR alpha-subunit 160-167 peptide demonstrated specific immunological cross-reactivity with a shared homologous domain on herpes simplex virus glycoprotein D, residues 286-293, by both binding and inhibition studies. Thus, HuAChR alpha-subunit, residues 160-167, elicits antibodies in myasthenic patients that binds to the native AChR protein and is capable of eliciting a biologic effect. Immunologic cross-reactivity of this "self" epitope with herpes simplex virus suggest that this virus may be associated with the initiation of some cases of myasthenia. Images PMID:2551924

  20. Beta3 subunits promote expression and nicotine-induced up-regulation of human nicotinic alpha6* nicotinic acetylcholine receptors expressed in transfected cell lines.

    PubMed

    Tumkosit, Prem; Kuryatov, Alexander; Luo, Jie; Lindstrom, Jon

    2006-10-01

    Nicotinic acetylcholine receptors (AChRs) containing alpha6 subunits are typically found at aminergic nerve endings where they play important roles in nicotine addiction and Parkinson's disease. alpha6* AChRs usually contain beta3 subunits. beta3 subunits are presumed to assemble only in the accessory subunit position within AChRs where they do not participate in forming acetylcholine binding sites. Assembly of subunits in the accessory position may be a critical final step in assembly of mature AChRs. Human alpha6 AChRs subtypes were permanently transfected into human tsA201 human embryonic kidney (HEK) cell lines. alpha6beta2beta3 and alpha6beta4beta3 cell lines were found to express much larger amounts of AChRs and were more sensitive to nicotine-induced increase in the amount of AChRs than were alpha6beta2 or alpha6beta4 cell lines. The increased sensitivity to nicotine-induced up-regulation was due not to a beta3-induced increase in affinity for nicotine but probably to a direct effect on assembly of AChR subunits. HEK cells express only a small amount of mature alpha6beta2 AChRs, but many of these subunits are on the cell surface. This contrasts with Xenopus laevis oocytes, which express a large amount of incorrectly assembled alpha6beta2 subunits that bind cholinergic ligands but form large amorphous intracellular aggregates. Monoclonal antibodies (mAbs) were made to the alpha6 and beta3 subunits to aid in the characterization of these AChRs. The alpha6 mAbs bind to epitopes C-terminal of the extracellular domain. These data demonstrate that both cell type and the accessory subunit beta3 can play important roles in alpha6* AChR expression, stability, and up-regulation by nicotine.

  1. Positive allosteric modulation of alpha 7 nicotinic acetylcholine receptors enhances recognition memory and cognitive flexibility in rats.

    PubMed

    Nikiforuk, Agnieszka; Kos, Tomasz; Potasiewicz, Agnieszka; Popik, Piotr

    2015-08-01

    A wide body of preclinical and clinical data suggests that alpha 7 nicotinic acetylcholine receptors (α7-nAChRs) may represent useful targets for cognitive improvement in schizophrenia and Alzheimer׳s disease. A promising recent approach is based on the use of positive allosteric modulators (PAMs) of α7-nAChRs due to their several advantages over the direct agonists. Nevertheless, the behavioural effects of this class of compounds, particularly with regard to higher-order cognitive functions, have not been broadly characterised. The aim of the present study was to evaluate the procognitive efficacies of type I and type II α7-nAChRs PAMs, N-(4-chlorophenyl)-[[(4-chlorophenyl)amino]methylene]-3-methyl-5-isoxazoleacet-amide (CCMI) and N-(5-Chloro-2,4-dimethoxyphenyl)-N'-(5-methyl-3-isoxazolyl)urea (PNU-120596) in the novel object recognition task (NORT), attentional set-shifting task (ASST) and five-choice serial reaction time task (5-CSRTT) in rats. Additionally, the effects of galantamine, an acetylcholinesterase inhibitor that also allosterically modulates nAChRs, were assessed. We report that CCMI (0.3-3mg/kg), PNU-120596 (0.3-3mg/kg) and galantamine (1-3mg/kg) attenuated the delay-induced impairment in NORT performance and facilitated cognitive flexibility in the ASST. Methyllycaconitine (3mg/kg) blocked the actions of CCMI, PNU-120596 and galantamine in the NORT and ASST, suggesting that the procognitive effects of these compounds are α7-nAChRs-dependent. However, none of the compounds tested affected the rats' attentional performance in the 5-CSRTT. The present findings confirm and extend the observations indicating that the positive allosteric modulation of α7-nAChRs enhances recognition memory and cognitive flexibility in preclinical tasks. Therefore, the present study supports the utility of α7-nAChRs PAMs as a potential cognitive enhancing therapy.

  2. Positive allosteric modulators of alpha 7 nicotinic acetylcholine receptors reverse ketamine-induced schizophrenia-like deficits in rats.

    PubMed

    Nikiforuk, Agnieszka; Kos, Tomasz; Hołuj, Małgorzata; Potasiewicz, Agnieszka; Popik, Piotr

    2016-02-01

    Alpha 7 nicotinic acetylcholine receptors (α7-nAChRs) have generated great interest as targets of new pharmacological treatments for cognitive dysfunction in schizophrenia. One promising recent approach is based on the use of positive allosteric modulators (PAMs) of α7-nAChRs, which demonstrate several advantages over direct agonists. Nevertheless, the efficacy of these newly introduced α7-nAChR agents has not been extensively characterised in animal models of schizophrenia. The aim of the present study was to evaluate the efficacy of type I and II PAMs, N-(5-chloro-2,4-dimethoxyphenyl)-N'-(5-methyl-3-isoxazolyl)urea (PNU-120596) and N-(4-chlorophenyl)-[[(4-chlorophenyl)amino]methylene]-3-methyl-5-isoxazoleacet-amide (CCMI), respectively, and galantamine, an acetylcholinesterase inhibitor (AChE) that also allosterically modulates nAChRs, against ketamine-induced cognitive deficits and social withdrawal in rats. The orthosteric α7-nAChR agonist octahydro-2-methyl-5-(6-phenyl-3-pyridazinyl)-pyrrolo[3,4-c]pyrrole (A-582941) was used as a positive control. Additionally, the antipsychotic activities of the tested compounds were assessed using the conditioned avoidance response (CAR) test. PNU-120596, CCMI, galantamine and A-582941 reversed ketamine-induced cognitive inflexibility, as assessed in the attentional set-shifting task (ASST). The tested compounds were also effective against ketamine-induced impairment in the novel object recognition task (NORT). PNU-120596, CCMI, and A-582941 ameliorated ketamine-induced social interaction deficits, whereas galantamine was ineffective. Moreover, all tested compounds selectively suppressed the CAR. The positive allosteric modulation of α7-nAChRs demonstrates preclinical efficacy not only against schizophrenia-like cognition impairments but also positive and negative symptoms. Therefore, the use of α7-nAChR PAMs as a potential treatment strategy in schizophrenia is supported.

  3. The interaction between maternal immune activation and alpha 7 nicotinic acetylcholine receptor in regulating behaviors in the offspring

    PubMed Central

    Wu, Wei-Li; Adams, Catherine E.; Stevens, Karen E.; Chow, Ke-Huan; Freedman, Robert; Patterson, Paul H.

    2015-01-01

    Mutation of human chromosome 15q13.3 increases the risk for autism and schizophrenia. One of the noteworthy genes in 15q13.3 is CHRNA7, which encodes the nicotinic acetylcholine receptor alpha 7 subunit (α7nAChR) associated with schizophrenia in clinical studies and rodent models. This study investigates the role of α7nAChR in maternal immune activation (MIA) mice model, a murine model of environmental risk factor for autism and schizophrenia. We provided choline, a selective α7nAChR agonist among its several developmental roles, in the diet of C57BL/6N wild-type dams throughout the gestation and lactation period and induced MIA at mid-gestation. The adult offspring behavior and gene expression profile in the maternal spleen-placenta-fetal brain axis at mid-gestation were investigated. We found that choline supplementation prevented several MIA-induced behavioral abnormalities in the wild-type offspring. Pro-inflammatory cytokine interleukin-6 (IL-6) and Chrna7 gene expression in the wild-type fetal brain were elevated by poly(I:C) injection and were suppressed by gestational choline supplementation. We further investigated the gene expression level of IL-6 in Chrna7 mutant mice. We found that the basal level of IL-6 was higher in Chrna7 mutant fetal brain, which suggests that α7nAChR may serve an anti-inflammatory role in the fetal brain during development. Lastly, we induced MIA in Chrna7+/− offspring. The Chrna7+/− offspring were more vulnerable to MIA, with increased behavioral abnormalities. Our study shows that α7nAChR modulates inflammatory response affecting the fetal brain and demonstrates its effects on offspring behavior development after MIA. PMID:25683697

  4. Binding of rabies virus to purified Torpedo acetylcholine receptor.

    PubMed

    Lentz, T L; Benson, R J; Klimowicz, D; Wilson, P T; Hawrot, E

    1986-12-01

    The binding of 125I- and 35S-labeled rabies virus (CVS strain) to affinity-purified acetylcholine receptor from Torpedo electric organ was demonstrated. The binding of rabies virus to the acetylcholine receptor increased with increasing receptor concentration, was dependent on the pH of the incubation medium, and was saturable with increasing virus concentration. Binding of radioactively labeled virus was effectively competed by unlabeled homologous virus particles. Binding of 35S-labeled rabies virus to the AChR was inhibited up to 50% by alpha-bungarotoxin and up to 30% by (+)-tubocurarine but was not affected by atropine. These results demonstrate direct binding of rabies virus to a well-defined neurotransmitter receptor, namely the acetylcholine receptor and indicate that at least a portion of the virus interaction occurs near the acetylcholine binding site on the receptor. These findings support the hypothesis that the acetylcholine receptor may serve as a rabies virus receptor in vivo.

  5. Alpha 7 nicotinic acetylcholine receptor-mediated protection against ethanol-induced neurotoxicity.

    PubMed

    de Fiebre, NancyEllen C; de Fiebre, Christopher M

    2003-11-01

    The alpha(7)-selective nicotinic partial agonist 3-[2,4-dimethoxybenzylidene]anabaseine (DMXB) was examined for its ability to modulate ethanol-induced neurotoxicity in primary cultures of rat neurons. Primary cultures of hippocampal neurons were established from Long-Evans, embryonic day (E)-18 rat fetuses and maintained for 7 days. Ethanol (0-150 mM), DMXB (0-56 microM), or both were subsequently co-applied to cultures. Ethanol was added two additional times to the cultures to compensate for evaporation. After 5 days, neuronal viability was assessed with the MTT cell proliferation assay. Results demonstrated that ethanol reduces neuronal viability in a concentration-dependent fashion and that DMXB protects against this ethanol-induced neurotoxicity, also in a concentration-dependent fashion. These results support the suggestion that nicotinic partial agonists may be useful in treating binge drinking-induced neurotoxicity and may provide clues as to why heavy drinkers are usually smokers.

  6. Acetylcholine and the alpha 7 nicotinic receptor: a potential therapeutic target for the treatment of periodontal disease?

    PubMed Central

    2013-01-01

    Objectives The aim of this review is to examine the evidence for a functional cholinergic system operating within the periodontium and determine the evidence for its role in periodontal immunity. Introduction Acetylcholine can influence the immune system via the ‘cholinergic anti-inflammatory pathway’. This pathway is mediated by the vagus nerve which releases acetylcholine to interact with the α7 subunit of the nicotinic acetylcholine receptor (α7nAChR) on proximate immuno-regulatory cells. Activation of the α7nAChR on these cells leads to down-regulated expression of pro-inflammatory mediators and thus regulates localised inflammatory responses. The role of the vagus nerve in periodontal pathophysiology is currently unknown. However, non-neuronal cells can also release acetylcholine and express the α7nAChR; these include keratinocytes, fibroblasts, T cells, B cells and macrophages. Therefore, by both autocrine and paracrine methods non-neuronal acetylcholine can also be hypothesised to modulate the localised immune response. Methods A Pubmed database search was performed for studies providing evidence for a functional cholinergic system operating in the periodontium. In addition, literature on the role of the ‘cholinergic anti-inflammatory pathway’ in modulating the immune response was extrapolated to hypothesise that similar mechanisms of immune regulation occur within the periodontium. Conclusion The evidence suggests a functional nonneuronal ‘cholinergic anti-inflammatory pathway’ may operate in the periodontium and that this may be targeted therapeutically to treat periodontal disease. PMID:22777144

  7. Alexa Fluor 546-ArIB[V11L;V16A] is a potent ligand for selectively labeling alpha 7 nicotinic acetylcholine receptors.

    PubMed

    Hone, Arik J; Whiteaker, Paul; Mohn, Jesse L; Jacob, Michele H; McIntosh, J Michael

    2010-08-01

    The alpha7* (*denotes the possible presence of additional subunits) nicotinic acetylcholine receptor (nAChR) subtype is widely expressed in the vertebrate nervous system and implicated in neuropsychiatric disorders that compromise thought and cognition. In this report, we demonstrate that the recently developed fluorescent ligand Cy3-ArIB[V11L;V16A] labels alpha7 nAChRs in cultured hippocampal neurons. However, photobleaching of this ligand during long image acquisition times prompted us to develop a new derivative. In photostability studies, this new ligand, Alexa Fluor 546-ArIB[V11L;V16A], was significantly more resistant to bleaching than the Cy3 derivative. The classic alpha7 ligand alpha-bungarotoxin binds to alpha1* and alpha9* nAChRs. In contrast, Alexa Fluor 546-ArIB[V11L;V16A] potently (IC(50) 1.8 nM) and selectively blocked alpha7 nAChRs but not alpha1* or alpha9* nAChRs expressed in Xenopus oocytes. Selectivity was further confirmed by competition binding studies of native nAChRs in rat brain membranes. The fluorescence properties of Alexa Fluor 546-ArIB[V11L;V16A] were assessed using human embryonic kidney-293 cells stably transfected with nAChRs; labeling was observed on cells expressing alpha7 but not cells expressing alpha3beta2, alpha3beta4, or alpha4beta2 nAChRs. Further imaging studies demonstrate that Alexa Fluor 546-ArIB[V11L;V16A] labels hippocampal neurons from wild-type mice but not from nAChR alpha7 subunit-null mice. Thus, Alexa Fluor 546-ArIB[V11L;V16A] represents a potent and selective ligand for imaging alpha7 nAChRs.

  8. Alpha-conotoxin MII-sensitive nicotinic acetylcholine receptors in the nucleus accumbens shell regulate progressive ratio responding maintained by nicotine.

    PubMed

    Brunzell, Darlene H; Boschen, Karen E; Hendrick, Elizabeth S; Beardsley, Patrick M; McIntosh, J Michael

    2010-02-01

    Beta2 subunit containing nicotinic acetylcholine receptors (beta2(*)nAChRs; asterisk ((*)) denotes assembly with other subunits) are critical for nicotine self-administration and nicotine-associated dopamine (DA) release that supports nicotine reinforcement. The alpha6 subunit assembles with beta2 on DA neurons where alpha6beta2(*)nAChRs regulate nicotine-stimulated DA release at neuron terminals. Using local infusion of alpha-conotoxin MII (alpha-CTX MII), an antagonist with selectivity for alpha6beta2(*)nAChRs, the purpose of these experiments was to determine if alpha6beta2(*)nAChRs in the nucleus accumbens (NAc) shell are required for motivation to self-administer nicotine. Long-Evans rats lever-pressed for 0.03 mg/kg, i.v., nicotine accompanied by light+tone cues (NIC) or for light+tone cues unaccompanied by nicotine (CUEonly). Following extensive training, animals were tested under a progressive ratio (PR) schedule that required an increasing number of lever presses for each nicotine infusion and/or cue delivery. Immediately before each PR session, rats received microinfusions of alpha-CTX MII (0, 1, 5, or 10 pmol per side) into the NAc shell or the overlying anterior cingulate cortex. alpha-CTX MII dose dependently decreased break points and number of infusions earned by NIC rats following infusion into the NAc shell but not the anterior cingulate cortex. Concentrations of alpha-CTX MII that were capable of attenuating nicotine self-administration did not disrupt locomotor activity. There was no effect of infusion on lever pressing in CUEonly animals and NAc infusion alpha-CTX MII did not affect locomotor activity in an open field. These data suggest that alpha6beta2(*)nAChRs in the NAc shell regulate motivational aspects of nicotine reinforcement but not nicotine-associated locomotor activation.

  9. Interaction of nuclear factors with the upstream region of the alpha-subunit gene of chicken muscle acetylcholine receptor: variations with muscle differentiation and denervation.

    PubMed Central

    Piette, J; Klarsfeld, A; Changeux, J P

    1989-01-01

    The region lying between nucleotides (nt) -110 and -45 of chicken acetylcholine receptor alpha-subunit gene 5' upstream sequence confers developmental control of expression in primary cultures of chicken myotubes. This region interacts with several nuclear factors present in muscle cells as shown by DNase I footprinting and gel-retardation experiments. An Sp1-like factor and a guanine stretch-binding protein were found to bind to overlapping sites immediately upstream of the TATA box. Several factors interacting in the same region with a domain similar to the SV40 enhancer core appeared during in vitro differentiation of myoblasts into myotubes. The concentration of some of these factors increased also after denervation of leg muscle in newborn chickens. The specific interaction of nuclear factors with this domain may thus play a critical role in the regulation of alpha-subunit gene expression by muscle differentiation and electrical activity. Images PMID:2721497

  10. Binding of quinolizidine alkaloids to nicotinic and muscarinic acetylcholine receptors.

    PubMed

    Schmeller, T; Sauerwein, M; Sporer, F; Wink, M; Müller, W E

    1994-09-01

    Fourteen quinolizidine alkaloids, isolated from Lupinus albus, L. mutabilis, and Anagyris foetida, were analyzed for their affinity for nicotinic and/or muscarinic acetylcholine receptors. Of the compounds tested, the alpha-pyridones, N-methylcytisine and cytisine, showed the highest affinities at the nicotinic receptor, while several quinolizidine alkaloid types were especially active at the muscarinic receptor.

  11. Structural determinants of alpha-bungarotoxin binding to the sequence segment 181-200 of the muscle nicotinic acetylcholine receptor. alpha. subunit: Effects of cysteine/cystine modification and species-specific amino acid substitution

    SciTech Connect

    McLane, K.E.; Wu, Xiadong; Diethelm, B.; Conti-Tronconi, B.M. )

    1991-05-21

    The sequence segment 181-200 of the Torpedo nicotinic acetylcholine receptor (nAChR) {alpha}subunit forms a binding site for {alpha}-bungarotoxin ({alpha}-BTX). Synthetic peptides corresponding to the homologous sequences of human, calf, mouse, chicken, frog, and cobra muscle nAChR {alpha}1 subunits were tested for their ability to bind {sup 125}I-{alpha}-BTX, and differences in {alpha}-BTX affinity were determined by using solution (IC{sub 50}s) and solid-phase (K{sub d}s) assays. Panels of overlapping peptides corresponding to the complete {alpha}1 subunit of mouse and human were also tested for {alpha}-BTX binding, but other sequence segments forming the {alpha}-BTX site were not consistently detectable. The role of a putative vicinal disulfide bound between Cys-192 and -193, relative to the Torpedo sequence, was determined by modifying the peptides with sulfhydryl reagents. Reduction and alkylation of the peptides decreased {alpha}-BTX binding, whereas oxidation of the peptides had little effect. These results indicate that while the adjacent cysteines are likely to be involved in forming the toxin/{alpha}1-subunit interface a vicinal disulfide bound was not required for {alpha}-BTX binding.

  12. Nicotine promotes cell proliferation via {alpha}7-nicotinic acetylcholine receptor and catecholamine-synthesizing enzymes-mediated pathway in human colon adenocarcinoma HT-29 cells

    SciTech Connect

    Wong, Helen Pui Shan; Yu Le; Lam, Emily Kai Yee; Tai, Emily Kin Ki; Wu, William Ka Kei; Cho, Chi Hin . E-mail: chcho@cuhk.edu.hk

    2007-06-15

    Cigarette smoking has been implicated in colon cancer. Nicotine is a major alkaloid in cigarette smoke. In the present study, we showed that nicotine stimulated HT-29 cell proliferation and adrenaline production in a dose-dependent manner. The stimulatory action of nicotine was reversed by atenolol and ICI 118,551, a {beta}{sub 1}- and {beta}{sub 2}-selective antagonist, respectively, suggesting the role of {beta}-adrenoceptors in mediating the action. Nicotine also significantly upregulated the expression of the catecholamine-synthesizing enzymes [tyrosine hydroxylase (TH), dopamine-{beta}-hydroxylase (D{beta}H) and phenylethanolamine N-methyltransferase]. Inhibitor of TH, a rate-limiting enzyme in the catecholamine-biosynthesis pathway, reduced the actions of nicotine on cell proliferation and adrenaline production. Expression of {alpha}7-nicotinic acetylcholine receptor ({alpha}7-nAChR) was demonstrated in HT-29 cells. Methyllycaconitine, an {alpha}7-nAChR antagonist, reversed the stimulatory actions of nicotine on cell proliferation, TH and D{beta}H expression as well as adrenaline production. Taken together, through the action on {alpha}7-nAChR nicotine stimulates HT-29 cell proliferation via the upregulation of the catecholamine-synthesis pathway and ultimately adrenaline production and {beta}-adrenergic activation. These data reveal the contributory role {alpha}7-nAChR and {beta}-adrenoceptors in the tumorigenesis of colon cancer cells and partly elucidate the carcinogenic action of cigarette smoke on colon cancer.

  13. Neuronal acetylcholine receptors in Drosophila: the ARD protein is a component of a high-affinity alpha-bungarotoxin binding complex.

    PubMed Central

    Schloss, P; Hermans-Borgmeyer, I; Betz, H; Gundelfinger, E D

    1988-01-01

    The ard gene of Drosophila melanogaster encodes a structural homologue of vertebrate nicotinic acetylcholine receptors (AChR) and is expressed exclusively in nervous tissue. To study the nature of the ARD protein, antibodies were raised against fusion constructs containing two regions of this polypeptide. One segment is putatively extracellular (amino acids 65-212), the other domain is exposed to the cytoplasm (amino acids 305-444). The ARD antisera obtained served to investigate the physical relationship between the ARD protein and alpha-bungarotoxin (alpha-Btx) binding sites occurring in Drosophila. Two different high-affinity binding sites for [125I]alpha-Btx, a highly potent antagonist of vertebrate muscle AChR, were detected in fly head membranes. Equilibrium binding and kinetic studies revealed Kd values of approximately 0.1 nM (site 1) and approximately 4 nM (site 2). The estimated maximal binding (Bmax) was approximately 240 and 1080 fmol/mg protein respectively. Both sites exhibited a nicotinic-cholinergic pharmacology. Immunoprecipitation experiments with the ARD antisera indicated that the ARD protein is associated with the [125I]alpha-Btx binding site 1 only. These data support the previously postulated hypothesis that the ARD protein is part of an alpha-Btx binding neuronal AChR of Drosophila. Furthermore, they indicate heterogeneity in nicotinic-cholinergic binding sites in the insect nervous system. PMID:3141150

  14. Nicotine facilitates long-term potentiation induction in oriens-lacunosum moleculare cells via Ca2+ entry through non-alpha7 nicotinic acetylcholine receptors.

    PubMed

    Jia, Yousheng; Yamazaki, Yoshihiko; Nakauchi, Sakura; Ito, Ken-Ichi; Sumikawa, Katumi

    2010-02-01

    Hippocampal inhibitory interneurons have a central role in the control of network activity, and excitatory synapses that they receive express Hebbian and anti-Hebbian long-term potentiation (LTP). Because many interneurons in the hippocampus express nicotinic acetylcholine receptors (nAChRs), we explored whether exposure to nicotine promotes LTP induction in these interneurons. We focussed on a subset of interneurons in the stratum oriens/alveus that were continuously activated in the presence of nicotine due to the expression of non-desensitizing non-alpha7 nAChRs. We found that, in addition to alpha2 subunit mRNAs, these interneurons were consistently positive for somatostatin and neuropeptide Y mRNAs, and showed morphological characteristics of oriens-lacunosum moleculare cells. Activation of non-alpha7 nAChRs increased intracellular Ca(2+) levels at least in part via Ca(2+) entry through their channels. Presynaptic tetanic stimulation induced N-methyl-D-aspartate receptor-independent LTP in voltage-clamped interneurons at -70 mV when in the presence, but not absence, of nicotine. Intracellular application of a Ca(2+) chelator blocked LTP induction, suggesting the requirement of Ca(2+) signal for LTP induction. The induction of LTP was still observed in the presence of ryanodine, which inhibits Ca(2+) -induced Ca(2+) release from ryanodine-sensitive intracellular stores, and the L-type Ca(2+) channel blocker nifedipine. These results suggest that Ca(2+) entry through non-alpha7 nAChR channels is critical for LTP induction. Thus, nicotine affects hippocampal network activity by promoting LTP induction in oriens-lacunosum moleculare cells via continuous activation of non-alpha7 nAChRs.

  15. Central Insulin Action Activates Kupffer Cells by Suppressing Hepatic Vagal Activation via the Nicotinic Alpha 7 Acetylcholine Receptor.

    PubMed

    Kimura, Kumi; Tanida, Mamoru; Nagata, Naoto; Inaba, Yuka; Watanabe, Hitoshi; Nagashimada, Mayumi; Ota, Tsuguhito; Asahara, Shun-ichiro; Kido, Yoshiaki; Matsumoto, Michihiro; Toshinai, Koji; Nakazato, Masamitsu; Shibamoto, Toshishige; Kaneko, Shuichi; Kasuga, Masato; Inoue, Hiroshi

    2016-03-15

    Central insulin action activates hepatic IL-6/STAT3 signaling, which suppresses the gene expression of hepatic gluconeogenic enzymes. The vagus nerve plays an important role in this centrally mediated hepatic response; however, the precise mechanism underlying this brain-liver interaction is unclear. Here, we present our findings that the vagus nerve suppresses hepatic IL-6/STAT3 signaling via α7-nicotinic acetylcholine receptors (α7-nAchR) on Kupffer cells, and that central insulin action activates hepatic IL-6/STAT3 signaling by suppressing vagal activity. Indeed, central insulin-mediated hepatic IL-6/STAT3 activation and gluconeogenic gene suppression were impeded in mice with hepatic vagotomy, pharmacological cholinergic blockade, or α7-nAchR deficiency. In high-fat diet-induced obese and insulin-resistant mice, control of the vagus nerve by central insulin action was disturbed, inducing a persistent increase of inflammatory cytokines. These findings suggest that dysregulation of the α7-nAchR-mediated control of Kupffer cells by central insulin action may affect the pathogenesis of chronic hepatic inflammation in obesity.

  16. Effects of cigarette smoke exposure on nicotinic acetylcholine receptor subunits {alpha}7 and {beta}2 in the sudden infant death syndrome (SIDS) brainstem

    SciTech Connect

    Machaalani, Rita; Say, Meichien; Waters, Karen A.

    2011-12-15

    It is postulated that nicotine, as the main neurotoxic constituent of cigarette smoke, influences SIDS risk through effects on nicotinic acetylcholine receptors (nAChRs) in brainstem nuclei that control respiration and arousal. This study compared {alpha}7 and {beta}2 nAChR subunit expression in eight nuclei of the caudal and rostral medulla and seven nuclei of the pons between SIDS (n = 46) and non-SIDS infants (n = 14). Evaluation for associations with known SIDS risk factors included comparison according to whether infants had a history of exposure to cigarette smoke in the home, and stratification for sleep position and gender. Compared to non-SIDS infants, SIDS infants had significantly decreased {alpha}7 in the caudal nucleus of the solitary tract (cNTS), gracile and cuneate nuclei, with decreased {beta}2 in the cNTS and increased {beta}2 in the facial. When considering only the SIDS cohort: 1-cigarette smoke exposure was associated with increased {alpha}7 in the vestibular nucleus and increased {beta}2 in the rostral dorsal motor nucleus of the vagus, rNTS and Cuneate, 2-there was a gender interaction for {alpha}7 in the gracile and cuneate, and {beta}2 in the cNTS and rostral arcuate nucleus, and 3-there was no effect of sleep position on {alpha}7, but prone sleep was associated with decreased {beta}2 in three nuclei of the pons. In conclusion, SIDS infants demonstrate differences in expression of {alpha}7 and {beta}2 nAChRs within brainstem nuclei that control respiration and arousal, which is independent on prior history of cigarette smoke exposure, especially for the NTS, with additional differences for smoke exposure ({beta}2), gender ({alpha}7 and {beta}2) and sleep position ({beta}2) evident. -- Highlights: Black-Right-Pointing-Pointer The 'normal' response to smoke exposure is decreased {alpha}7 and {beta}2 in certain nuclei. Black-Right-Pointing-Pointer SIDS infants have decreased {alpha}7 in cNTS, Grac and Cun. Black-Right-Pointing-Pointer SIDS

  17. Non-charged amino acids from three different domains contribute to link agonist binding to channel gating in alpha7 nicotinic acetylcholine receptors.

    PubMed

    Aldea, Marcos; Mulet, José; Sala, Salvador; Sala, Francisco; Criado, Manuel

    2007-10-01

    Binding of agonists to nicotinic acetylcholine receptors results in channel opening. Previously, we have shown that several charged residues at three different domains of the alpha7 nicotinic receptor are involved in coupling binding and gating, probably through a network of electrostatic interactions. This network, however, could also be integrated by other residues. To test this hypothesis, non-charged amino acids were mutated and expression levels and electrophysiological responses of mutant receptors were determined. Mutants at positions Asn47 and Gln48 (loop 2), Ile130, Trp134, and Gln140 (loop 7), and Thr264 (M2-M3 linker) showed poor or null functional responses, despite significant membrane expression. By contrast, mutants F137A and S265A exhibited a gain of function effect. In all cases, changes in dose-response relationships were small, EC(50) values being between threefold smaller and fivefold larger, arguing against large modifications of agonist binding. Peak currents decayed at the same rate in all receptors except two, excluding large effects on desensitization. Thus, the observed changes could be mostly caused by alterations of the gating characteristics. Moreover, analysis of double mutants showed an interconnection between some residues in these domains, especially Gln48 with Ile130, suggesting a potential coupling between agonist binding and channel gating through these amino acids.

  18. Attenuation of ethanol-induced ataxia by alpha(4)beta(2) nicotinic acetylcholine receptor subtype in mouse cerebellum: a functional interaction.

    PubMed

    Taslim, N; Al-Rejaie, S; Saeed Dar, M

    2008-11-11

    Many epidemiological studies support the notion that people who drink alcohol also smoke cigarettes and vice versa thereby suggesting a possible functional interaction between these two most widely used psychoactive substances. We have earlier demonstrated that direct intracerebellar (ICB) microinfusion of nicotine dose-dependently antagonizes ethanol-induced ataxia and further that this antagonism occurs in a glutamate-nitric oxide-cyclic guanylyl monophosphate (cGMP) sensitive manner. The present study was designed to determine the possible involvement of specific nicotinic acetylcholine receptor (nAChR) subtype alpha(4)beta(2) in nicotine-induced attenuation of ethanol ataxia. Using the Rotorod test and direct ICB microinfusion technique in stereotaxically cannulated CD-1 male mice, we performed the Rotorod test following ICB administration of the alpha(4)beta(2)-selective agonist, (E)-N-methyl-4-(3-pyridinyl)-3-buten-1-amine (RJR-2403; 31.25, 62.5, 125 ng) on ethanol (2 g/kg; i.p.) ataxia at 15, 30, 45, 60 min post-ethanol injection. RJR-2403 dose-dependently attenuated ethanol ataxia suggesting a role of alpha(4)beta(2) subtype in ameliorating ethanol-induced ataxia. Pretreatment with ICB dihydro-beta-erythroidine (DHbetaE: 125, 250, 500, 750 ng), a potent alpha(4)beta(2)-selective antagonist, significantly reduced RJR-2403's effect further supporting the alpha(4)beta(2) involvement. DHbetaE (ICB) also antagonized ICB nicotine-induced attenuation of ethanol ataxia again reinforcing the role of alpha(4)beta(2) subtype. Additional evidence for the role of alpha(4)beta(2) subtype was provided when ICB alpha(4)beta(2) antisense oligodeoxynucleotide treatment markedly antagonized RJR 2403-induced attenuation of ethanol ataxia compared with missense-treated animals. This was confirmed with an associated decrease in the expression of alpha(4)beta(2) subtypes indicated by immunoblot experiments. In conclusion, the results of the present investigation support an

  19. Alpha7 Nicotinic Acetylcholine Receptors Modulate Motivation to Self-Administer Nicotine: Implications for Smoking and Schizophrenia

    PubMed Central

    Brunzell, Darlene H; McIntosh, J Michael

    2012-01-01

    Individuals diagnosed with schizophrenia have an exceptionally high risk for tobacco dependence. Postmortem studies show that these individuals have significant reductions in α7 nicotinic acetylcholine receptors (nAChRs) in several brain areas. Decreased α7-mediated function might not only be linked to schizophrenia but also to increased tobacco consumption. The purpose of this study was to determine whether pharmacological blockade of α7 nAChRs would increase motivation of rats to intravenously self-administer nicotine (NIC) during a progressive ratio schedule of reinforcement (PR). Before PR, rats received local infusions of 0, 10, or 20 pmol of a selective α7 nAChR antagonist, α-conotoxin ArIB [V11L,V16D] (ArIB) into the nucleus accumbens (NAc) shell or the anterior cingulate cortex, brain areas that contribute to motivation for drug reward. We additionally sought to determine whether local infusion of 0, 10, or 40 nmol of a selective α7 nAChR agonist, PNU 282987, into these brain areas would decrease motivation for NIC use. Infusion of ArIB into the NAc shell and anterior cingulate cortex resulted in a significant increase in active lever pressing, breakpoints, and NIC intake, suggesting that a decrease in α7 nAChR function increases motivation to work for NIC. In contrast, PNU 282987 infusion resulted in reductions in these measures when administered into the NAc shell, but had no effect after administration into the anterior cingulate cortex. These data identify reduction of α7 nAChR function as a potential mechanism for elevated tobacco use in schizophrenia and also identify activation of α7 nAChRs as a potential strategy for tobacco cessation therapy. PMID:22169946

  20. Modal gating of muscle nicotinic acetylcholine receptors

    NASA Astrophysics Data System (ADS)

    Vij, Ridhima

    Many ion channels exhibit multiple patterns of kinetic activity in single-channel currents. This behavior is rare in WT mouse muscle nicotinic acetylcholine receptors (AChRs), where A2C↔A2O gating events are well-described by single exponentials. Also, single-channel open probability (PO) is essentially homogeneous at a given agonist concentration in the WT receptors. Here I report that perturbations of almost all the residues in loop C (alpha188-alpha199, at the agonist binding site) generate heterogeneity in PO ('modes'). Such unsettled activity was apparent with an alanine substitution at all positions in loop C (except alphaY190 and alphaY198) and with different side chain substitutions at alphaP197 for both adult- and fetal-type AChRs. I used single channel electrophysiology along with site-directed mutagenesis to study modal gating in AChRs consequent to mutations/deletions in loop C. The multiple patterns of kinetic activity arose from the difference in agonist affinity rather than in intrinsic AChR gating. Out of the four different agonists used to study the modal behavior, acetylcholine (ACh) showed a higher degree of kinetic heterogeneity compared to others. The time constant for switching between modes was long (~mins), suggesting that they arise from alternative, stable protein conformations. By studying AChRs having only 1 functional binding site, I attempted to find the source of the affinity difference, which was traced mainly to the alphadelta agonist site. Affinity at the neurotransmitter binding site is mainly determined by a core of five aromatic residues (alphaY93, alphaW149, alphaY190, alphaY198 and deltaW57). Phenylalanine substitutions at all aromatic residues except alphaY93 resulted in elimination of modes. Modes were also eliminated by alanine mutation at deltaW57 on the complementary side but not at other aromatics. Also, by substituting four gamma subunit residues into the delta subunit on the complementary beta sheet, I found that

  1. Acetylcholine receptor extracellular domain determines sensitivity to nicotine-induced inactivation.

    PubMed

    Kuryatov, A; Olale, F A; Choi, C; Lindstrom, J

    2000-03-30

    We have shown previously that chronic exposure to submicromolar concentrations of nicotine permanently inactivates alpha4beta2 and alpha7 neuronal nicotinic acetylcholine receptors while alpha3beta2 acetylcholine receptors are resistant to inactivation. Phosphorylation of the large cytoplasmic domain has been proposed to mediate functional inactivation. Chimeric subunits consisting of human alpha4 sequence from their N-terminus to either the beginning of the first transmembrane domain or the large cytoplasmic domain and alpha3 sequences thereafter formed acetylcholine receptors with beta2 subunits which were as susceptible to nicotine-induced inactivation as wild-type alpha4 acetylcholine receptors. The converse chimeras, containing the N-terminal parts of the alpha3 subunit and the C-terminal parts of the alpha4 subunit, formed acetylcholine receptors with beta2 subunits which were as resistant to nicotine-induced inactivation as wild-type alpha3beta2 acetylcholine receptors. Thus, inactivation of acetylcholine receptors produced by chronic exposure to nicotine results primarily from effects of the agonist on the extracellular and transmembrane domains of the alpha subunit.

  2. Lack of modulation of nicotinic acetylcholine alpha-7 receptor currents by kynurenic acid in adult hippocampal interneurons.

    PubMed

    Dobelis, Peter; Staley, Kevin J; Cooper, Donald C

    2012-01-01

    Kynurenic acid (KYNA), a classical ionotropic glutamate receptor antagonist is also purported to block the α7-subtype nicotinic acetylcholine receptor (α7* nAChR). Although many published studies cite this potential effect, few have studied it directly. In this study, the α7*-selective agonist, choline, was pressure-applied to interneurons in hippocampal subregions, CA1 stratum radiatum and hilus of acute brain hippocampal slices from adolescent to adult mice and adolescent rats. Stable α7* mediated whole-cell currents were measured using voltage-clamp at physiological temperatures. The effects of bath applied KYNA on spontaneous glutamatergic excitatory postsynaptic potentials (sEPSC) as well as choline-evoked α7* currents were determined. In mouse hilar interneurons, KYNA totally blocked sEPSC whole-cell currents in a rapid and reversible manner, but had no effect on choline-evoked α7* whole-cell currents. To determine if this lack of KYNA effect on α7* function was due to regional and/or species differences in α7* nAChRs, the effects of KYNA on choline-evoked α7* whole-cell currents in mouse and rat stratum radiatum interneurons were tested. KYNA had no effect on either mouse or rat stratum radiatum interneuron choline-evoked α7* whole-cell currents. Finally, to test whether the lack of effect of KYNA was due to unlikely slow kinetics of KYNA interactions with α7* nAChRs, recordings of a7*-mediated currents were made from slices that were prepared and stored in the presence of 1 mM KYNA (>90 minutes exposure). Under these conditions, KYNA had no measurable effect on α7* nAChR function. The results show that despite KYNA-mediated blockade of glutamatergic sEPSCs, two types of hippocampal interneurons that express choline-evoked α7* nAChR currents fail to show any degree of modulation by KYNA. Our results indicate that under our experimental conditions, which produced complete KYNA-mediated blockade of sEPSCs, claims of KYNA effects on choline

  3. Association of polymorphisms in nicotinic acetylcholine receptor alpha 4 subunit gene (CHRNA4), mu-opioid receptor gene (OPRM1), and ethanol-metabolizing enzyme genes with alcoholism in Korean patients.

    PubMed

    Kim, Soon Ae; Kim, Jong-Woo; Song, Ji-Young; Park, Sunny; Lee, Hee Jae; Chung, Joo-Ho

    2004-01-01

    Findings obtained from several studies indicate that ethanol enhances the activity of alpha4beta2 neuronal nicotinic acetylcholine receptor and support the possibility that a polymorphism of the nicotinic acetylcholine receptor alpha4 subunit gene (CHRNA4) modulates enhancement of nicotinic receptor function by ethanol. To identify the association between the CfoI polymorphism of the CHRNA4 and alcoholism, we examined distribution of genotypes and allele frequencies in Korean patients diagnosed with alcoholism (n = 127) and Korean control subjects without alcoholism (n = 185) with polymerase chain reaction-restriction fragment length polymorphism methods. We were able to detect the association between the CfoI polymorphism of the CHRNA4 and alcoholism in Korean patients (genotype P = .023; allele frequency P = .047). The genotypes and allele frequencies of known polymorphisms in other alcoholism candidate genes, such as alcohol metabolism-related genes [alcohol dehydrogenase 2 (ADH2), aldehyde dehydrogenase 2 (ALDH2), alcohol dehydrogenase 3 (ADH3), and cytochrome P450 2E1 (CYP2E1)] and mu-opioid receptor gene (OPRM1), were studied. The polymorphisms of ADH2, ALDH2, and CYP2E1 were significantly different in Korean patients with alcoholism and Korean control subjects without alcoholism, but ADH3 and OPRM1 did not differ between the two groups.

  4. (R)-3'-(3-methylbenzo[b]thiophen-5-yl)spiro[1-azabicyclo[2,2,2]octane-3,5'-oxazolidin]-2'-one, a novel and potent alpha7 nicotinic acetylcholine receptor partial agonist displays cognitive enhancing properties.

    PubMed

    Tatsumi, Ryo; Fujio, Masakazu; Takanashi, Shin-ichi; Numata, Atsushi; Katayama, Jiro; Satoh, Hiroyuki; Shiigi, Yasuyuki; Maeda, Jun-ichi; Kuriyama, Makoto; Horikawa, Takashi; Murozono, Takahiro; Hashimoto, Kenji; Tanaka, Hiroshi

    2006-07-13

    Recent studies have suggested that the alpha7 nicotinic acetylcholine receptors play important roles in learning and memory. Herein, we describe our research of the structure-activity relationships (SAR) in a series of (S)-spiro[1-azabicyclo[2.2.2]octane-3,5'-oxazolidin]-2'-ones bearing various bicyclic moieties to discover novel alpha7 receptor agonists. Through a number of SAR studies on the series, we have found out that inhibition of CYP 2D6 isozyme, which was a primary obstacle for the previously identified compound, was avoidable by the introduction of bicyclic moieties. Chemical optimization of the series led to the identification of a novel and potent alpha7 nicotinic acetylcholine receptor partial agonist 23. This compound not only possessed high binding affinity (K(i) = 3 nmol/L) toward the alpha7 receptor but also showed agonistic activity even at a concentration of 0.1 micromol/L. In addition, compound 23 improved cognition in several rat models, which might suggest the potential of the alpha7 receptor partial agonist for the treatment of neurological disorders including cognitive dysfunction.

  5. Nicotinic Acetylcholine Receptors in Sensory Cortex

    ERIC Educational Resources Information Center

    Metherate, Raju

    2004-01-01

    Acetylcholine release in sensory neocortex contributes to higher-order sensory function, in part by activating nicotinic acetylcholine receptors (nAChRs). Molecular studies have revealed a bewildering array of nAChR subtypes and cellular actions; however, there is some consensus emerging about the major nAChR subtypes and their functions in…

  6. Regulation of acetylcholine receptor alpha subunit variants in human myasthenia gravis. Quantification of steady-state levels of messenger RNA in muscle biopsy using the polymerase chain reaction.

    PubMed Central

    Guyon, T; Levasseur, P; Truffault, F; Cottin, C; Gaud, C; Berrih-Aknin, S

    1994-01-01

    Myasthenia gravis (MG) is an autoimmune disease mediated by auto-antibodies that attack the nicotinic acetylcholine receptor (AChR). To elucidate the molecular mechanisms underlying the decrease in AChR levels at the neuromuscular junction, we investigated the regulation of AChR expression by analyzing mRNA of the two AChR alpha subunit isoforms (P3A+ and P3A-) in muscle samples from myasthenic patients relative to controls. We applied a quantitative method based on reverse transcription of total RNA followed by polymerase chain reaction (PCR), using an internal standard we constructed by site-directed mutagenesis. An increased expression of mRNA coding for the alpha subunit of the AChR isoforms was observed in severely affected patients (P < 0.003 versus controls) but not in moderately affected patients, independently of the anti-AChR antibody titer. Study of mRNA precursor levels indicates a higher expression in severely affected patients compared to controls, suggesting an enhanced rate of transcription of the message coding for the alpha subunit isoforms in these patients. We have also reported that mRNA encoding both isoforms are expressed at an approximate 1:1 ratio in controls and in patients. We have thus identified a new biological parameter correlated with disease severity, and provide evidence of a compensatory mechanism to balance the loss of AChR in human myasthenia gravis, which is probably triggered only above a certain degree of AChR loss. Images PMID:8040257

  7. Alpha7 nicotinic acetylcholine receptor is required for amyloid pathology in brain endothelial cells induced by Glycoprotein 120, methamphetamine and nicotine

    PubMed Central

    Liu, Liqun; Yu, Jingyi; Li, Li; Zhang, Bao; Liu, Lingjuan; Wu, Chun-Hua; Jong, Ambrose; Mao, Ding-An; Huang, Sheng-He

    2017-01-01

    One of the most challenging issues in HIV-associated neurocognitive disorders (HAND) caused by HIV-1 virotoxins and drug abuse is the lack of understanding the underlying mechanisms that are commonly associated with disorders of the blood-brain barrier (BBB), which mainly consists of brain microvascular endothelial cells (BMEC). Here, we hypothesized that Glycoprotein 120 (gp120), methamphetamine (METH) and nicotine (NT) can enhance amyloid-beta (Aβ) accumulation in BMEC through Alpha7 nicotinic acetylcholine receptor (α7 nAChR). Both in vitro (human BMEC) (HBMEC) and in vivo (mice) models of BBB were used to dissect the role of α7 nAChR in up-regulation of Aβ induced by gp120, METH and NT. Aβ release from and transport across HBMEC were significantly increased by these factors. Methyllycaconitine (MLA), an antagonist of α7 nAChR, could efficiently block these pathogenic effects. Furthermore, our animal data showed that these factors could significantly increase the levels of Aβ, Tau and Ubiquitin C-Terminal Hydrolase L1 (UCHL1) in mouse cerebrospinal fluid (CSF) and Aβ in the mouse brains. These pathogenicities were significantly reduced by MLA, suggesting that α7 nAChR may play an important role in neuropathology caused by gp120, METH and NT, which are the major pathogenic factors contributing to the pathogenesis of HAND. PMID:28074940

  8. Antigen-specific modulation of experimental myasthenia gravis: nasal tolerization with recombinant fragments of the human acetylcholine receptor alpha-subunit.

    PubMed

    Barchan, D; Souroujon, M C; Im, S H; Antozzi, C; Fuchs, S

    1999-07-06

    Myasthenia gravis (MG) and experimental autoimmune myasthenia gravis (EAMG) are antibody-mediated autoimmune diseases in which the nicotinic acetylcholine receptor (AcChoR) is the major autoantigen. The immune response in these diseases is heterogeneous and is directed to a wide variety of T and B cell epitopes of AcChoR. Candidate molecules for specific immunotherapy of MG should, therefore, have a broad specificity. We used recombinant fragments of the human AcChoR, encompassing the extracellular domain of the alpha-subunit, or shorter fragments derived from it, in experiments to modulate EAMG. We have demonstrated that intranasal administration of these recombinant fragments, which represent a major portion of epitopes involved in MG, prevents the induction of EAMG in rats and immunosuppresses an ongoing disease, as assessed by clinical symptoms, weight loss, and muscle AcChoR content. These effects on EAMG were accompanied by a marked reduction in the proliferative T-cell response and IL-2 production in response to AcChoR, in reduced anti-self AcChoR antibody titers and in an isotype switch of AcChoR-specific antibodies, from IgG2 to IgG1. We conclude that nasal tolerance induced by appropriate recombinant fragments of human AcChoR is effective in suppressing EAMG and might possibly be considered as a therapeutic modality for MG.

  9. Serotonergic modulation of muscle acetylcholine receptors of different subunit composition.

    PubMed Central

    García-Colunga, J; Miledi, R

    1996-01-01

    Modulation of muscle acetylcholine (AcCho) receptors (AcChoRs) by serotonin [5-hydroxytryptamine (5HT)] and other serotonergic compounds was studied in Xenopus laevis oocytes. Various combinations of alpha, beta, gamma, and delta subunit RNAs were injected into oocytes, and membrane currents elicited by AcCho were recorded under voltage clamp. Judging by the amplitudes of AcCho currents generated, the levels of functional receptor expression were: alpha beta gamma delta > alpha beta delta > alpha beta gamma > alpha gamma delta. The alpha beta gamma delta and alpha beta delta AcChoR Subtypes were strongly blocked by 5HT, whereas the alpha beta gamma receptor was blocked only slightly. The order of blocking potency of AcChoRs by 5HT was: alpha beta delta > alpha beta gamma delta > alpha beta gamma. 5HT receptor antagonists, such as methysergide and spiperone, were even more potent blockers of AcChoRs than 5HT but did not show much subunit selectivity. Blockage of alpha beta gamma delta and alpha beta delta receptors by 5HT was voltage-dependent, and the voltage dependence was abolished when the delta subunit was omitted. These findings may need to be taken into consideration when trying to elucidate the mode of action of many clinically important serotonergic compounds. Images Fig. 3 PMID:8633003

  10. Spinal activation of alpha7-nicotinic acetylcholine receptor attenuates posttraumatic stress disorder-related chronic pain via suppression of glial activation.

    PubMed

    Sun, Rao; Zhang, Wei; Bo, Jinhua; Zhang, Zuoxia; Lei, Yishan; Huo, Wenwen; Liu, Yue; Ma, Zhengliang; Gu, Xiaoping

    2017-03-06

    The high prevalence of chronic pain in posttraumatic stress disorder (PTSD) individuals has been widely reported by clinical studies, which emphasized an urgent need to uncover the underlying mechanisms and identify potential therapeutic targets. Recent studies suggested that targeting activated glia and their pro-inflammatory products may provide a novel and effective therapy for the stress-related pain. In this study, we investigated whether activation of alpha-7 nicotinic acetylcholine receptor (α7 nAChR), a novel anti-inflammatory target, could attenuate PTSD-related chronic pain. The experiments were conducted in a rat model of single prolonged stress (SPS), an established model of PTSD-pain comorbidity. We found that SPS exposure produced persistent mechanical allodynia. Immunohistochemical and enzyme-linked immuno sorbent assay analysis showed that SPS also induced elevated activation of glia cells (including microglia and astrocytes) and accumulation of pro-inflammatory cytokines in spinal cord. In another experiment, we found that intrathecal injection of PHA-543613, a selective α7 nAchR agonist, attenuated the SPS-evoked allodynia in a dose dependent manner. However, this anti-hyperalgesic effect was blocked by pretreatment with methyllycaconitine (MLA), a selective α7 nAchR antagonist. Further analyses showed that PHA-543613 suppressed SPS-induced spinal glial activation and SPS-elevated spinal pro-inflammatory cytokines, and these were abolished by MLA. Taken together, the present study showed that spinal activation of α7 nAChR by PHA-543613 attenuated mechanical allodynia induced by PTSD-like stress, and the suppression of spinal glial activation may underlie this anti-hyperalgesic effect. Our study demonstrated the therapeutic potential of targeting α7 nAChR in the treatment of PTSD-related chronic pain.

  11. Deletion of the Alpha7 Nicotinic Acetylcholine Receptor Gene Improves Cognitive Deficits and Synaptic Pathology in a Mouse Model of Alzheimer's Disease

    PubMed Central

    Dziewczapolski, Gustavo; Glogowski, Carolina M.; Masliah, Eliezer; Heinemann, Stephen F.

    2009-01-01

    It has been recently shown that the Alzheimer's disease (AD) pathogenic peptide amyloid-β1-42 (Aβ1-42) binds to the alpha7 nicotinic acetylcholine receptor (α7nAChR) with high affinity and the α7nAChR and Aβ1-42 are both found co-localized in neuritic plaques of human brains with AD. Moreover, the intraneuronal accumulation of Aβ1-42 was shown to be facilitated by its high-affinity binding to the α7nAChR and α7nAChR activation mediates Aβ-induced tau protein phosphorylation. To test the hypothesis that α7nAChRs are involved in AD pathogenesis, we used a transgenic mouse model of AD over-expressing a mutated form of the human amyloid precursor protein (APP) and lacking the α7nAChR gene (APPα7KO). We have shown that, despite the presence of high amounts of APP and amyloid deposits, deleting the α7nAChR subunit in the mouse model of AD leads to a protection from the dysfunction in synaptic integrity (pathology and plasticity) and learning and memory behavior. Specifically, APPα7KO mice express APP and Aβ at similar levels to APP mice and yet they were able to solve a cognitive challenge such as the Morris water maze test significantly better than APP, with performances comparable to control groups. Moreover, deleting the α7nAChR subunit protected the brain from loss of the synaptic markers, synaptophysin and MAP2, reduced the gliosis, and preserved the capacity to elicit LTP otherwise deficient in APP mice. These results are consistent with the hypothesis that the α7nAChR plays a role in AD and suggests that interrupting α7nAChR function could be beneficial in the treatment of AD. PMID:19587288

  12. Progesterone Modulates a Neuronal Nicotinic Acetylcholine Receptor

    NASA Astrophysics Data System (ADS)

    Valera, S.; Ballivet, M.; Bertrand, D.

    1992-10-01

    The major brain nicotinic acetylcholine receptor is assembled from two subunits termed α 4 and nα 1. When expressed in Xenopus oocytes, these subunits reconstitute a functional acetylcholine receptor that is inhibited by progesterone levels similar to those found in serum. In this report, we show that the steroid interacts with a site located on the extracellular part of the protein, thus confirming that inhibition by progesterone is not due to a nonspecific perturbation of the membrane bilayer or to the activation of second messengers. Because inhibition by progesterone does not require the presence of agonist, is voltage-independent, and does not alter receptor desensitization, we conclude that the steroid is not an open channel blocker. In addition, we show that progesterone is not a competitive inhibitor but may interact with the acetylcholine binding site and that its effect is independent of the ionic permeability of the receptor.

  13. The alpha7 nicotinic acetylcholine receptor-selective antagonist, methyllycaconitine, partially protects against beta-amyloid1-42 toxicity in primary neuron-enriched cultures.

    PubMed

    Martin, Shelley E; de Fiebre, Nancy Ellen C; de Fiebre, Christopher M

    2004-10-01

    Studies have suggested that the neuroprotective actions of alpha7 nicotinic agonists arise from activation of receptors and not from the extensive desensitization which rapidly follows activation. Here, we report that the alpha7-selective nicotinic antagonist, methyllycaconitine (MLA), protects against beta-amyloid-induced neurotoxicity; whereas the alpha4beta2-selective antagonist, dihydro-beta-erythroidine, does not. These findings suggest that neuroprotective actions of alpha7-acting agents arise from receptor inhibition/desensitization and that alpha7 antagonists may be useful neuroprotective agents.

  14. Primary Structure of Nicotinic Acetylcholine Receptor

    DTIC Science & Technology

    1986-08-01

    quantities of starting material (for reviews of receptor, see Popot and Changeux, 1984; Stroud and Finer-Moore, 1985). This work led to the...Cloning of the Acetylcholine Receptor. Cold Spring Harbor Symp. on Quant. Biol. XLVIH: 71-78. 15. Popot , J-L. and Changeux, J-P. (1984) The

  15. Effect of nicotinic acetylcholine receptor alpha 1 (nAChRα1) peptides on rabies virus infection in neuronal cells.

    PubMed

    Sajjanar, Basavaraj; Saxena, Shikha; Bisht, Deepika; Singh, Arvind Kumar; Manjunatha Reddy, G B; Singh, Rajendra; Singh, R P; Kumar, Satish

    2016-06-01

    Rabies virus (RABV) is neurotropic and causes acute progressive encephalitis. Herein, we report the interaction of nAChRα1-subunit peptides with RABV and the effect of these peptides on RABV infection in cultured neuronal cells. Peptide sequences derived from torpedo, bovine, human and rats were synthesized and studied for their interactions with RABV using virus capture ELISA and peptide immunofluorescence. The results showed specific binding of the nAChRα1-subunit peptides to the RABV. In the virus adsorption assay, these peptides were found to inhibit the attachment of the RABV to the neuronal cells. The nAChRα1-subunit peptides inhibited the RABV infection and reduced viral gene expression in the cultured neuroblastoma (N2A) cells. Torpedo peptide sequence (T-32) had highest antiviral effect (IC50=14±3.01μM) compared to the other peptides studied. The results of the study indicated that nAChRα1-subunit peptides may act as receptor decoy molecules and inhibit the binding of virus to the native host cell receptors and hence may reduce viral infection.

  16. Histamine H3 receptors regulate acetylcholine release from the guinea pig ileum myenteric plexus

    SciTech Connect

    Poli, E.; Coruzzi, G.; Bertaccini, G. )

    1991-01-01

    The effect of selective histamine H3-receptor agonists and antagonists on the acetylcholine release from peripheral nerves was evaluated in the guinea pig longitudinal muscle-myenteric plexus preparations, preloaded with ({sup 3}H)choline. In the presence of H1 and H2 blockade, histamine and (R)-{alpha}-methylhistamine inhibited the electrically-evoked acetylcholine release, being (R)-{alpha}-methylhistamine more active than histamine, but behaving as a partial agonist. The effect of histamine was completely reversed by selective H3-blocking drugs, thioperamide and impromidine, while only submaximal doses of (R)-{alpha}-methylhistamine were antagonized. Furthermore, thioperamide and impromidine enhanced the electrically-evoked acetylcholine release. On the contrary, the new H3-blocker, HST-7, was found substantially ineffective, both as histamine antagonist and as acetylcholine overflow enhancer. These data suggest that histamine exerts an inhibitory control on the acetylcholine release from intestinal cholinergic nerves through the activation of H3 receptors.

  17. Turnover of Acetylcholine Receptors: Mechanisms of Regulation

    DTIC Science & Technology

    1988-12-01

    ME, Whittingham S, and Duane DD (1976) Antibody to acetylcholine receptor in myasthenia gravis : prevalance, clinical correlates and diagnostic value...transferred to nitorcellulose. Proc Natl Acad Sci 77:5201-5205. Weinberg CB and Hall ZW (1979) Antibodies from patients with myasthenia gravis recognize

  18. Neural regulation of MRNA for the alpha-subunit of acetylcholine receptors: Role of neuromuscular transmission. (Reannouncement with new availability information)

    SciTech Connect

    Lipsky, N.G.; Drachman, D.B.; Pestronk, A.; Shih, P.J.

    1989-12-31

    Levels of mRNA for acetylcholine receptor (AChR) subunits are relatively low in innervated skeletal muscles. Following denervation they rise rapidly, leading to increased AChR synthesis. The mechanism by which motor nerves normally regulate these mRNA levels is not yet known. In order to determine the possible role of synaptic transmission in this process, the authors have compared the effect of blockade of cholinergic ACh transmission with that of surgical denervation. Blockade of quantal ACh transmission was produced by injection of type A botulinum toxin into the soleus muscles of rats.

  19. Neuronal Nicotinic Acetylcholine Receptors and Epilepsy

    PubMed Central

    Bertrand, Daniel

    2002-01-01

    The identification of a genetically transmissible form of epilepsy that is associated with a mutation in CHRNA4, the gene that encodes the α4 subunit of the high-affinity nicotinic acetylcholine receptor, was the first demonstration that an alteration in a ligand-gated ion channel can cause seizures. Since then, nine mutations have been found, and analysis of their physiologic properties has revealed that all of them enhance receptor function. PMID:15309115

  20. External Imaging of Cerebral Muscarinic Acetylcholine Receptors

    NASA Astrophysics Data System (ADS)

    Eckelman, William C.; Reba, Richard C.; Rzeszotarski, Waclaw J.; Gibson, Raymond E.; Hill, Thomas; Holman, B. Leonard; Budinger, Thomas; Conklin, James J.; Eng, Robert; Grissom, Michael P.

    1984-01-01

    A radioiodinated ligand that binds to muscarinic acetylcholine receptors was shown to distribute in the brain by a receptor-mediated process. With single-photon-emission imaging techniques, radioactivity was detected in the cerebrum but not in the cerebellum, whereas with a flow-limited radiotracer, radioactivity was detected in cerebrum and cerebellum. Single-photon-emission computed tomography showed good definition of the caudate putamen and cortex in man.

  1. External imaging of cerebral muscarinic acetylcholine receptors

    SciTech Connect

    Eckelman, W.C.; Reba, R.C.; Rzeszotarski, W.J.; Gibson, R.E.; Hill, T.; Holman, B.L.; Budinger, T.; Conklin, J.J.; Eng, R.; Grissom, M.P.

    1984-01-20

    A radioiodinated ligand that binds to muscarinic acetylcholine receptors was shown to distribute in the brain by a receptor-mediated process. With single-photon-emission imaging techniques, radioactivity was detected in the cerebrum but not in the cerebellum, whereas with a flow-limited radiotracer, radioactivity was detected in cerebrum and cerebellum. Single-photon-emission computed tomography showed good definition of the caudate putamen and cortex in man.

  2. Similarities between the Binding Sites of SB-206553 at Serotonin Type 2 and Alpha7 Acetylcholine Nicotinic Receptors: Rationale for Its Polypharmacological Profile.

    PubMed

    Möller-Acuña, Patricia; Contreras-Riquelme, J Sebastián; Rojas-Fuentes, Cecilia; Nuñez-Vivanco, Gabriel; Alzate-Morales, Jans; Iturriaga-Vásquez, Patricio; Arias, Hugo R; Reyes-Parada, Miguel

    2015-01-01

    Evidence from systems biology indicates that promiscuous drugs, i.e. those that act simultaneously at various protein targets, are clinically better in terms of efficacy, than those that act in a more selective fashion. This has generated a new trend in drug development called polypharmacology. However, the rational design of promiscuous compounds is a difficult task, particularly when the drugs are aimed to act at receptors with diverse structure, function and endogenous ligand. In the present work, using docking and molecular dynamics methodologies, we established the most probable binding sites of SB-206553, a drug originally described as a competitive antagonist of serotonin type 2B/2C metabotropic receptors (5-HT2B/2CRs) and more recently as a positive allosteric modulator of the ionotropic α7 nicotinic acetylcholine receptor (nAChR). To this end, we employed the crystal structures of the 5-HT2BR and acetylcholine binding protein as templates to build homology models of the 5-HT2CR and α7 nAChR, respectively. Then, using a statistical algorithm, the similarity between these binding sites was determined. Our analysis showed that the most plausible binding sites for SB-206553 at 5-HT2Rs and α7 nAChR are remarkably similar, both in size and chemical nature of the amino acid residues lining these pockets, thus providing a rationale to explain its affinity towards both receptor types. Finally, using a computational tool for multiple binding site alignment, we determined a consensus binding site, which should be useful for the rational design of novel compounds acting simultaneously at these two types of highly different protein targets.

  3. Similarities between the Binding Sites of SB-206553 at Serotonin Type 2 and Alpha7 Acetylcholine Nicotinic Receptors: Rationale for Its Polypharmacological Profile

    PubMed Central

    Möller-Acuña, Patricia; Contreras-Riquelme, J. Sebastián; Rojas-Fuentes, Cecilia; Nuñez-Vivanco, Gabriel; Alzate-Morales, Jans; Iturriaga-Vásquez, Patricio; Arias, Hugo R.; Reyes-Parada, Miguel

    2015-01-01

    Evidence from systems biology indicates that promiscuous drugs, i.e. those that act simultaneously at various protein targets, are clinically better in terms of efficacy, than those that act in a more selective fashion. This has generated a new trend in drug development called polypharmacology. However, the rational design of promiscuous compounds is a difficult task, particularly when the drugs are aimed to act at receptors with diverse structure, function and endogenous ligand. In the present work, using docking and molecular dynamics methodologies, we established the most probable binding sites of SB-206553, a drug originally described as a competitive antagonist of serotonin type 2B/2C metabotropic receptors (5-HT2B/2CRs) and more recently as a positive allosteric modulator of the ionotropic α7 nicotinic acetylcholine receptor (nAChR). To this end, we employed the crystal structures of the 5-HT2BR and acetylcholine binding protein as templates to build homology models of the 5-HT2CR and α7 nAChR, respectively. Then, using a statistical algorithm, the similarity between these binding sites was determined. Our analysis showed that the most plausible binding sites for SB-206553 at 5-HT2Rs and α7 nAChR are remarkably similar, both in size and chemical nature of the amino acid residues lining these pockets, thus providing a rationale to explain its affinity towards both receptor types. Finally, using a computational tool for multiple binding site alignment, we determined a consensus binding site, which should be useful for the rational design of novel compounds acting simultaneously at these two types of highly different protein targets. PMID:26244344

  4. A role for acetylcholine receptors in the fusion of chick myoblasts

    PubMed Central

    1988-01-01

    The role of acetylcholine receptors in the control of chick myoblast fusion in culture has been explored. Spontaneous fusion of myoblasts was inhibited by the nicotinic acetylcholine receptor antagonists alpha- bungarotoxin, Naja naja toxin and monoclonal antibody mcAb 5.5. The muscarinic antagonists QNB and n-methyl scopolamine were without effect. Atropine had no effect below 1 microM, where it blocks muscarinic receptors; at higher concentrations, when it blocks nicotinic receptors also, atropine inhibited myoblast fusion. The inhibitions imposed by acetylcholine receptor antagonists lasted for approximately 12 h; fusion stimulated by other endogenous substances then took over. The inhibition was limited to myoblast fusion. The increases in cell number, DNA content, the level of creatine phosphokinase activity (both total and muscle-specific isozyme) and the appearance of heavy chain myosin, which accompany muscle differentiation, followed a normal time course. Pre-fusion myoblasts, fusing myoblasts, and young myotubes specifically bound labeled alpha- bungarotoxin, indicating the presence of acetylcholine receptors. The nicotinic acetylcholine receptor agonist, carbachol, induced uptake of [14C]Guanidinium through the acetylcholine receptor. Myoblasts, aligned myoblasts and young myotubes expressed the synthetic enzyme Choline acetyltransferase and stained positively with antibodies against acetylcholine. The appearance of ChAT activity in myogenic cultures was prevented by treatment with BUDR; nonmyogenic cells in the cultures expressed ChAT at a level which was too low to account for the activity in myogenic cultures. We conclude that activation of the nicotinic acetylcholine receptor is part of the mechanism controlling spontaneous myoblast fusion and that myoblasts synthesize an endogenous, fusion- inducing agent that activates the nicotinic ACh receptor. PMID:3372592

  5. Agonists block currents through acetylcholine receptor channels.

    PubMed Central

    Sine, S M; Steinbach, J H

    1984-01-01

    We have examined the effects of high concentrations of cholinergic agonists on currents through single acetylcholine receptor (AChR) channels on clonal BC3H1 cells. We find that raised concentrations of acetylcholine (ACh; above 300 microM) or carbamylcholine (Carb; above 1,000 microM) produce a voltage- and concentration-dependent reduction in the mean single-channel current. Raised concentrations of suberyldicholine (Sub; above 3 microM) produce a voltage- and concentration-dependent increase in the number of brief duration low-conductance interruptions of open-channel currents. These observations can be quantitatively described by a model in which agonist molecules enter and transiently occlude the ion-channel of the AChR. PMID:6478036

  6. Identification of a novel nicotinic acetylcholine receptor structural subunit expressed in goldfish retina

    PubMed Central

    1989-01-01

    A new non-alpha (n alpha) member of the nicotinic acetylcholine receptor (nAChR) gene family designated GFn alpha-2 has been identified in goldfish retina by cDNA cloning. This cDNA clone encodes a protein with structural features common to all nAChR subunits sequenced to date; however, unlike all known alpha-subunits of the receptor, it lacks the cysteine residues believed to be involved in acetylcholine binding. Northern blot analysis shows multiple transcripts hybridizing to the GFn alpha-2 cDNA in goldfish retina but undetectable levels of hybridizable RNA in brain, muscle, or liver. S1 nuclease protection experiments indicate that multiple mRNAs are expressed in retina with regions identical or very similar to the GFn alpha-2 sequence. In situ hybridization shows that the gene encoding GFn alpha-2 is expressed predominantly in the ganglion cell layer of the retina. PMID:2465296

  7. Naturally occurring and synthetic peptides acting on nicotinic acetylcholine receptors.

    PubMed

    Kasheverov, Igor E; Utkin, Yuri N; Tsetlin, Victor I

    2009-01-01

    Nicotinic acetylcholine receptors (nAChRs) are pentameric membrane-bound proteins belonging to the large family of ligand-gated ion channels. nAChRs possess various binding sites which interact with compounds of different chemical nature, including peptides. Historically first peptides found to act on nAChR were synthetic fragments of snake alpha-neurotoxins, competitive receptor antagonists. Later it was shown that fragments of glycoprotein from rabies virus, having homology to alpha-neurotoxins, and polypeptide neurotoxins waglerins from the venom of Wagler's pit viper Trimeresurus (Tropidolaemus) wagleri bind in a similar way, waglerins being efficient blockers of muscle-type nAChRs. Neuropeptide substance P appears to interact with the channel moiety of nAChR. beta-Amyloid, a peptide forming senile plaques in Alzheimer's disease, also can bind to nAChR, although the mode of binding is still unclear. However, the most well-studied peptides interacting with the ligand-binding sites of nAChRs are so-called alpha-conotoxins, peptide neurotoxins from marine snails of Conus genus. First alpha-conotoxins were discovered in the late 1970s, and now it is a rapidly growing family due to isolation of peptides from multiple Conus species, as well as to cloning, and chemical synthesis of new analogues. Because of their unique selectivity towards distinct nAChR subtypes, alpha-conotoxins became valuable tools in nAChR research. Recent X-ray structures of alpha-conotoxin complexes with acetylcholine-binding protein, a model of nAChR ligand-binding domains, revealed the details of the nAChR ligand-binding sites and provided the basis for design of novel ligands.

  8. Homology modeling of human muscarinic acetylcholine receptors.

    PubMed

    Thomas, Trayder; McLean, Kimberley C; McRobb, Fiona M; Manallack, David T; Chalmers, David K; Yuriev, Elizabeth

    2014-01-27

    We have developed homology models of the acetylcholine muscarinic receptors M₁R-M₅R, based on the β₂-adrenergic receptor crystal as the template. This is the first report of homology modeling of all five subtypes of acetylcholine muscarinic receptors with binding sites optimized for ligand binding. The models were evaluated for their ability to discriminate between muscarinic antagonists and decoy compounds using virtual screening using enrichment factors, area under the ROC curve (AUC), and an early enrichment measure, LogAUC. The models produce rational binding modes of docked ligands as well as good enrichment capacity when tested against property-matched decoy libraries, which demonstrates their unbiased predictive ability. To test the relative effects of homology model template selection and the binding site optimization procedure, we generated and evaluated a naïve M₂R model, using the M₃R crystal structure as a template. Our results confirm previous findings that binding site optimization using ligand(s) active at a particular receptor, i.e. including functional knowledge into the model building process, has a more pronounced effect on model quality than target-template sequence similarity. The optimized M₁R-M₅R homology models are made available as part of the Supporting Information to allow researchers to use these structures, compare them to their own results, and thus advance the development of better modeling approaches.

  9. Alcohol's actions on neuronal nicotinic acetylcholine receptors.

    PubMed

    Davis, Tiffany J; de Fiebre, Christopher M

    2006-01-01

    Although it has been known for many years that alcoholism and tobacco addiction often co-occur, relatively little information is available on the biological factors that regulate the co-use and abuse of nicotine and alcohol. In the brain, nicotine acts at several different types of receptors collectively known as nicotinic acetylcholine receptors (nAChRs). Alcohol also acts on at least some of these receptors, enhancing the function of some nAChR subtypes and inhibiting the activity of others. Chronic alcohol and nicotine administration also lead to changes in the numbers of nAChRs. Natural variations (i.e., polymorphisms) in the genes encoding different nAChR subunits may be associated with individual differences in the sensitivity to some of alcohol's and nicotine's effects. Finally, at least one subtype of nAChR may help protect cells against alcohol-induced neurotoxicity.

  10. [Desensitization of the nicotinic acetylcholine receptor].

    PubMed

    Quiñonez, M; Rojas, L

    1994-01-01

    In biological membranes, ionic channels act speeding up ion movements. Each ionic channel is excited by a specific stimulus (i.e. electric, mechanical, chemical, etc.). Chemically activated ionic channels (CAIC), such as the nicotinic acetylcholine receptor (nAChR), suffer desensitization when the receptor site is still occupied by the agonist molecule. The desensitized CAIC is a non functional channel state regarded as a particular case of receptors rundown. CAIC desensitization only involve reduced activity and not their membrane elimination. Desensitization is important to control synaptic transmission and the development of the nervous system. In this review we discuss results related to its production, modulation and some aspects associated to models that consider it. Finally, an approach combining molecular biology and electrophysiology techniques to understand desensitization and its importance in biological systems is presented.

  11. Inflammation decreases the level of alpha7 nicotinic acetylcholine receptors in the brain mitochondria and makes them more susceptible to apoptosis induction.

    PubMed

    Lykhmus, Olena; Gergalova, Galyna; Zouridakis, Marios; Tzartos, Socrates; Komisarenko, Sergiy; Skok, Maryna

    2015-11-01

    α7 nicotinic acetylcholine receptors (α7 nAChRs) are involved in regulating inflammatory reactions, as well as the cell viability. They are expressed in both the plasma membrane and mitochondria of eukaryotic cells. Previously we found that neuroinflammation resulted in the decrease of α7 nAChR density in the brain of mice and was accompanied by accumulation of amyloid-beta (Aβ) peptides and memory impairment. In the present paper, it is shown that inflammation induced by either regular bacterial lipopolysaccharide (LPS) injections or immunizations with α7 nAChR extracellular domain (1-208) affected also the brain cell mitochondria. Using various modifications of sandwich ELISA, we observed the decrease of α7 nAChRs and accumulation of Aβ(1-40) and Aβ(1-42) in mitochondria of immunized or LPS-treated mice compared to control ones. Mitochondria of treated mice responded with cytochrome c release to lower Ca(2+) concentrations than mitochondria of control mice and were less sensitive to its attenuation with α7 nAChR agonist PNU282987. It is concluded that inflammation decreases α7 nAChR expression in both mitochondria and cell plasma membrane and makes mitochondria more susceptible to apoptosis induction.

  12. Modulation of nicotinic acetylcholine receptors by strychnine

    PubMed Central

    García-Colunga, Jesús; Miledi, Ricardo

    1999-01-01

    Strychnine, a potent and selective antagonist at glycine receptors, was found to inhibit muscle (α1β1γδ, α1β1γ, and α1β1δ) and neuronal (α2β2 and α2β4) nicotinic acetylcholine receptors (AcChoRs) expressed in Xenopus oocytes. Strychnine alone (up to 500 μM) did not elicit membrane currents in oocytes expressing AcChoRs, but, when applied before, concomitantly, or during superfusion of acetylcholine (AcCho), it rapidly and reversibly inhibited the current elicited by AcCho (AcCho-current). Although in the three cases the AcCho-current was reduced to the same level, its recovery was slower when the oocytes were preincubated with strychnine. The amount of AcCho-current inhibition depended on the receptor subtype, and the order of blocking potency by strychnine was α1β1γδ > α2β4 > α2β2. With the three forms of drug application, the Hill coefficient was close to one, suggesting a single site for the receptor interaction with strychnine, and this interaction appears to be noncompetitive. The inhibitory effects on muscle AcChoRs were voltage-independent, and the apparent dissociation constant for AcCho was not appreciably changed by strychnine. In contrast, the inhibitory effects on neuronal AcChoRs were voltage-dependent, with an electrical distance of ≈0.35. We conclude that strychnine regulates reversibly and noncompetitively the embryonic type of muscle AcChoR and some forms of neuronal AcChoRs. In the former case, strychnine presumably inhibits allosterically the receptor by binding at an external domain whereas, in the latter case, it blocks the open receptor-channel complex. PMID:10097172

  13. The conformation of acetylcholine at its target site in the membrane-embedded nicotinic acetylcholine receptor

    PubMed Central

    Williamson, P. T. F.; Verhoeven, A.; Miller, K. W.; Meier, B. H.; Watts, A.

    2007-01-01

    The conformation of the neurotransmitter acetylcholine bound to the fully functional nicotinic acetylcholine receptor embedded in its native membrane environment has been characterized by using frequency-selective recoupling solid-state NMR. Six dipolar couplings among five resolved 13C-labeled atoms of acetylcholine were measured. Bound acetylcholine adopts a bent conformation characterized with a quaternary ammonium-to-carbonyl distance of 5.1 Å. In this conformation, and with its orientation constrained to that previously determined by us, the acetylcholine could be docked satisfactorily in the agonist pocket of the agonist-bound, but not the agonist-free, crystal structure of a soluble acetylcholine-binding protein from Lymnaea stagnali. The quaternary ammonium group of the acetylcholine was determined to be within 3.9 Å of five aromatic residues and its acetyl group close to residues C187/188 of the principle and residue L112 of the complementary subunit. The observed >CO chemical shift is consistent with H bonding to the nicotinic acetylcholine receptor residues γY116 and δT119 that are homologous to L112 in the soluble acetylcholine-binding protein. PMID:17989232

  14. The noncompetitive blocker ( sup 3 H)chlorpromazine labels three amino acids of the acetylcholine receptor gamma subunit: Implications for the alpha-helical organization of regions MII and for the structure of the ion channel

    SciTech Connect

    Revah, F.; Galzi, J.L.; Giraudat, J.; Haumont, P.Y.; Lederer, F.; Changeux, J.P. )

    1990-06-01

    Labeling studies of Torpedo marmorata nicotinic acetylcholine receptor with the noncompetitive channel blocker ({sup 3}H)chlorpromazine have led to the initial identification of amino acids plausibly participating to the walls of the ion channel on the alpha, beta, and delta subunits. We report here results obtained with the gamma subunit, which bring additional information on the structure of the channel. After photolabeling of the membrane-bound receptor under equilibrium conditions in the presence of agonist and with or without phencyclidine (a specific ligand for the high-affinity site for noncompetitive blockers), the purified labeled gamma subunit was digested with trypsin, and the resulting fragments were fractionated by HPLC. Sequence analysis of peptide mixtures containing various amounts of highly hydrophobic fragments showed that three amino acids are labeled by ({sup 3}H)chlorpromazine in a phencyclidine-sensitive manner: Thr-253, Ser-257, and Leu-260. These residues all belong to the hydrophobic and putative transmembrane region MII of the gamma subunit. Their distribution along the sequence is consistent with an alpha-helical organization of this segment. The ({sup 3}H)chlorpromazine-labeled amino acids are conserved at homologous positions in the known sequences of other ligand-gated ion channels and may, thus, play a critical role in ion-transport mechanisms.

  15. Mutational analysis of muscle nicotinic acetylcholine receptor subunit assembly

    PubMed Central

    1990-01-01

    The structural elements required for normal maturation and assembly of the nicotinic acetylcholine receptor alpha subunit were investigated by expression of mutated subunits in transfected fibroblasts. Normally, the wild-type alpha subunit acquires high affinity alpha bungarotoxin binding in a time-dependent manner; however, mutation of the 128 and/or 142 cysteines to either serine or alanine, as well as deletion of the entire 14 amino acids in this region abolished all detectable high affinity binding. Nonglycosylated subunits that had a serine to glycine mutation in the consensus sequence also did not efficiently attain high affinity binding to toxin. In contrast, mutation of the proline at position 136 to glycine or alanine, or a double mutation of the cysteines at position 192 and 193 to serines had no effect on the acquisition of high affinity toxin binding. These data suggest that a disulfide bridge between cysteines 128 and 142 and oligosaccharide addition at asparagine 141 are required for the normal maturation of alpha subunit as assayed by high affinity toxin binding. The unassembled wild-type alpha subunit expressed in fibroblasts is normally degraded with a t1/2 of 2 h; upon assembly with the delta subunit, the degradation rate slows significantly (t1/2 greater than 13 h). All mutated alpha subunits retained the capacity to assemble with a delta subunit coexpressed in fibroblasts; however, mutated alpha subunits that were not glycosylated or did not acquire high affinity toxin binding were rapidly degraded (t1/2 = 20 min to 2 h) regardless of whether or not they assembled with the delta subunit. Assembly and rapid degradation of nonglycosylated acetylcholine receptor (AChR) subunits and subunit complexes were also observed in tunicamycin- treated BC3H-1 cells, a mouse musclelike cell line that normally expresses functional AChR. Hence, rapid degradation may be one form of regulation assuring that only correctly processed and assembled subunits

  16. Modulation of the anti-acetylcholine receptor response and experimental autoimmune myasthenia gravis by recombinant fragments of the acetylcholine receptor.

    PubMed

    Barchan, D; Asher, O; Tzartos, S J; Fuchs, S; Souroujon, M C

    1998-02-01

    Myasthenia gravis (MG) is a neuromuscular disorder of man caused by a humoral response to the acetylcholine receptor (AChR). Most of the antibodies in MG and in experimental autoimmune myasthenia gravis (EAMG) are directed to the extracellular portion of the AChR alpha subunit, and within it, primarily to the main immunogenic region (MIR). We have cloned and expressed recombinant fragments, corresponding to the entire extracellular domain of the AChR alpha subunit (H alpha1-210), and to portions of it that encompass either the MIR (H alpha1-121) or the ligand binding site of AChR (H alpha122-210), and studied their ability to interfere with the immunopathological anti-AChR response in vitro and in vivo. All fragments were expressed as fusion proteins with glutathione S-transferase. Fragments H alpha1-121 and H alpha1-210 protected AChR in TE671 cells against accelerated degradation induced by the anti-MIR monoclonal antibody (mAb)198 in a dose-dependent manner. Moreover, these fragments had a similar effect on the antigenic modulation of AChR by other anti-MIR mAb and by polyclonal rat anti-AChR antibodies. Fragments H alpha1-121 and H alpha1-210 were also able to modulate in vivo muscle AChR loss and development of clinical symptoms of EAMG, passively transferred to rats by mAb 198. Fragment H alpha122-210 did not have such a protective activity. Our results suggest that the appropriate recombinant fragments of the human AChR may be employed in the future for antigen-specific therapy of myasthenia.

  17. Impulsive behavior and nicotinic acetylcholine receptors.

    PubMed

    Ohmura, Yu; Tsutsui-Kimura, Iku; Yoshioka, Mitsuhiro

    2012-01-01

    Higher impulsivity is thought to be a risk factor for drug addiction, criminal involvement, and suicide. Excessive levels of impulsivity are often observed in several psychiatric disorders including attention-deficit/hyperactivity disorder and schizophrenia. Previous studies have demonstrated that nicotinic acetylcholine receptors (nAChRs) are involved in impulsive behavior. Here, we introduce recent advances in this field and describe the role of the following nAChR-related brain mechanisms in modulating impulsive behavior: dopamine release in the ventral striatum; α4β2 nAChRs in the infralimbic cortex, which is a ventral part of the medial prefrontal cortex (mPFC); and dopamine release in the mPFC. We also suggest several potential therapeutic drugs to address these mechanisms in impulsivity-related disorders and explore future directions to further elucidate the roles of central nAChRs in impulsive behavior.

  18. Conotoxins Targeting Nicotinic Acetylcholine Receptors: An Overview

    PubMed Central

    Lebbe, Eline K. M.; Peigneur, Steve; Wijesekara, Isuru; Tytgat, Jan

    2014-01-01

    Marine snails of the genus Conus are a large family of predatory gastropods with an unparalleled molecular diversity of pharmacologically active compounds in their venom. Cone snail venom comprises of a rich and diverse cocktail of peptide toxins which act on a wide variety of ion channels such as voltage-gated sodium- (NaV), potassium- (KV), and calcium- (CaV) channels as well as nicotinic acetylcholine receptors (nAChRs) which are classified as ligand-gated ion channels. The mode of action of several conotoxins has been the subject of investigation, while for many others this remains unknown. This review aims to give an overview of the knowledge we have today on the molecular pharmacology of conotoxins specifically interacting with nAChRs along with the structure–function relationship data. PMID:24857959

  19. Suitability of Nicotinic Acetylcholine Receptor α7 and Muscarinic Acetylcholine Receptor 3 Antibodies for Immune Detection

    PubMed Central

    Rommel, Frank R.; Raghavan, Badrinarayanan; Paddenberg, Renate; Kummer, Wolfgang; Tumala, Susanne; Lochnit, Günter; Gieler, Uwe

    2015-01-01

    Recent evidence reveals a crucial role for acetylcholine and its receptors in the regulation of inflammation, particularly of nicotinic acetylcholine receptor α7 (Chrna7) and muscarinic acetylcholine receptor 3 (Chrm3). Immunohistochemistry is a key tool for their cellular localization in functional tissues. We evaluated nine different commercially available antibodies on back skin tissue from wild-type (Wt) and gene-deficient (KO) mice. In the immunohistochemical analysis, we focused on key AChR-ligand sensitive skin cells (mast cells, nerve fibers and keratinocytes). All five antibodies tested for Chrm3 and the first three Chrna7 antibodies stained positive in both Wt and respective KO skin. With the 4th antibody (ab23832) nerve fibers were unlabeled in the KO mice. By western blot analysis, this antibody detected bands in both Wt and Chrna7 KO skin and brain. qRT-PCR revealed mRNA amplification with a primer set for the undeleted region in both Wt and KO mice, but none with a primer set for the deleted region in KO mice. By 2D electrophoresis, we found β-actin and β-enolase cross reactivity, which was confirmed by double immunolabeling. In view of the present results, the tested antibodies are not suitable for immunolocalization in skin and suggest thorough control of antibody specificity is required if histomorphometry is intended. PMID:25673288

  20. Alpha2-adrenoceptor-independent inhibition of acetylcholine receptor channel and sodium channel by dexmedetomidine in rat superior cervical ganglion neurons.

    PubMed

    Yang, L; Tang, J; Dong, J; Zheng, J

    2015-03-19

    Both central and peripheral sympathetic nervous systems contribute to the cardiovascular effects of dexmedetomidine (DMED), a highly selective and widely used a2-adrenoceptor agonist for sedation, analgesia, and stress management. The central sympatholytic effects are augmented by peripheral inhibition of sympathetic ganglion transmission. The mechanism is not clear. In this research, using conventional patch-clamp recordings we investigated the direct effects of DMED on sodium (Na(+)) channel currents (INa) and nicotinic acetylcholine (ACh) receptor (nAChRs) channel currents (IACh) in rat superior cervical ganglion (SCG) neurons to explore the possible mechanisms of sympathetic ganglion transmission inhibition by DMED. DMED voltage-dependently suppressed INa with half maximal inhibitory concentration (IC50) values of 67.2±9.6μM and 26.1±5.3μM at holding potentials of -80mV and -60mV, respectively. The inhibition of Na(+) channels by DMED was also frequency dependent. 100μM DMED shifted the Na(+) channel inactivation curves to the hyperpolarizing direction by 9.8mV (P<0.01) and slowed the recovery from inactivation by 8.9ms (P<0.01), but no effects were seen on the shape of the current-voltage relationship or Na(+) channels activation curves. DMED dose-dependently inhibited IACh with an IC50 value of 5.5±2.4μM in SCG neurons, and this inhibition was voltage-independent. DMED pretreatment followed by fast co-application of DMED and ACh produced a significantly larger IACh inhibition than without DMED pretreatment. Yohimbine, phentolamine, and atropine pretreatment did not alter the inhibitory effects of DMED on INa and IACh. In conclusion, DMED dose-dependently inhibits INa and IACh in rat SCG neurons by preferential binding to the inactivated state of the Na(+) channels and the closed state (resting) of nAChR channels respectively. Both inhibitions are a2-adrenoceptor independent. Furthermore, the nAChR channels in rat SCG neurons are much more sensitive to

  1. Computer modeling of the neurotoxin binding site of acetylcholine receptor spanning residues 185 through 196

    NASA Technical Reports Server (NTRS)

    Garduno-Juarez, R.; Shibata, M.; Zielinski, T. J.; Rein, R.

    1987-01-01

    A model of the complex between the acetylcholine receptor and the snake neurotoxin, cobratoxin, was built by molecular model building and energy optimization techniques. The experimentally identified functionally important residues of cobratoxin and the dodecapeptide corresponding to the residues 185-196 of acetylcholine receptor alpha subunit were used to build the model. Both cis and trans conformers of cyclic L-cystine portion of the dodecapeptide were examined. Binding residues independently identified on cobratoxin are shown to interact with the dodecapeptide AChR model.

  2. Genes expressed in the brain define three distinct neuronal nicotinic acetylcholine receptors.

    PubMed Central

    Nef, P; Oneyser, C; Alliod, C; Couturier, S; Ballivet, M

    1988-01-01

    Four genes encode the related protein subunits that assemble to form the nicotinic acetylcholine receptor (nAChR) at the motor endplate of vertebrates. We have isolated from the chicken genome four additional members of the same gene family whose protein products, termed alpha 2, alpha 3, alpha 4 and n alpha (non-alpha) probably define three distinct neuronal nAChR subtypes. The neuronal nAChR genes have identical structures consisting of six protein-coding exons and specify proteins that are best aligned with the chicken endplate alpha subunit, whose gene we have also characterized. mRNA transcripts encoding alpha 4 and n alpha are abundant in embryonic and in adult avian brain, whereas alpha 2 and alpha 3 transcripts are much scarcer. The same set of neuronal genes probably exists in all vertebrates since their counterparts have also been identified in the rat genome. Images PMID:3267226

  3. Allosteric binding sites on muscarinic acetylcholine receptors.

    PubMed

    Wess, Jürgen

    2005-12-01

    In this issue of Molecular Pharmacology, Tränkle et al. (p. 1597) present new findings regarding the existence of a second allosteric site on the M2 muscarinic acetylcholine receptor (M2 mAChR). The M2 mAChR is a prototypic class A G protein-coupled receptor (GPCR) that has proven to be a very useful model system to study the molecular mechanisms involved in the binding of allosteric GPCR ligands. Previous studies have identified several allosteric muscarinic ligands, including the acetylcholinesterase inhibitor tacrine and the bis-pyridinium derivative 4,4'-bis-[(2,6-dichloro-benzyloxy-imino)-methyl]-1,1'-propane-1,3-diyl-bis-pyridinium dibromide (Duo3), which, in contrast to conventional allosteric muscarinic ligands, display concentration-effect curves with slope factors >1. By analyzing the interactions of tacrine and Duo3 with other allosteric muscarinic agents predicted to bind to the previously identified ;common' allosteric binding site, Tränkle et al. provide evidence suggesting that two allosteric agents and one orthosteric ligand may be able to bind to the M2 mAChR simultaneously. Moreover, studies with mutant mAChRs indicated that the M2 receptor epitopes involved in the binding of tacrine and Duo3 may not be identical. Molecular modeling and ligand docking studies suggested that the additional allosteric site probably represents a subdomain of the receptor's allosteric binding cleft. Because allosteric binding sites have been found on many other GPCRs and drugs interacting with these sites are thought to have great therapeutic potential, the study by Tränkle et al. should be of considerable general interest.

  4. Alpha5 nicotinic acetylcholine receptor mediates nicotine-induced HIF-1α and VEGF expression in non-small cell lung cancer

    SciTech Connect

    Ma, Xiaoli; Jia, Yanfei; Zu, Shanshan; Li, Ruisheng; Jia, Ying; Zhao, Yun; Xiao, Dongjie; Dang, Ningning; Wang, Yunshan

    2014-07-15

    By binding to nicotinic acetylcholine receptors (nAChRs), nicotine induces the proliferation and apoptosis of non-small cell lung cancer (NSCLC). Previous studies have indicated that α5-nAChR is highly associated with lung cancer risk and nicotine dependence. However, the mechanisms through which α5-nAChRs may influence lung carcinogenesis are far from clear. In the present study, we investigated the roles of α5-nAChR in the nicotine-induced expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF). Immunohistochemistry was used to detect the expression of α5-nAChR and HIF-1α in 60 specimens of lung cancer and para-carcinoma tissue. The correlations between the expression levels of α5-nAChR and HIF-1α and other clinicopathological data were analyzed. In a cell line that highly expressed α5-nAChR, the loss of α5-nAChR function by siRNA was used to study whether α5-nAChR is involved in the nicotine-induced expression of HIF-1α and VEGF through the activation of the ERK1/2 and PI3K/Akt signaling pathways. Cell growth was detected using the cell counting kit-8 (CCK-8). α5-nAChR (78.3%) and HIF-1α (88.3%) were both overexpressed in NSCLC, and their expression levels were found to be correlated with each other (P < 0.05). In the A549 cell line, α5-nAChR and HIF-1α were found to be expressed under normal conditions, and their expression levels were significantly increased in response to nicotine treatment. The silencing of α5-nAChR significantly inhibited the nicotine-induced cell proliferation compared with the control group and attenuated the nicotine-induced upregulation of HIF-1α and VEGF, and these effects required the cooperation of the ERK1/2 and PI3K/Akt signaling pathways. These results show that the α5-nAChR/HIF-1α/VEGF axis is involved in nicotine-induced tumor cell proliferation, which suggests that α5-nAChR may serve as a potential anticancer target in nicotine-associated lung cancer. - Highlights

  5. The acetylcholine receptor as a cellular receptor for rabies virus.

    PubMed

    Lentz, T L; Burrage, T G; Smith, A L; Tignor, G H

    1983-01-01

    Characterization of specific host cell receptors for enveloped viruses is a difficult problem because many enveloped viruses bind to a variety of substrates which are not obviously related to tissue tropisms in the intact host. Viruses with a limited cellular tropism in infected animals present useful models for studying the mechanisms by which virus attachment regulates the disease process. Rabies virus is a rhabdovirus which exhibits a marked neuronotropism in infected animals. Limited data suggest that spread occurs by transsynaptic transfer of virus. The results of recent experiments at Yale suggest that viral antigen is localized very soon after injection at neuromuscular junctions, the motor nerve endings on muscle tissue. On cultured muscle cells, similar co-localization with the acetylcholine receptor is seen both before and after virus multiplication. Pretreatment of these cells with some ligands of the acetylcholine receptor results in reduced viral infection. These findings suggest that a neurotransmitter receptor or a closely associated molecule may serve as a specific host cell receptor for rabies virus and thus may be responsible for the tissue tropism exhibited by this virus. In addition to clarifying aspects of rabies virus pathogenesis, these studies have broad implications regarding the mechanism by which other viruses or viral immunizations might mediate autoimmune diseases such as myasthenia gravis.

  6. Diversity of insect nicotinic acetylcholine receptor subunits.

    PubMed

    Jones, Andrew K; Sattelle, David B

    2010-01-01

    Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that mediate fast synaptic transmission in the insect nervous system and are targets of a major group of insecticides, the neonicotinoids. They consist of five subunits arranged around a central ion channeL Since the subunit composition determines the functional and pharmacological properties of the receptor the presence of nAChR families comprising several subunit-encodinggenes provides a molecular basis for broad functional diversity. Analyses of genome sequences have shown that nAChR gene families remain compact in diverse insect species, when compared to their nematode andvertebrate counterparts. Thus, the fruit fly (Drosophila melanogaster), malaria mosquito (Anopheles gambiae), honey bee (Apis mellifera), silk worm (Bombyx mon) and the red flour beetle (Tribolium castaneum) possess 10-12 nAChR genes while human and the nematode Caenorhabditis elegans have 16 and 29 respectively. Although insect nAChRgene families are amongst the smallest known, receptor diversity can be considerably increased by the posttranscriptional processes alternative splicing and mRNA A-to-I editingwhich can potentially generate protein products which far outnumber the nAChR genes. These two processes can also generate species-specific subunit isoforms. In addition, each insect possesses at least one highly divergent nAChR subunit which may perform species-specific functions. Species-specific subunit diversification may offer promising targets for future rational design of insecticides that target specific pest insects while sparing beneficial species.

  7. Molecular forms and subunit structure of the acetylcholine receptor in the central nervous system of insects.

    PubMed

    Breer, H; Kleene, R; Hinz, G

    1985-12-01

    The nicotinic acetylcholine receptor as probed by alpha-bungarotoxin binding has been isolated from detergent-solubilized ganglionic membrane preparations from the insect, Locusta migratoria. The isolation and characterization of the receptor protein was achieved by preparation of membrane fragments, extraction by sodium deoxycholate, centrifugation on sucrose density gradient, affinity chromatography, gel electrophoresis, and immunoblotting. The purified receptor protein migrated as a single band on polyacrylamide when native (Mr = 250,000 to 300,000) but also under denaturing conditions (Mr = 65,000) and cross-reacted with some monoclonal antibodies against the Torpedo receptor. In immunohistochemical approaches using polyclonal antibodies the acetylcholine receptor antigenic sites could topochemically be identified at very distinct zones in the neuropil of locust ganglia. The results suggest that the acetylcholine receptor in the central nervous system of insects represents an oligomeric complex composed of four identical or very similar subunits and thus may represent a prototype of the recently proposed homo-oligomeric ancestral acetylcholine receptor.

  8. Synthetic peptides in the study of the interaction of rabies virus and the acetylcholine receptor.

    PubMed

    Lentz, T L; Hawrot, E; Donnelly-Roberts, D; Wilson, P T

    1988-01-01

    The neurotropism of some viruses may be explained in part by the attachment of these viruses to host cell receptors that are present on or even largely restricted to neurons. Rabies virus is an RNA virus that, after a period of replication in muscle, gains access to the central nervous system, where it selectively infects certain neuronal populations. The nicotinic acetylcholine receptor occurs in high density at the neuromuscular junction and is present in the central nervous system. Although several different cell surface constituents may act as attachment determinants for rabies, direct binding of radioactively labeled virus to affinity-purified acetylcholine receptor has been demonstrated. Binding of virus to the receptor was saturable and inhibited by up to 50% by alpha-bungarotoxin, a snake venom neurotoxin that binds at or near the acetylcholine binding site on the receptor. The molecular basis for the virus-receptor interaction may lie in an amino acid sequence similarity between the snake venom neurotoxins and a segment of the rabies virus glycoprotein. Two peptides (10 and 13 residues) of the rabies virus glycoprotein and homologous bungarotoxin peptides were synthesized and tested for ability to compete with labeled alpha-bungarotoxin for binding to the acetylcholine receptor. The peptides were found to compete with toxin binding with affinities comparable to those of the cholinergic ligands d-tubocurarine and nicotine. These findings indicate that a segment of the rabies virus glycoprotein interacts with the acetylcholine receptor at or near the acetylcholine binding site of the receptor. The similarity between the virus glycoprotein and the neurotoxin was further evidenced by the cross reaction of antibody raised against the virus 10-mer with the bungarotoxin 10-mer. Binding of rabies virus to the acetylcholine receptor or to other neuronal bungarotoxin-binding proteins may be related to the neurotropism of this virus. In addition, knowledge of both

  9. Chronic nicotine blunts hypoxic sensitivity in perinatal rat adrenal chromaffin cells via upregulation of KATP channels: role of alpha7 nicotinic acetylcholine receptor and hypoxia-inducible factor-2alpha.

    PubMed

    Buttigieg, Josef; Brown, Stephen; Holloway, Alison C; Nurse, Colin A

    2009-06-03

    Fetal nicotine exposure blunts hypoxia-induced catecholamine secretion from neonatal adrenomedullary chromaffin cells (AMCs), providing a link between maternal smoking, abnormal arousal responses, and risk of sudden infant death syndrome. Here, we show that the mechanism is attributable to upregulation of K(ATP) channels via stimulation of alpha7 nicotinic ACh receptors (AChRs). These K(ATP) channels open during hypoxia, thereby suppressing membrane excitability. After in utero exposure to chronic nicotine, neonatal AMCs show a blunted hypoxic sensitivity as determined by inhibition of outward K(+) current, membrane depolarization, rise in cytosolic Ca(2+), and catecholamine secretion. However, hypoxic sensitivity could be unmasked in nicotine-exposed AMCs when glibenclamide, a blocker of K(ATP) channels, was present. Both K(ATP) current density and K(ATP) channel subunit (Kir 6.2) expression were significantly enhanced in nicotine-exposed cells relative to controls. The entire sequence could be reproduced in culture by exposing neonatal rat AMCs or immortalized fetal chromaffin (MAH) cells to nicotine for approximately 1 week, and was prevented by coincubation with selective blockers of alpha7 nicotinic AChRs. Additionally, coincubation with inhibitors of protein kinase C and CaM kinase, but not protein kinase A, prevented the effects of chronic nicotine in vitro. Interestingly, chronic nicotine failed to blunt hypoxia-evoked responses in MAH cells bearing short hairpin knockdown (>90%) of the transcription factor, hypoxia-inducible factor-2alpha (HIF-2alpha), suggesting involvement of the HIF pathway. The therapeutic potential of K(ATP) channel blockers was validated in experiments in which hypoxia-induced neonatal mortality in nicotine-exposed pups was significantly reduced after pretreatment with glibenclamide.

  10. Identification of a family of muscarinic acetylcholine receptor genes

    SciTech Connect

    Bonner, T.I.; Buckley, N.J.; Young, A.C.; Brann, M.R.

    1987-07-31

    Complementary DNAs for three different muscarinic acetylcholine receptors were isolated from a rat cerebral cortex library, and the cloned receptors were expressed in mammalian cells. Analysis of human and rat genomic clones indicates that there are at least four functional muscarinic receptor genes and that these genes lack introns in the coding sequence. This gene family provides a new basis for evaluating the diversity of muscarinic mechanisms in the nervous system.

  11. The ligand binding domain of the nicotinic acetylcholine receptor. Immunological analysis.

    PubMed

    Kachalsky, S G; Aladjem, M; Barchan, D; Fuchs, S

    1993-03-08

    The interaction of the acetylcholine receptor (AChR) binding site domain with specific antibodies and with alpha-bungarotoxin (alpha-BTX) has been compared. The cloned and expressed ligand binding domain of the mouse AChR alpha-subunit binds alpha-BTX, whereas the mongoose-expressed domain is not recognized by alpha-BTX. On the other hand, both the mouse and mongoose domains bind to the site-specific monoclonal antibody 5.5. These results demonstrate that the structural requirements for binding of alpha-BTX and mcAb 5.5, both of which interact with the AChR binding site, are distinct from each other.

  12. Endoplasmic reticulum stress contributes to acetylcholine receptor degradation by promoting endocytosis in skeletal muscle cells.

    PubMed

    Du, Ailian; Huang, Shiqian; Zhao, Xiaonan; Zhang, Yun; Zhu, Lixun; Ding, Ji; Xu, Congfeng

    2016-01-15

    After binding by acetylcholine released from a motor neuron, a nicotinic acetylcholine receptor at the neuromuscular junction produces a localized end-plate potential, which leads to muscle contraction. Improper turnover and renewal of acetylcholine receptors contributes to the pathogenesis of myasthenia gravis. In the present study, we demonstrate that endoplasmic reticulum (ER) stress contributes to acetylcholine receptor degradation in C2C12 myocytes. We further show that ER stress promotes acetylcholine receptor endocytosis and lysosomal degradation, which was dampened by blocking endocytosis or treating with lysosome inhibitor. Knockdown of ER stress proteins inhibited acetylcholine receptor endocytosis and degradation, while rescue assay restored its endocytosis and degradation, confirming the effects of ER stress on promoting endocytosis-mediated degradation of junction acetylcholine receptors. Thus, our studies identify ER stress as a factor promoting acetylcholine receptor degradation through accelerating endocytosis in muscle cells. Blocking ER stress and/or endocytosis might provide a novel therapeutic approach for myasthenia gravis.

  13. Influence of subunit composition on desensitization of neuronal acetylcholine receptors at low concentrations of nicotine.

    PubMed

    Fenster, C P; Rains, M F; Noerager, B; Quick, M W; Lester, R A

    1997-08-01

    The influence of alpha and beta subunits on the properties of nicotine-induced activation and desensitization of neuronal nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes was examined. Receptors containing alpha4 subunits were more sensitive to activation by nicotine than alpha3-containing receptors. At low concentrations of nicotine, nAChRs containing beta2 subunits reached near-maximal desensitization more rapidly than beta4-containing receptors. The concentration of nicotine producing half-maximal desensitization was influenced by the particular alpha subunit expressed; similar to results for activation, alpha4-containing receptors were more sensitive to desensitizing levels of nicotine than alpha3-containing receptors. The alpha subunit also influenced the rate of recovery from desensitization; this rate was approximately inversely proportional to the apparent nicotine affinity for the desensitized state. The homomeric alpha7 receptor showed the lowest sensitivity to nicotine for both activation and desensitization; alpha7 nAChRs also demonstrated the fastest desensitization kinetics. These subunit-dependent properties remained in the presence of external calcium, although subtle, receptor subtype-specific effects on both the apparent affinities for activation and desensitization and the desensitization kinetics were noted. These data imply that the subunit composition of various nAChRs determines the degree to which receptors are desensitized and/or activated by tobacco-related levels of nicotine. The subtype-specific balance between receptor activation and desensitization should be considered important when the cellular and behavioral actions of nicotine are interpreted.

  14. Acetylcholine receptors in the retinas of the α7 nicotinic acetylcholine receptor knockout mouse

    PubMed Central

    Souza, Fred G. Oliveira; Bruce, Kady S.; Strang, Christianne E.; Morley, Barbara J.; Keyser, Kent T.

    2014-01-01

    Purpose The α7 nicotinic acetylcholine receptor (nAChR) is widely expressed in the nervous system, including in the inner retinal neurons in all species studied to date. Although reductions in the expression of α7 nAChRs are thought to contribute to the memory and visual deficits reported in Alzheimer’s disease (AD) and schizophrenia , the α7 nAChR knockout (KO) mouse is viable and has only slight visual dysfunction. The absence of a major phenotypic abnormality may be attributable to developmental mechanisms that serve to compensate for α7 nAChR loss. We hypothesized that the upregulation of genes encoding other nAChR subunits or muscarinic acetylcholine receptor (mAChR) subtypes during development partially accounts for the absence of major deficiencies in the α7 nAChR KO mouse. The purpose of this study was to determine whether the deletion of the α7 nAChR subunit in a mouse model resulted in changes in the regulation of other cholinergic receptors or other ion channels in an α7 nAChR KO mouse when compared to a wild-type (WT) mouse. Methods To examine gene expression changes, we employed a quantitative real-time polymerase chain reaction (qPCR) using whole retina RNA extracts as well as RNA extracted from selected regions of the retina. These extracts were collected using laser capture microdissection (LCM). The presence of acetylcholine receptor (AChR) subunit and subtype proteins was determined via western blotting. To determine any differences in the number and distribution of choline acetyltransferase (ChAT) amacrine cells, we employed wholemount and vertical immunohistochemistry (IHC) and cell counting. Additionally, in both WT and α7 nAChR KO mouse retinas, the distribution of the nAChR subunit and mAChR subtype proteins were determined via IHC for those KO mice that experienced mRNA changes. Results In the whole retina, there was a statistically significant upregulation of α2, α9, α10, β4, nAChR subunit, and m1 and m4 mAChR subtype

  15. Looking below the surface of nicotinic acetylcholine receptors

    PubMed Central

    Stokes, Clare; Treinin, Millet; Papke, Roger L.

    2015-01-01

    The amino acid sequences of nicotinic acetylcholine receptors (nAChRs) from diverse species can be compared across extracellular, transmembrane, and intracellular domains. The intracellular domains are most divergent among subtypes, yet relatively consistent among species. The diversity indicates that each nAChR subtype possesses a unique language for communication with its host cell. The conservation across species also suggests that the intracellular domains may play defining functional roles for each subtype. Secondary structure prediction indicates two relatively conserved alpha helices within the intracellular domains of all nAChRs. Among all subtypes, the intracellular domain of α7 nAChR is one of the most-well conserved, and α7 nAChRs have effects in non-neuronal cells independent of generating ion currents, making it likely that the α7 intracellular domain directly mediates signal transduction. There are potential phosphorylation and protein binding sites in the α7 intracellular domain, which are conserved and may be the basis for α7-mediated signal transduction. PMID:26067101

  16. Mechanisms of Action of Anticholinesterases and Oximes on Acetylcholine Receptors

    DTIC Science & Technology

    1988-07-23

    J.F. and D.B. Sanders. The management of patients with myasthenia gravis , in Myasthenia Gravis (E.X. Albuquerque and A.T. Eldefrawi, eds.), Chapman...Eldefrawi. Affinity of myasthenia drugs to acetylcholinesterase and acetylcholine receptor. Biochem. Med. 10:258-265 (1974). 9. Carpenter, D.O., L.A

  17. Action of nereistoxin on recombinant neuronal nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes.

    PubMed

    Raymond Delpech, Valérie; Ihara, Makoto; Coddou, Claudio; Matsuda, Kazuhiko; Sattelle, David B

    2003-11-01

    Nereistoxin (NTX), a natural neurotoxin from the salivary glands of the marine annelid worm Lumbriconereis heteropoda, is highly toxic to insects. Its synthetic analogue, Cartap, was the first commercial insecticide based on a natural product. We have used voltage-clamp electrophysiology to compare the actions of NTX on recombinant nicotinic acetylcholine receptors (nicotinic AChRs) expressed in Xenopus laevis oocytes following nuclear injection of cDNAs. The recombinant nicotinic AChRs investigated were chicken alpha7, chicken alpha4beta2 and the Drosophila melanogaster/chicken hybrid receptors SAD/beta2 and ALS/beta2. No agonist action of NTX (0.1-100 microM) was observed on chicken alpha7, chicken alpha4beta2 and the Drosophila/chicken hybrid nicotinic AChRs. Currents elicited by ACh were reduced in amplitude by NTX in a dose-dependent manner. The toxin was slightly more potent on recombinant Drosophila/vertebrate hybrid receptors than on vertebrate homomeric (alpha7) or heteromeric (alpha4beta2) nicotinic AChRs. Block by NTX of the chicken alpha7, chicken alpha4beta2 and the SAD/beta2 and ALS/beta2 Drosophila/chicken hybrid receptors is in all cases non-competitive. Thus, the site of action on nicotinic AChRs of NTX, to which the insecticide Cartap is metabolised in insects, differs from that of the major nicotinic AChR-active insecticide, imidacloprid.

  18. Neuronal nicotinic acetylcholine receptors are modulated by zinc.

    PubMed

    Vázquez-Gómez, Elizabeth; García-Colunga, Jesús

    2009-01-01

    It is known that zinc modulates nicotinic acetylcholine receptors (nAChRs). Here, we studied the effects of zinc on neuronal alpha4beta4 nAChRs, expressed in Xenopus oocytes and activated by nicotine. Membrane ion currents elicited by nicotine (10 nM to 100 microM) were enhanced by zinc (100 microM). Maximal zinc potentiation of the nicotine-activated current (2530%) occurred at 50 nM nicotine, and potentiation gradually decreased as the nicotine concentration increased. The EC(50) and IC(50) for the nicotine-activated current were 639 nM and 14.7 microM nicotine, respectively. Both parameters decreased in the presence of zinc to 160 nM and 4.6 microM, respectively, probably due to an increase of sensitivity of nAChRs for nicotine. We used different concentrations and durations of exposure to nicotine, due to desensitization of nAChRs directly depends on both these factors. With 500 nM nicotine and 20 min washing periods between nicotine applications, zinc potentiation remained constant, 901% for 2 min and 813% for 20 min of nicotine exposure. With continuous application of nicotine, zinc potentiation decreased as the time of nicotine exposure increased, 721% for 2 min and 254% for 48 min of nicotine exposure. Our results indicate that zinc-potentiating effects on alpha4beta4 nAChRs strongly depend on both concentration and time of exposure to nicotine, suggesting that zinc potentiation depends on the degree of desensitization.

  19. Characterization of a putative acetylcholine receptor in chick ciliary ganglion neurons

    SciTech Connect

    Stollberg, J.

    1985-01-01

    Monoclonal antibodies to the main immunogenic region on the alpha subunit of acetylcholine receptors in muscle and electric organ recognize membrane components in chick brain and ciliary ganglia that are candidates for the neuronal receptor. The component in chick brain has been purified by immunoaffinity chromatography. It specifically binds nicotine but not alpha-bungarotoxin, and can be affinity labeled with (/sup 3/H)bromoacetylcholine. The cross-reacting component in ciliary ganglion neurons is concentrated in synaptic membrane, and can be modulated by exposure of the cells to cholinergic ligands in culture. The cross-reacting component in ciliary ganglion neurons is an integral membrane component that binds concanavalin A, and it is distinct from the alpha-bungarotoxin binding component. The acetylcholine receptor function in these neurons can be locked by affinity alkylation with bromoacetylcholine, indicating similarity in this respect to receptors from muscle and electric organ. Antisera raised against the partially purified component from chick brain also block receptor function on ciliary ganglion neurons. The subcellular distribution of the ganglion component in culture is assessed, and it is shown that approximately 2/3 of the cross-reacting components are intracellular; the majority of these seem not to be destined for insertion into the plasma membrane.

  20. Pharmacological and biochemical characterization of the D-1 dopamine receptor mediating acetylcholine release in rabbit retina

    SciTech Connect

    Hensler, J.G.; Cotterell, D.J.; Dubocovich, M.L.

    1987-12-01

    Superfusion with dopamine (0.1 microM-10 mM) evokes calcium-dependent (/sup 3/H)acetylcholine release from rabbit retina labeled in vitro with (/sup 3/H)choline. This effect is antagonized by the D-1 dopamine receptor antagonist SCH 23390. Activation or blockade of D-2 dopamine, alpha-2 or beta receptors did not stimulate or attenuate the release of (/sup 3/H)acetylcholine from rabbit retina. Dopamine receptor agonists evoke the release of (/sup 3/H)acetylcholine with the following order of potency: apomorphine less than or equal to SKF(R)82526 < SKF 85174 < SKF(R)38393 less than or equal to pergolide less than or equal to dopamine (EC50 = 4.5 microM) < SKF(S)82526 less than or equal to SKF(S)38393. Dopamine receptor antagonists inhibited the dopamine-evoked release of (/sup 3/H)acetylcholine: SCH 23390 (IC50 = 1 nM) < (+)-butaclamol less than or equal to cis-flupenthixol < fluphenazine < perphenazine < trans-flupenthixol < R-sulpiride. The potencies of dopamine receptor agonists and antagonists at the dopamine receptor mediating (/sup 3/H)acetylcholine release is characteristic of the D-1 dopamine receptor. These potencies were correlated with the potencies of dopamine receptor agonists and antagonists at the D-1 dopamine receptor in rabbit retina as labeled by (/sup 3/H)SCH 23390, or as determined by adenylate cyclase activity. (/sup 3/H)SCH 23390 binding in rabbit retinal membranes was stable, saturable and reversible. Scatchard analysis of (/sup 3/H)SCH 23390 saturation data revealed a single high affinity binding site (Kd = 0.175 +/- 0.002 nM) with a maximum binding of 482 +/- 12 fmol/mg of protein. The potencies of dopamine receptor agonists to stimulate (/sup 3/H)acetylcholine release were correlated with their potencies to stimulate adenylate cyclase (r = 0.784, P less than .05, n = 7) and with their affinities at (/sup 3/H)SCH 23390 binding sites (r = 0.755, P < .05, n = 8).

  1. Optochemical control of genetically engineered neuronal nicotinic acetylcholine receptors

    NASA Astrophysics Data System (ADS)

    Tochitsky, Ivan; Banghart, Matthew R.; Mourot, Alexandre; Yao, Jennifer Z.; Gaub, Benjamin; Kramer, Richard H.; Trauner, Dirk

    2012-02-01

    Advances in synthetic chemistry, structural biology, molecular modelling and molecular cloning have enabled the systematic functional manipulation of transmembrane proteins. By combining genetically manipulated proteins with light-sensitive ligands, innately ‘blind’ neurobiological receptors can be converted into photoreceptors, which allows them to be photoregulated with high spatiotemporal precision. Here, we present the optochemical control of neuronal nicotinic acetylcholine receptors (nAChRs) with photoswitchable tethered agonists and antagonists. Using structure-based design, we produced heteromeric α3β4 and α4β2 nAChRs that can be activated or inhibited with deep-violet light, but respond normally to acetylcholine in the dark. The generation of these engineered receptors should facilitate investigation of the physiological and pathological functions of neuronal nAChRs and open a general pathway to photosensitizing pentameric ligand-gated ion channels.

  2. Spin labeled acetylcholine analogs: studies of cholinergic receptor.

    PubMed

    Rosen, G M; Abou-Donia, M B; Yeh, J Z; Menzel, D B

    1975-10-01

    Some spin-labeled acetylcholine analogs, in which the number of methylene groups between the quaternary nitrogen and the ether oxygen ranged between 1-5, were synthesized to study drug interacitons with acetylcholine receptors. None of the compounds tested, with the exception of the one that contained 2 methylene groups (SL-2) had any cholinergic activity. SL-2 was not capable of producing any nicotinic cholinomimetic activity. On the other hand it proved to have a very weak nicotinic cholinolytic activity on the receptors of the frog satorius muscle. This compound exhibited strong antagonism against muscarinic receptors of the isolated frog heart. The muscarinic cholinolytic action of the spin-label ACh analog is discussed in terms of the molecular perturbation theory of drug action.

  3. Synthesis, Trafficking, and Localization of Muscarinic Acetylcholine Receptors

    PubMed Central

    Nathanson, Neil M.

    2008-01-01

    Muscarinic acetylcholine receptors are members of the G-protein coupled receptor superfamily that are expressed in and regulate the function of neurons, cardiac and smooth muscle, glands, and many other cell types and tissues. The correct trafficking of membrane proteins to the cell surface and their subsequent localization at appropriate sites in polarized cells are required for normal cellular signaling and physiological responses. This review will summarize work on the synthesis and trafficking of muscarinic receptors to the plasma membrane and their localization at the cell surface. PMID:18558434

  4. Nicotinic acetylcholine receptors: from basic science to therapeutics.

    PubMed

    Hurst, Raymond; Rollema, Hans; Bertrand, Daniel

    2013-01-01

    Substantial progress in the identification of genes encoding for a large number of proteins responsible for various aspects of neurotransmitter release, postsynaptic detection and downstream signaling, has advanced our understanding of the mechanisms by which neurons communicate and interact. Nicotinic acetylcholine receptors represent a large and well-characterized family of ligand-gated ion channels that is expressed broadly throughout the central and peripheral nervous system, and in non-neuronal cells. With 16 mammalian genes identified that encode for nicotinic receptors and the ability of the subunits to form heteromeric or homomeric receptors, the repertoire of conceivable receptor subtype combinations is enormous and offers unique possibilities for the design and development of new therapeutics that target nicotinic acetylcholine receptors. The aim of this review is to provide the reader with recent insights in nicotinic acetylcholine receptors from genes, structure and function to diseases, and with the latest findings on the pharmacology of these receptors. Although so far only a few nicotinic drugs have been marketed or are in late stage development, much progress has been made in the design of novel chemical entities that are being explored for the treatment of various diseases, including addiction, depression, ADHD, cognitive deficits in schizophrenia and Alzheimer's disease, pain and inflammation. A pharmacological analysis of these compounds, including those that were discontinued, can improve our understanding of the pharmacodynamic and pharmacokinetic requirements for nicotinic 'drug-like' molecules and will reveal if hypotheses on therapies based on targeting specific nicotinic receptor subtypes have been adequately tested in the clinic.

  5. Binding of Alpha-Bungarotoxin to Single Identified Neurons of ’Aplysia’ which have Different Ionic Responses to Acetylcholine,

    DTIC Science & Technology

    1976-09-01

    Identifiable Aplysia neurons have one or more of three different ionic responses to acetylcholine, due to Na, Cl, and K conductance increases... Aplysia acetylcholine receptors. Thus the inhibition of the Na response by hexamethonium may be a result of the binding to a site which prevent the conductance change rather than preventing acetylcholine from binding to its receptor.

  6. Role of dopamine receptor and muscarinic acetylcholine receptor blockade in the antiapomorphine action of neuroleptics

    SciTech Connect

    Zharkovskii, A.M.; Langel, Yu.L.; Chereshka, K.S.; Zharkovskaya, T.A.

    1987-08-01

    The authors analyze the role of dopamine and muscarinic acetylcholine receptor blocking components in the antistereotypic action of neuroleptics with different chemical structure. To determine dopamine-blocking activity in vitro, binding of /sup 3/H-spiperone with membranes of the rat striatum was measured. To study the blocking action of the substances on muscarinic acetylcholine receptors, binding of /sup 3/H-quinuclidinyl benzylate with brain membranes was chosen.

  7. Nicotinic Acetylcholine Receptor Signaling in Tumor Growth and Metastasis

    PubMed Central

    Singh, Sandeep; Pillai, Smitha; Chellappan, Srikumar

    2011-01-01

    Cigarette smoking is highly correlated with the onset of a variety of human cancers, and continued smoking is known to abrogate the beneficial effects of cancer therapy. While tobacco smoke contains hundreds of molecules that are known carcinogens, nicotine, the main addictive component of tobacco smoke, is not carcinogenic. At the same time, nicotine has been shown to promote cell proliferation, angiogenesis, and epithelial-mesenchymal transition, leading to enhanced tumor growth and metastasis. These effects of nicotine are mediated through the nicotinic acetylcholine receptors that are expressed on a variety of neuronal and nonneuronal cells. Specific signal transduction cascades that emanate from different nAChR subunits or subunit combinations facilitate the proliferative and prosurvival functions of nicotine. Nicotinic acetylcholine receptors appear to stimulate many downstream signaling cascades induced by growth factors and mitogens. It has been suggested that antagonists of nAChR signaling might have antitumor effects and might open new avenues for combating tobacco-related cancer. This paper examines the historical data connecting nicotine tumor progression and the recent efforts to target the nicotinic acetylcholine receptors to combat cancer. PMID:21541211

  8. Expression of a Drosophila melanogaster acetylcholine receptor-related gene in the central nervous system

    SciTech Connect

    Wadsworth, S.C.; Rosenthal, L.S.; Kammermeyer, K.L.; Potter, M.B.; Nelson, D.J.

    1988-02-01

    The authors isolated Drosophila melanogaster genomic sequences with nucleotide and amino acid sequence homology to subunits of vertebrate acetylcholine receptor by hybridization with a Torpedo acetylcholine receptor subunit cDNA probe. Five introns are present in the portion of the Drosophila gene encoding the unprocessed protein and are positionally conserved relative to the human acetylcholine receptor alpha-subunit gene. The Drosophila genomic clone hybridized to salivary gland polytene chromosome 3L within region 64B and was termed AChR64B. A 3-kilobasae poly(A)-containing transcript complementary to the AChR64B clone was readily detectable by RNA blot hybridizations during midembryogenesis, during metamorphosis, and in newly enclosed adults. AChR64B transcripts were localized to the cellular regions of the central nervous system during embryonic, larval, pupal, and adult stages of development. During metamorphosis, a temporal relationship between the morphogenesis of the optic lobe and expression of AChR64B transcripts was observed.

  9. Role of acetylcholine receptors in proliferation and differentiation of P19 embryonal carcinoma cells

    SciTech Connect

    Resende, R.R.; Alves, A.S.; Britto, L.R.G; Ulrich, H.

    2008-04-15

    Coordinated proliferation and differentiation of progenitor cells is the base for production of appropriate numbers of neurons and glia during neuronal development in order to establish normal brain functions. We have used murine embryonal carcinoma P19 cells as an in vitro model for early differentiation to study participation of nicotinic (nAChR) and muscarinic acetylcholine (mAChR) receptors in the proliferation of neural progenitor cells and their differentiation to neurons. We have previously shown that functional nicotinic acetylcholine receptors (nAChRs) already expressed in embryonic cells mediate elevations in cytosolic free calcium concentration ([Ca{sup 2+}]{sub i}) via calcium influx through nAChR channels whereas intracellular stores contribute to nAChR- and mAChR-mediated calcium fluxes in differentiated cells [Resende et al., Cell Calcium 43 (2008) 107-121]. In the present study, we have demonstrated that nicotine provoked inhibition of proliferation in embryonic cells as determined by BrdU labeling. However, in neural progenitor cells nicotine stimulated proliferation which was reversed in the presence of inhibitors of calcium mobilization from intracellular stores, indicating that liberation of intracellular calcium contributed to this proliferation induction. Muscarine induced proliferation stimulation in progenitor cells by activation of G{alpha}{sub q/11}-coupled M{sub 1}, M{sub 3} and M{sub 5} receptors and intracellular calcium stores, whereas G{alpha}{sub i/o}-protein coupled M{sub 2} receptor activity mediated neuronal differentiation.

  10. Acetylcholine Promotes Binding of α-Conotoxin MII for α3β2 Nicotinic Acetylcholine Receptors

    PubMed Central

    Sambasivarao, Somisetti V.; Roberts, Jessica; Bharadwaj, Vivek S.; Slingsby, Jason G.; Rohleder, Conrad; Mallory, Chris; Groome, James R.

    2014-01-01

    α-Conotoxin MII (α-CTxMII) is a 16 amino acid peptide with the sequence GCCSNPVCHLEHSNLC containing disulfide bonds between Cys2-Cys8 and Cys3-Cys16. This peptide, isolated from the venom of the marine cone snail Conus magus, is a potent and selective antagonist of neuronal nicotinic acetylcholine receptors (nAChRs). To evaluate the impact of channel-ligand interactions on ligand binding affinity, homology models of the heteropentameric α3β2-nAChR were constructed. The models were created in MODELLER using crystal structures of the Torpedo marmorata-nAChR (Tm-nAChR, PDB ID: 2BG9) and the Aplysia californica-acetylcholine binding protein (Ac-AChBP, PDB ID: 2BR8) as templates for the α3 and β2 subunit isoforms derived from rat neuronal nAChR primary amino acid sequences. Molecular docking calculations were performed with AutoDock to evaluate interactions of the heteropentameric nAChR homology models with the ligands acetylcholine (ACh) and α-CTxMII. The nAChR homology models described here bind ACh with commensurate binding energies to previously reported systems, and identify critical interactions that facilitate both ACh and α-CTxMII ligand binding. The docking calculations revealed an increased binding affinity of the α3β2-nAChR for α-CTxMII with ACh bound to the receptor, which was confirmed through two-electrode voltage clamp experiments on oocytes from Xenopus laevis. These findings provide insights into the inhibition and mechanism of electrostatically driven antagonist properties of the α-CTxMIIs on nAChRs. PMID:24420650

  11. Immunological studies on the structure and function of the nicotinic acetylcholine receptor in mammalian muscle

    SciTech Connect

    Gu, Y.

    1989-01-01

    The specificity of the antibodies in the serum of a patient with myasthenia gravis for a the {alpha}-bungarotoxin binding sites of the acetylcholine receptor (AChR) was examined using AChRs in the C2 mouse muscle cell line as a model. The antibodies were shown to be specific for one of the two toxin-binding sites. The effect of the antibodies in this myasthenic serum on the functional response of the receptor to cholinergic agonists was also examined using carbamylcholine-induced {sup 22}Na uptake into C2 myotubes as a measured of the receptor function. Antibodies specific for the {gamma}, {delta}, and {epsilon} subunit, respectively, of mammalian muscle AChRs were developed using subunit-specific synthetic peptides as antigens. Using these antibodies and monoclonal antibodies for other subunits as probes, I have identified four ({alpha}, {beta}, {gamma}, and {delta}) subunits of mammalian muscle AChRs on immunoblots. When AChRs from embryonic, neonatal, normal and denervated adult muscles were compared on immunoblots, the {alpha}, {beta}, and {delta} subunits were identical in all four receptor preparations, with or without endoglycosidase digestion. The spatial and temporal distribution of the {gamma}- and {epsilon}- AChRs in developing and in denervated muscles corresponds to the distribution of AChRs with slow and fast channels, respectively, and that the development changes in the channel properties of the receptor arise from a change in the subunit composition of the receptor, in which the {gamma} is replaced by {epsilon}.

  12. A model of the human M2 muscarinic acetylcholine receptor

    NASA Astrophysics Data System (ADS)

    Jöhren, Kirstin; Höltje, Hans-Dieter

    2002-11-01

    The M2 muscarinic acetylcholine receptor belongs to the family of rhodopsin like G-Protein Coupled Receptors. This subtype of muscarinic receptors is of special interest because it bears, aside from an orthosteric binding site, also an allosteric binding site. Based on the X-ray structure of bovine rhodopsin a complete homology model of the human M2 receptor was developed. For the orthosteric binding site point mutations and binding studies with different agonists and antagonists are available. This knowledge was utilized for an initial verification of the M2 model. Allosteric modulation of activity is mediated by structurally different ligands such as gallamine, caracurine V salts or W84 (a hexamethonium-derivative). Caracurine V derivatives with different affinities to M2 were docked using GRID-fields. Subsequent molecular dynamics simulations yielded different binding energies based on diverse electrostatic and lipophilic interactions. The calculated affinities are in good agreement to experimentally determined affinities.

  13. Alternative splicing in nicotinic acetylcholine receptor subunits from Locusta migratoria and its influence on acetylcholine potencies.

    PubMed

    Zhang, Yixi; Liu, Yang; Bao, Haibo; Sun, Huahua; Liu, Zewen

    2017-01-18

    Due to the great abundance within insect central nervous system (CNS), nicotinic acetylcholine receptors (nAChRs) play key roles in insect CNS, which makes it to be the targets of several classes of insecticides, such as neonicotinoids. Insect nAChRs are pentameric complexes consisting of five subunits, and a dozen subunits in one insect species can theoretically comprise diverse nAChRs. The alternative splicing in insect nAChR subunits may increase the diversity of insect nAChRs. In the oriental migratory locust (Locusta migratoria manilensis Meyen), a model insect species with agricultural importance, the alternative splicing was found in six α subunits among nine α and two β subunits, such as missing conserved residues in Loop D from Locα1, Locα6 and Locα9, a 34-residue insertion in Locα8 cytoplasmic loop, and truncated transcripts for Locα4, Locα7 and Locα9. Hybrid nAChRs were successfully constructed in Xenopus oocytes through co-expression with rat β2 and one α subunit from L. migratoria, which included Locα1, Locα2, Locα3, Locα4, Locα5, Locα8 and Locα9. Influences of alternative splicing in Locα1, Locα8 and Locα9 on acetylcholine potency were tested on hybrid nAChRs. The alternative splicing in Locα1 and Locα9 could increase acetylcholine sensitivities on recombinant receptors, while the splicing in Locα8 showed significant influences on the current amplitudes of oocytes. The results revealed that the alternative splicing at or close to the ligand-binding sites, as well as at cytoplasmic regions away from the ligand-binding sites, in insect nAChR subunits would change the agonist potencies on the receptors, which consequently increased nAChR diversity in functional and pharmacological properties.

  14. Biochemical and immunological studies of the Muscarinic acetylcholine receptor

    SciTech Connect

    Gainer, M.W.

    1985-01-01

    Muscarinic acetylcholine receptors were solubilized from bovine brain membranes with 3(3-cholamidopropyl)dimethylammonio)propanesulfonate (CHAPS). A combination of 10 mM CHAPS and 1 M NaCl solubilized 15-40% of the specific receptor binding sites from these membranes. The solubilized receptors displayed high affinity binding of the muscarinic antagonist, (/sup 3/H)quinuclidinyl benzilate with a K/sub D/ = 300 pM. In addition, the solubilized and retained guanyl nucleotide regulation of agonist binding characteristic of membrane bound receptors. Gel filtration experiments showed that solubilized receptors from cortex and cerebellum had different elution profiles. Analysis by sucrose density gradient centrifugation showed that receptors in the lower molecular weight peak sedimented with a coefficient of 5S. Receptors in the larger molecular weight peak sedimented to the bottom of the gradient. Attempts to purify receptors by chromatography on propylbenzilycholine Sepharose were unsuccessful. The technique used to attach the ligand to the solid support, however, was used to synthesize a PrBCM-BSA conjugate and the conjugate used as an antigen in the production of anti-ligand antibodies. Two anti-PrBCM monoclonal antibodies were isolated that recognize muscarinic but not nicotinic cholinergic ligands. The abilities of the antibodies to recognize other muscarinic ligands indicated the antibodies recognized a portion of PrBCM involved in binding to the receptor. Construction of an antibody affinity resin resulted in the purification of this fragment a minimum of 170 fold.

  15. Molecular alteration of a muscarinic acetylcholine receptor system during synaptogenesis

    SciTech Connect

    Large, T.H.; Cho, N.J.; De Mello, F.G.; Klein, W.L.

    1985-07-25

    Biochemical properties of the muscarinic acetylcholine receptor system of the avian retina were found to change during the period when synapses form in ovo. Comparison of ligand binding to membranes obtained before and after synaptogenesis showed a significant increase in the affinity, but not proportion, of the high affinity agonist-binding state. There was no change in receptor sensitivity to antagonists during this period. Pirenzepine binding, which can discriminate muscarinic receptor subtypes, showed the presence of a single population of low affinity sites (M2) before and after synaptogenesis. The change in agonist binding was not due to the late development of receptor function. However, detergent-solubilization of membranes eliminated differences in agonist binding between receptors from embryos and hatched chicks, suggesting a developmental change in interactions of the receptor with functionally related membrane components. A possible basis for altered interactions was obtained from isoelectric point data showing that the muscarinic receptor population underwent a transition from a predominantly low pI form (4.25) in 13 day embryos to a predominantly high pI form (4.50) in newly hatched chicks. The possibility that biochemical changes in the muscarinic receptor play a role in differentiation of the system by controlling receptor position on the surface of nerve cells is discussed.

  16. Structure and dynamics of the M3 muscarinic acetylcholine receptor

    SciTech Connect

    Kruse, Andrew C.; Hu, Jianxin; Pan, Albert C.; Arlow, Daniel H.; Rosenbaum, Daniel M.; Rosemond, Erica; Green, Hillary F.; Liu, Tong; Chae, Pil Seok; Dror, Ron O.; Shaw, David E.; Weis, William I.; Wess, Jürgen; Kobilka, Brian K.

    2012-03-01

    Acetylcholine, the first neurotransmitter to be identified, exerts many of its physiological actions via activation of a family of G-protein-coupled receptors (GPCRs) known as muscarinic acetylcholine receptors (mAChRs). Although the five mAChR subtypes (M1-M5) share a high degree of sequence homology, they show pronounced differences in G-protein coupling preference and the physiological responses they mediate. Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences. We describe here the structure of the G{sub q/11}-coupled M3 mAChR ('M3 receptor', from rat) bound to the bronchodilator drug tiotropium and identify the binding mode for this clinically important drug. This structure, together with that of the G{sub i/o}-coupled M2 receptor, offers possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows a structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en route to the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and provide additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors.

  17. Neural Systems Governed by Nicotinic Acetylcholine Receptors: Emerging Hypotheses

    PubMed Central

    Miwa, Julie M.; Freedman, Robert; Lester, Henry A.

    2015-01-01

    Cholinergic neurons and nicotinic acetylcholine receptors (nAChRs) in the brain participate in diverse functions: reward, learning and memory, mood, sensory processing, pain, and neuroprotection. Nicotinic systems also have well-known roles in drug abuse. Here, we review recent insights into nicotinic function, linking exogenous and endogenous manipulations of nAChRs to alterations in synapses, circuits, and behavior. We also discuss how these contemporary advances can motivate attempts to exploit nicotinic systems therapeutically in Parkinson’s disease, cognitive decline, epilepsy, and schizophrenia. PMID:21482353

  18. Antigenic role of single residues within the main immunogenic region of the nicotinic acetylcholine receptor.

    PubMed Central

    Papadouli, I; Potamianos, S; Hadjidakis, I; Bairaktari, E; Tsikaris, V; Sakarellos, C; Cung, M T; Marraud, M; Tzartos, S J

    1990-01-01

    The target of most of the autoantibodies against the acetylcholine receptor (AChR) in myasthenic sera is the main immunogenic region (MIR) on the extracellular side of the AChR alpha-subunit. Binding of anti-MIR monoclonal antibodies (mAbs) has been recently localized between residues alpha 67 and alpha 76 of Torpedo californica electric organ (WNPADYGGIK) and human muscle (WNPDDYGGVK) AChR. In order to evaluate the contribution of each residue to the antigenicity of the MIR, we synthesized peptides corresponding to residues alpha 67-76 from Torpedo and human AChRs, together with 13 peptide analogues. Nine of these analogues had one residue of the Torpedo decapeptide replaced by L-alanine, three had a structure which was intermediate between those of the Torpedo and human alpha 67-76 decapeptides, and one had D-alanine in position 73. Binding studies employing six anti-MIR mAbs and all 15 peptides revealed that some residues (Asn68 and Asp71) are indispensable for binding by all mAbs tested, whereas others are important only for binding by some mAbs. Antibody binding was mainly restricted to residues alpha 68-74, the most critical sequence being alpha 68-71. Fish electric organ and human MIR form two distinct groups of strongly overlapping epitopes. Some peptide analogues enhanced mAb binding compared with Torpedo and human peptides, suggesting that the construction of a very antigenic MIR is feasible. PMID:1695844

  19. How the mongoose can fight the snake: the binding site of the mongoose acetylcholine receptor.

    PubMed Central

    Barchan, D; Kachalsky, S; Neumann, D; Vogel, Z; Ovadia, M; Kochva, E; Fuchs, S

    1992-01-01

    The ligand binding site of the nicotinic acetylcholine receptor (AcChoR) is within a short peptide from the alpha subunit that includes the tandem cysteine residues at positions 192 and 193. To elucidate the molecular basis of the binding properties of the AcChoR, we chose to study nonclassical muscle AcChoRs from animals that are resistant to alpha-neurotoxins. We have previously reported that the resistance of snake AcChoR to alpha-bungarotoxin (alpha-BTX) may be accounted for by several major substitutions in the ligand binding site of the receptor. In the present study, we have analyzed the binding site of AcChoR from the mongoose, which is also resistant to alpha-neurotoxins. It was shown that mongoose AcChoR does not bind alpha-BTX in vivo or in vitro. cDNA fragments of the alpha subunit of mongoose AcChoR corresponding to codons 122-205 and including the presumed ligand binding site were cloned, sequenced, and expressed in Escherichia coli. The expressed protein fragments of the mongoose, as well as of snake receptors, do not bind alpha-BTX. The mongoose fragment is highly homologous (greater than 90%) to the respective mouse fragment. Out of the seven amino acid differences between the mongoose and mouse in this region, five cluster in the presumed ligand binding site, close to cysteines 192 and 193. These changes are at positions 187 (Trp----Asn), 189 (Phe----Thr), 191 (Ser----Ala), 194 (Pro----Leu), and 197 (Pro----His). The mongoose like the snake AcChoR has a potential glycosylation site in the binding site domain. Sequence comparison between species suggests that substitutions at positions 187, 189, and 194 are important in determining the resistance of mongoose and snake AcChoR to alpha-BTX. In addition, it was shown that amino acid residues that had been reported to be necessary for acetylcholine binding are conserved in the toxin-resistant animals as well. Images PMID:1380164

  20. How the mongoose can fight the snake: the binding site of the mongoose acetylcholine receptor.

    PubMed

    Barchan, D; Kachalsky, S; Neumann, D; Vogel, Z; Ovadia, M; Kochva, E; Fuchs, S

    1992-08-15

    The ligand binding site of the nicotinic acetylcholine receptor (AcChoR) is within a short peptide from the alpha subunit that includes the tandem cysteine residues at positions 192 and 193. To elucidate the molecular basis of the binding properties of the AcChoR, we chose to study nonclassical muscle AcChoRs from animals that are resistant to alpha-neurotoxins. We have previously reported that the resistance of snake AcChoR to alpha-bungarotoxin (alpha-BTX) may be accounted for by several major substitutions in the ligand binding site of the receptor. In the present study, we have analyzed the binding site of AcChoR from the mongoose, which is also resistant to alpha-neurotoxins. It was shown that mongoose AcChoR does not bind alpha-BTX in vivo or in vitro. cDNA fragments of the alpha subunit of mongoose AcChoR corresponding to codons 122-205 and including the presumed ligand binding site were cloned, sequenced, and expressed in Escherichia coli. The expressed protein fragments of the mongoose, as well as of snake receptors, do not bind alpha-BTX. The mongoose fragment is highly homologous (greater than 90%) to the respective mouse fragment. Out of the seven amino acid differences between the mongoose and mouse in this region, five cluster in the presumed ligand binding site, close to cysteines 192 and 193. These changes are at positions 187 (Trp----Asn), 189 (Phe----Thr), 191 (Ser----Ala), 194 (Pro----Leu), and 197 (Pro----His). The mongoose like the snake AcChoR has a potential glycosylation site in the binding site domain. Sequence comparison between species suggests that substitutions at positions 187, 189, and 194 are important in determining the resistance of mongoose and snake AcChoR to alpha-BTX. In addition, it was shown that amino acid residues that had been reported to be necessary for acetylcholine binding are conserved in the toxin-resistant animals as well.

  1. Nicotinic acetylcholine receptors control acetylcholine and noradrenaline release in the rodent habenulo-interpeduncular complex

    PubMed Central

    Beiranvand, F; Zlabinger, C; Orr-Urtreger, A; Ristl, R; Huck, S; Scholze, P

    2014-01-01

    Background and purpose Nicotinic acetylcholine receptors (nACh receptors) play a central role in the habenulo-interpeduncular system. We studied nicotine-induced release of NA and ACh in the habenula and interpeduncular nucleus (IPN). Experimental approach The habenula and IPN were loaded with [3H]-choline or [3H]-NA and placed in superfusion chambers. [3H]-ACh release was also stimulated using nicotinic agonists, electrical pulses and elevated [KCl]o in hippocampal and cortical slices from rats, wild-type mice and mice lacking α5, α7, β2, or β4 nACh receptor subunits. Finally, we analysed nACh receptor subtypes in the IPN using immunoprecipitation. Key results Nicotine induced release of [3H]-ACh in the IPN of rats and mice. This release was calcium-dependent but not blocked by tetrodotoxin (TTX); moreover, [3H]-ACh release was abolished in β4-knockout mice but was unaffected in β2- and α5-knockout mice. In contrast, nicotine-induced release of [3H]-NA in the IPN and habenula was blocked by TTX and reduced in both β2-knockout and β4-knockout mice, and dose–response curves were right-shifted in α5-knockout mice. Although electrical stimuli triggered the release of both transmitters, [3H]-ACh release required more pulses delivered at a higher frequency. Conclusions and implications Our results confirm previous findings that β4-containing nACh receptors are critical for [3H]-ACh release in the mouse IPN. Experiments using α5-knockout mice also revealed that unlike in the hippocampus, nicotine-induced [3H]-NA release in the habenulo-interpeduncular system is altered in this knockout model. As α5-containing nACh receptors play a key role in nicotine intake, our results add NA to the list of transmitters involved in this mechanism. PMID:25041479

  2. Synthetic peptides corresponding to sequences of snake venom neurotoxins and rabies virus glycoprotein bind to the nicotinic acetylcholine receptor.

    PubMed

    Lentz, T L; Hawrot, E; Wilson, P T

    1987-01-01

    Peptides corresponding to portions of loop 2 of snake venom curare-mimetic neurotoxins and to a structurally similar region of rabies virus glycoprotein were synthesized. Interaction of these peptides with purified Torpedo electric organ acetylcholine receptor was tested by measuring their ability to block the binding of 125I-labeled alpha-bungarotoxin to the receptor. In addition, inhibition of alpha-bungarotoxin binding to a 32-residue synthetic peptide corresponding to positions 173-204 of the alpha-subunit was determined. Neurotoxin and glycoprotein peptides corresponding to toxin loop 2 inhibited labeled toxin binding to the receptor with IC50 values comparable to those of nicotine and the competitive antagonist d-tubocurarine and to the alpha-subunit peptides with apparent affinities between those of d-tubocurarine and alpha-cobratoxin. Substitution of neurotoxin residue Arg37, the proposed counterpart of the quaternary ammonium of acetylcholine, with a negatively charged Glu residue reduced the apparent affinity about 10-fold. Peptides containing the neurotoxin invariant residue Trp29 and 10- to 100-fold higher affinities than peptides lacking this residue. These results demonstrate that relatively short synthetic peptides retain some of the binding ability of the native protein from which they are derived, indicating that such peptides are useful in the study of protein-protein interactions. The ability of the peptides to compete alpha-bungarotoxin binding to the receptor with apparent affinities comparable to those of other cholinergic ligands indicates that loop 2 of the neurotoxins and the structurally similar segment of the rabies virus glycoprotein act as recognition sites for the acetylcholine receptor. Invariant toxin residues Arg37 and Trp29 and their viral homologs play important, although not essential, roles in binding, possibly by interaction with complementary anionic and hydrophobic subsites on the acetylcholine receptor. The alpha

  3. Acetylcholine receptors and cholinergic ligands: biochemical and genetic aspects in Torpedo californica and Drosophila melanogaster

    SciTech Connect

    Rosenthal, L.S.

    1987-01-01

    This study evaluates the biochemical and genetic aspects of the acetylcholine receptor proteins and cholinergic ligands in Drosophila melanogaster and Torpedo californica. Included are (1) a comparative study of nicotinic ligand-induced cation release from acetylcholine receptors isolated from Torpedo californica and from Drosophila melanogaster, (2) solution studies of the cholinergic ligands, nikethamide and ethamivan, aimed at measuring internal molecular rotational barriers in solvents of different polarity; and (3) the isolation and characterization of the gene(s) for the acetylcholine receptor in Drosophila melasogaster. Acetylcholine receptor proteins isolated from Drosphila melanogaster heads were found to behave kinetically similar (with regards to cholinergic ligand-induced /sup 155/Eu:/sup 3 +/ displacement from prelabeled proteins) to receptor proteins isolated from Torpedo californica electric tissue, providing additional biochemical evidence for the existence of a Drosophila acetylcholine receptor.

  4. Expression of neuronal nicotinic acetylcholine receptor subunit mRNAs in rat hippocampal GABAergic interneurons.

    PubMed

    Son, Jong-Hyun; Winzer-Serhan, Ursula H

    2008-11-10

    Hippocampal inhibitory interneurons are a diverse population of cells widely scattered in the hippocampus, where they regulate hippocampal circuit activity. The hippocampus receives cholinergic projections from the basal forebrain, and functional studies have suggested the presence of different subtypes of nicotinic acetylcholine receptors (AChRs) on gamma-aminobutyric acid (GABA)ergic interneurons. Single-cell polymerase chain reaction analysis had confirmed that several nAChR subunit mRNAs are co-expressed with glutamate decarboxylase 67 (GAD67), the marker for GABAergic interneurons. In this anatomical study, we systematically investigated the co-expression of GAD67 with different nAChR subunits by using double in situ hybridization with a digoxigenin-labeled GAD67 probe and (35)S-labeled probes for nAChR subunits (alpha2, alpha3, alpha4, alpha5, alpha6, alpha7, beta2, beta3, and beta4). The results revealed that most GAD67-positive interneurons expressed beta2, and 67 % also expressed alpha7 mRNA. In contrast, mRNA expression of other subunits was limited; only 13 % of GAD67-positive neurons co-expressed alpha4, and less than 10% expressed transcripts for alpha2, alpha3, alpha5, or beta4. Most GAD67/alpha2 co-expression was located in CA1/CA3 stratum oriens, and GAD67/alpha5 co-expression was predominantly detected in CA1/CA3 stratum radiatum/lacunosum moleculare and the dentate gyrus. Expression of alpha6 and beta3 mRNAs was rarely detected in the hippocampus, and mRNAs were not co-expressed with GAD67. These findings suggest that the majority of nicotinic responses in GABAergic interneurons should be mediated by a homomeric alpha7 or heteromeric alpha7*-containing nAChRs. Other possible combinations such as alpha2beta2*, alpha4beta2*, or alpha5beta2* heteromeric nAChRs could contribute to functional nicotinic response in subsets of GABAergic interneurons but overall would have a minor role.

  5. Single acetylcholine receptor channel currents recorded at high hydrostatic pressures.

    PubMed Central

    Heinemann, S H; Stühmer, W; Conti, F

    1987-01-01

    A technique for performing patch-clamp experiments under high hydrostatic (oil) pressure is described. The method allows the transfer of whole cell or membrane patches in a recording configuration into a pressure vessel, where pressure can be increased up to 60 MPa (approximately equal to 600 bar). We have studied in this way the pressure dependence of single acetylcholine receptor channels in excised "outside-out" membrane patches from cultured rat muscle cells. In the range of 0.1 to 60 MPa the open channel conductance in 140 mM NaCl solutions did not vary by more than 2%, which implies that the translocation of sodium ions through the channel pore does not involve steps with significant activation volumes. At high acetylcholine concentrations (20 microM) bursts of single-channel activity allowed measurements of the mean open and mean closed times of the channel. Pressurization to 40 MPa increased both mean open and mean closed times giving apparent activation volumes of about 59 and 139 A3, respectively. This implies a net volume increase of 80 A3, associated with the transition from the agonist-free state to the open state of the channel, which may be partially associated with the agonist-binding step. All the observed pressure effects were reversible. The activation volumes for the gating of acetylcholine receptor channels are comparable to those of sodium and potassium channels in the squid giant axon, suggesting that there is some basic common mechanism in the operation of ion-channel proteins. Images PMID:2437577

  6. Cell-surface translational dynamics of nicotinic acetylcholine receptors

    PubMed Central

    Barrantes, Francisco J.

    2014-01-01

    Synapse efficacy heavily relies on the number of neurotransmitter receptors available at a given time. In addition to the equilibrium between the biosynthetic production, exocytic delivery and recycling of receptors on the one hand, and the endocytic internalization on the other, lateral diffusion and clustering of receptors at the cell membrane play key roles in determining the amount of active receptors at the synapse. Mobile receptors traffic between reservoir compartments and the synapse by thermally driven Brownian motion, and become immobilized at the peri-synaptic region or the synapse by: (a) clustering mediated by homotropic inter-molecular receptor–receptor associations; (b) heterotropic associations with non-receptor scaffolding proteins or the subjacent cytoskeletal meshwork, leading to diffusional “trapping,” and (c) protein-lipid interactions, particularly with the neutral lipid cholesterol. This review assesses the contribution of some of these mechanisms to the supramolecular organization and dynamics of the paradigm neurotransmitter receptor of muscle and neuronal cells -the nicotinic acetylcholine receptor (nAChR). Currently available information stemming from various complementary biophysical techniques commonly used to interrogate the dynamics of cell-surface components is critically discussed. The translational mobility of nAChRs at the cell surface differs between muscle and neuronal receptors in terms of diffusion coefficients and residence intervals at the synapse, which cover an ample range of time regimes. A peculiar feature of brain α7 nAChR is its ability to spend much of its time confined peri-synaptically, vicinal to glutamatergic (excitatory) and GABAergic (inhibitory) synapses. An important function of the α7 nAChR may thus be visiting the territories of other neurotransmitter receptors, differentially regulating the dynamic equilibrium between excitation and inhibition, depending on its residence time in each domain. PMID

  7. Expression of cloned α6* nicotinic acetylcholine receptors.

    PubMed

    Wang, Jingyi; Kuryatov, Alexander; Lindstrom, Jon

    2015-09-01

    Nicotinic acetylcholine receptors (AChRs) are ACh-gated ion channels formed from five homologous subunits in subtypes defined by their subunit composition and stoichiometry. Some subtypes readily produce functional AChRs in Xenopus oocytes and transfected cell lines. α6β2β3* AChRs (subtypes formed from these subunits and perhaps others) are not easily expressed. This may be because the types of neurons in which they are expressed (typically dopaminergic neurons) have unique chaperones for assembling α6β2β3* AChRs, especially in the presence of the other AChR subtypes. Because these relatively minor brain AChR subtypes are of major importance in addiction to nicotine, it is important for drug development as well as investigation of their functional properties to be able to efficiently express human α6β2β3* AChRs. We review the issues and progress in expressing α6* AChRs. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'.

  8. Acetylcholine elongates neuronal growth cone filopodia via activation of nicotinic acetylcholine receptors.

    PubMed

    Zhong, Lei Ray; Estes, Stephen; Artinian, Liana; Rehder, Vincent

    2013-07-01

    In addition to acting as a classical neurotransmitter in synaptic transmission, acetylcholine (ACh) has been shown to play a role in axonal growth and growth cone guidance. What is not well understood is how ACh acts on growth cones to affect growth cone filopodia, structures known to be important for neuronal pathfinding. We addressed this question using an identified neuron (B5) from the buccal ganglion of the pond snail Helisoma trivolvis in cell culture. ACh treatment caused pronounced filopodial elongation within minutes, an effect that required calcium influx and resulted in the elevation of the intracellular calcium concentration ([Ca]i ). Whole-cell patch clamp recordings showed that ACh caused a reduction in input resistance, a depolarization of the membrane potential, and an increase in firing frequency in B5 neurons. These effects were mediated via the activation of nicotinic acetylcholine receptors (nAChRs), as the nAChR agonist dimethylphenylpiperazinium (DMPP) mimicked the effects of ACh on filopodial elongation, [Ca]i elevation, and changes in electrical activity. Moreover, the nAChR antagonist tubucurarine blocked all DMPP-induced effects. Lastly, ACh acted locally at the growth cone, because growth cones that were physically isolated from their parent neuron responded to ACh by filopodial elongation with a similar time course as growth cones that remained connected to their parent neuron. Our data revealed a critical role for ACh as a modulator of growth cone filopodial dynamics. ACh signaling was mediated via nAChRs and resulted in Ca influx, which, in turn, caused filopodial elongation.

  9. [Nicotine effects on mitochondria membrane potential: participation of nicotinic acetylcholine receptors].

    PubMed

    Gergalova, G L; Skok, M V

    2011-01-01

    The effect of nicotine on the mouse liver mitochondria was studied by fluorescent flow cytometry. Mice consumed nicotine during 65 days; alternatively, nicotine was added to isolated mitochondria. Mitochondria of nicotine-treated mice had significantly lower basic levels of membrane potential and granularity as compared to those of the control group. Pre-incubation of the isolated mitochondria with nicotine prevented from dissipation of their membrane potential stimulated with 0.8 microM CaCl2 depending on the dose, and this effect was strengthened by the antagonist of alpha7 nicotinic receptors (alpha7 nAChR) methyllicaconitine. Mitochondria of mice intravenously injected with the antibodies against alpha7 nAChR demonstrated lower levels of membrane potential. Introduction of nicotine, choline, acetylcholine or synthetic alpha7 nAChR agonist PNU 282987 into the incubation medium inhibited Ca2+ accumulation in mitochondria, although the doses of agonists were too low to activate the alpha7 nAChR ion channel. It is concluded that nicotine consumption worsens the functional state of mitochondria by affecting their membrane potential and granularity, and this effect, at least in part, is mediated by alpha7 nAChR desensitization.

  10. Subtype Differences in Pre-Coupling of Muscarinic Acetylcholine Receptors

    PubMed Central

    Jakubík, Jan; Janíčková, Helena; Randáková, Alena; El-Fakahany, Esam E.; Doležal, Vladimír

    2011-01-01

    Based on the kinetics of interaction between a receptor and G-protein, a myriad of possibilities may result. Two extreme cases are represented by: 1/Collision coupling, where an agonist binds to the free receptor and then the agonist-receptor complex “collides” with the free G-protein. 2/Pre-coupling, where stable receptor/G-protein complexes exist in the absence of agonist. Pre-coupling plays an important role in the kinetics of signal transduction. Odd-numbered muscarinic acetylcholine receptors preferentially couple to Gq/11, while even-numbered receptors prefer coupling to Gi/o. We analyzed the coupling status of the various subtypes of muscarinic receptors with preferential and non-preferential G-proteins. The magnitude of receptor-G-protein coupling was determined by the proportion of receptors existing in the agonist high-affinity binding conformation. Antibodies directed against the C-terminus of the α-subunits of the individual G-proteins were used to interfere with receptor-G-protein coupling. Effects of mutations and expression level on receptor-G-protein coupling were also investigated. Tested agonists displayed biphasic competition curves with the antagonist [3H]-N-methylscopolamine. Antibodies directed against the C-terminus of the α-subunits of the preferential G-protein decreased the proportion of high-affinity sites, and mutations at the receptor-G-protein interface abolished agonist high-affinity binding. In contrast, mutations that prevent receptor activation had no effect. Expression level of preferential G-proteins had no effect on pre-coupling to non-preferential G-proteins. Our data show that all subtypes of muscarinic receptors pre-couple with their preferential classes of G-proteins, but only M1 and M3 receptors also pre-couple with non-preferential Gi/o G-proteins. Pre-coupling is not dependent on agonist efficacy nor on receptor activation. The ultimate mode of coupling is therefore dictated by a combination of the receptor subtype

  11. Characterization and photoaffinity labeling of the muscarinic acetylcholine receptor

    SciTech Connect

    Cremo, C.R.

    1983-01-01

    The muscarinic acetylcholine receptor, identified by tritiated L-quinuclidinyl benzilate (L-(/sup 3/H)QNB) binding, was solubilized from porcine atrial membranes using a 5:1 (w/w) ratio of digitonin and cholate. Specific binding activities of the solubilized receptor solutions usually exceeded 1.0 nmol L-(/sup 3/H)QNB sites per gram of protein, representing 75-98% total site recovery and a two- to three-fold enrichment over untreated atrial membranes. Two rapid assays for measuring the binding activities of detergent extracts were devised and compared with equilibrium dialysis. All three methods gave similar results. The equilibrium dissociation constant of the solubilized receptor for L-(/sup 3/H)QNB as determined by the three methods varied from 230 to 450 pM depending on the method and temperature. The interaction of alkyl quanidines and decahydrohistrionicotoxin with the membrane-bound and solubilized muscarinic acetylcholine receptor (mAcChR) from porcine atria was described. Alkyl guanidines with alkyl chain lengths from one to ten carbons displaced (/sup 3/H)L-quinuclidinyl bensilate ((/sup 3/H)L-QNB) competitively from a single class of sites for the membrane-bound mAcChR. From a plot of -1n K/sub i/ versus alkyl carbon chain number, a value of -(473 +/- 30) cal/mol was estimated as the energetic contribution per methylene group to the total binding energy. The synthesis and properties of a radiolabeled muscarinic antagonist photoaffinity probe, (/sup 3/H) p-azidoatropine methyl iodide were reported.

  12. Regulation of phosphorylation of nicotinic acetylcholine receptors in mouse BC3H1 myocytes

    SciTech Connect

    Smith, M.M.; Merlie, J.P.; Lawrence, J.C. Jr.

    1987-09-01

    By using /sup 32/P-labeling methods and performing immunoprecipitations with specific antibodies, the authors have found that three subunits of the nicotinic acetylcholine receptor and phosphorylated in mouse skeletal muscle cells. In nonstimulated cells, the molar ratios of phosphate estimated in ..cap alpha.., ..beta.., and delta subunits were 0.02, 0.05, and 0.5, respectively. All three subunits contained predominantly phosphoserine with some phosphothreonine; the ..beta.., subunit also contained phosphotyrosine. Incubating cells with agents that stimulate cAMP-dependent pathways (isoproterenol, forskolin, 8-Br-cAMP) increased the phosphorylation of the delta subunit by 50%, but phosphate labeling of the ..beta.. subunit was depressed by a third. In contrast, when cells were incubated with the divalent cation ionophores A-23187 or ionomycin, phosphorylation of both the delta and ..beta.. subunits increased. The results indicate that acetylcholine receptors are phosphorylated to significant levels in skeletal muscle cells and that cAMP-dependent and Ca/sup 2 +/-dependent pathways exist for controlling the phosphorylation state of the receptor subunits.

  13. Nicotine induces fibrogenic changes in human liver via nicotinic acetylcholine receptors expressed on hepatic stellate cells

    SciTech Connect

    Soeda, Junpei; Morgan, Maelle; McKee, Chad; Mouralidarane, Angelina; Lin, ChingI; Roskams, Tania; Oben, Jude A.

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Cigarette smoke may induce liver fibrosis via nicotine receptors. Black-Right-Pointing-Pointer Nicotine induces proliferation of hepatic stellate cells (HSCs). Black-Right-Pointing-Pointer Nicotine activates hepatic fibrogenic pathways. Black-Right-Pointing-Pointer Nicotine receptor antagonists attenuate HSC proliferation. Black-Right-Pointing-Pointer Nicotinic receptor antagonists may have utility as novel anti-fibrotic agents. -- Abstract: Background and aims: Cigarette smoke (CS) may cause liver fibrosis but possible involved mechanisms are unclear. Among the many chemicals in CS is nicotine - which affects cells through nicotinic acetylcholine receptors (nAChR). We studied the effects of nicotine, and involved pathways, on human primary hepatic stellate cells (hHSCs), the principal fibrogenic cells in the liver. We then determined possible disease relevance by assaying nAChR in liver samples from human non-alcoholic steatohepatitis (NASH). Methods: hHSC were isolated from healthy human livers and nAChR expression analyzed - RT-PCR and Western blotting. Nicotine induction of hHSC proliferation, upregulation of collagen1-{alpha}2 and the pro-fibrogenic cytokine transforming growth factor beta 1 (TGF-{beta}1) was determined along with involved intracellular signaling pathways. nAChR mRNA expression was finally analyzed in whole liver biopsies obtained from patients diagnosed with non-alcoholic steatohepatitis (NASH). Results: hHSCs express muscle type ({alpha}1, {beta}1, delta and epsilon) and neuronal type ({alpha}3, {alpha}6, {alpha}7, {beta}2 and {beta}4) nAChR subunits at the mRNA level. Among these subunits, {alpha}3, {alpha}7, {beta}1 and {epsilon} were predominantly expressed as confirmed by Western blotting. Nicotine induced hHSC proliferation was attenuated by mecamylamine (p < 0.05). Additionally, collagen1-{alpha}2 and TGF-{beta}1 mRNA expression were significantly upregulated by nicotine and inhibited by

  14. Nicotinic Acetylcholine Receptors at the Single-Channel Level.

    PubMed

    Bouzat, Cecilia; Sine, Steven M

    2017-03-05

    Over the past four decades, the patch clamp technique and nicotinic acetylcholine (nACh) receptors have established an enduring partnership. Like all good partnerships, each partner has proven significant in its own right, while their union has spurred innumerable advances in life science research. A member and prototype of the superfamily of pentameric ligand-gated ion channels, the nACh receptor is a chemo-electric transducer, binding nerve-released ACh and rapidly opening its channel to cation flow to elicit cellular excitation. A subject of a Nobel Prize in Physiology or Medicine, the patch clamp technique provides unprecedented resolution of currents through single ion channels in their native cellular environments. Here, focusing on muscle and α7 nACh receptors, we describe the extraordinary contribution of the patch clamp technique toward understanding how they activate in response to neurotransmitter, how subtle structural and mechanistic differences among nACh receptor subtypes translate into significant physiological differences, and how nACh receptors are being exploited as therapeutic drug targets.

  15. Caenorhabditis elegans nicotinic acetylcholine receptors are required for nociception

    PubMed Central

    Cohen, Emiliano; Chatzigeorgiou, Marios; Husson, Steven J.; Steuer-Costa, Wagner; Gottschalk, Alexander; Schafer, William R.; Treinin, Millet

    2014-01-01

    Polymodal nociceptors sense and integrate information on injurious mechanical, thermal, and chemical stimuli. Chemical signals either activate nociceptors or modulate their responses to other stimuli. One chemical known to activate or modulate responses of nociceptors is acetylcholine (ACh). Across evolution nociceptors express subunits of the nicotinic acetylcholine receptor (nAChR) family, a family of ACh-gated ion channels. The roles of ACh and nAChRs in nociceptor function are, however, poorly understood. Caenorhabditis elegans polymodal nociceptors, PVD, express nAChR subunits on their sensory arbor. Here we show that mutations reducing ACh synthesis and mutations in nAChR subunits lead to defects in PVD function and morphology. A likely cause for these defects is a reduction in cytosolic calcium measured in ACh and nAChR mutants. Indeed, overexpression of a calcium pump in PVD mimics defects in PVD function and morphology found in nAChR mutants. Our results demonstrate, for the first time, a central role for nAChRs and ACh in nociceptor function and suggest that calcium permeating via nAChRs facilitates activity of several signaling pathways within this neuron. PMID:24518198

  16. Physiological characterization of human muscle acetylcholine receptors from ALS patients.

    PubMed

    Palma, Eleonora; Inghilleri, Maurizio; Conti, Luca; Deflorio, Cristina; Frasca, Vittorio; Manteca, Alessia; Pichiorri, Floriana; Roseti, Cristina; Torchia, Gregorio; Limatola, Cristina; Grassi, Francesca; Miledi, Ricardo

    2011-12-13

    Amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of motor neurons leading to muscle paralysis. Research in transgenic mice suggests that the muscle actively contributes to the disease onset, but such studies are difficult to pursue in humans and in vitro models would represent a good starting point. In this work we show that tiny amounts of muscle from ALS or from control denervated muscle, obtained by needle biopsy, are amenable to functional characterization by two different technical approaches: "microtransplantation" of muscle membranes into Xenopus oocytes and culture of myogenic satellite cells. Acetylcholine (ACh)-evoked currents and unitary events were characterized in oocytes and multinucleated myotubes. We found that ALS acetylcholine receptors (AChRs) retain their native physiological characteristics, being activated by ACh and nicotine and blocked by α-bungarotoxin (α-BuTX), d-tubocurarine (dTC), and galantamine. The reversal potential of ACh-evoked currents and the unitary channel behavior were also typical of normal muscle AChRs. Interestingly, in oocytes injected with muscle membranes derived from ALS patients, the AChRs showed a significant decrease in ACh affinity, compared with denervated controls. Finally, riluzole, the only drug currently used against ALS, reduced, in a dose-dependent manner, the ACh-evoked currents, indicating that its action remains to be fully characterized. The two methods described here will be important tools for elucidating the role of muscle in ALS pathogenesis and for developing drugs to counter the effects of this disease.

  17. Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: ( sup 3 H)nicotine as an agonist photoaffinity label

    SciTech Connect

    Middleton, R.E.; Cohen, J.B. )

    1991-07-16

    The agonist ({sup 3}H)nicotine was used as a photoaffinity label for the acetylcholine binding sties on the Torpedo nicotinic acetylcholine receptor (AChR). ({sup 3}H)Nicotine binds at equilibrium with K{sub eq} = 0.6 {mu}M to the agonist binding sites. Irradiation with 254-nm light of AChR-rich membranes equilibrated with ({sup 3}H)nicotine resulted in covalent incorporation into the {alpha}- and {gamma}-subunits, which was inhibited by agonists and competitive antagonists but not by noncompetitive antagonists. Inhibition of labeling by d-tubocurarine demonstrated that the {alpha}-subunit was labeled via both agonist sites but the {gamma}-subunit was labeled only via the site that binds d-tubocurarine with high affinity. Chymotryptic digestion of the {alpha}-subunit confirmed that Try-198 was the principal amino acid labeled by ({sup 3}H)nicotine. This confirmation required a novel radiosequencing strategy employing o-phthalaldehyde ({sup 3}H)Nicotine, which is the first photoaffinity agonist used, labels primarily Tyr-198 in contrast to competitive antagonist affinity labels, which label primarily Tyr-190 and Cys-192/Cys-193.

  18. Regulation of nicotinic acetylcholine receptor phosphorylation in rat myotubes by forskolin and cAMP

    SciTech Connect

    Miles, K.; Anthony, D.T.; Rubin, L.L.; Greengard, P.; Huganir, R.L.

    1987-09-01

    The nicotinic acetylcholine receptor (Ac-ChoR) from rat myotubes prelabeled in culture with (/sup 32/P)orthophosphate was isolated by acetylcholine affinity chromatography followed by immunoaffinity chromatography. Under basal conditions, the nicotinic AcChoR was shown to be phosphorylated in situ on the ..beta.. and delta subunits. Regulation of AcChoR phosphorylation by cAMP-dependent protein kinase was explored by the addition of forskolin or cAMP analogues to prelabeled cell cultures. Forskolin, an activator of adenylate cyclase, stimulated the phosphorylation of the delta subunit 20-fold over basal phosphorylation and induced phosphorylation of the ..cap alpha.. subunit. The effect of forskolin was dose dependent with a half-maximal response at 8 ..mu..M in the presence of 35 ..mu..M Ro 20-1724, a phosphodiesterase inhibitor. Stimulation of delta subunit phosphorylation was almost maximal within 5 min, whereas stimulation of ..cap alpha.. subunit phosphorylation was not maximal until 45 min after forskolin treatment. Stimulation of AcChoR phosphorylation by 8-benzylthioadenosine 3',5'-cyclic monophosphate was identical to that obtained by forskolin. Two-dimensional thermolytic phosphopeptide maps of the delta subunit revealed a single major phosphopeptide. These results correlate closely with the observed effects of forskolin on AcChoR desensitization in muscle and suggest that cAMP-dependent phosphorylation of the delta subunit increases the rate of AcChoR desensitization in rat myotubes.

  19. Microtransplantation of acetylcholine receptors from normal or denervated rat skeletal muscles to frog oocytes

    PubMed Central

    Bernareggi, Annalisa; Reyes-Ruiz, Jorge Mauricio; Lorenzon, Paola; Ruzzier, Fabio; Miledi, Ricardo

    2011-01-01

    Cell membranes, carrying neurotransmitter receptors and ion channels, can be ‘microtransplanted’ into frog oocytes. This technique allows a direct functional characterization of the original membrane proteins, together with any associated molecules they may have, still embedded in their natural lipid environment. This approach has been previously demonstrated to be very useful to study neurotransmitter receptors and ion channels contained in cell membranes isolated from human brains. Here, we examined the possibility of using the microtransplantation method to study acetylcholine receptors from normal and denervated rat skeletal muscles. We found that the muscle membranes, carrying their fetal or adult acetylcholine receptor isoforms, could be efficiently microtransplanted to the oocyte membrane, making the oocytes become sensitive to acetylcholine. These results show that oocytes injected with skeletal muscle membranes efficiently incorporate functional acetylcholine receptors, thus making the microtransplantation approach a valuable tool to further investigate receptors and ion channels of human muscle diseases. PMID:21224230

  20. Separate fractions of mRNA from Torpedo electric organ induce chloride channels and acetylcholine receptors in Xenopus oocytes.

    PubMed Central

    Sumikawa, K; Parker, I; Amano, T; Miledi, R

    1984-01-01

    Poly(A)+ mRNA extracted from the electric organ of Torpedo was fractionated by sucrose density gradient centrifugation. After injection into Xenopus oocytes one mRNA fraction induced the appearance of chloride channels in the oocyte membrane. Many of these channels were normally open, and the ensuing chloride current kept the resting potential of injected oocytes close to the chloride equilibrium potential. When the membrane was hyperpolarized, the chloride current was reduced. A separate fraction of mRNA induced the incorporation of acetylcholine receptors into the oocyte membrane. When translated in a cell-free system this fraction directed the synthesis of the alpha, beta, gamma, and delta subunits of the acetylcholine receptor. In contrast, the mRNA fraction that induced the chloride channels caused the synthesis of the delta subunit, a very small amount of alpha, and no detectable beta or gamma subunits. This suggests that the size of the mRNA coding for the chloride channel is similar to the preponderant species of mRNA coding for the delta subunit of the acetylcholine receptor. Images Fig. 1. PMID:6094179

  1. Quinuclidine compounds differently act as agonists of Kenyon cell nicotinic acetylcholine receptors and induced distinct effect on insect ganglionic depolarizations.

    PubMed

    Mathé-Allainmat, Monique; Swale, Daniel; Leray, Xavier; Benzidane, Yassine; Lebreton, Jacques; Bloomquist, Jeffrey R; Thany, Steeve H

    2013-12-01

    We have recently demonstrated that a new quinuclidine benzamide compound named LMA10203 acted as an agonist of insect nicotinic acetylcholine receptors. Its specific pharmacological profile on cockroach dorsal unpaired median neurons (DUM) helped to identify alpha-bungarotoxin-insensitive nAChR2 receptors. In the present study, we tested its effect on cockroach Kenyon cells. We found that it induced an inward current demonstrating that it bounds to nicotinic acetylcholine receptors expressed on Kenyon cells. Interestingly, LMA10203-induced currents were completely blocked by the nicotinic antagonist α-bungarotoxin. We suggested that LMA10203 effect occurred through the activation of α-bungarotoxin-sensitive receptors and did not involve α-bungarotoxin-insensitive nAChR2, previously identified in DUM neurons. In addition, we have synthesized two new compounds, LMA10210 and LMA10211, and compared their effects on Kenyon cells. These compounds were members of the 3-quinuclidinyl benzamide or benzoate families. Interestingly, 1 mM LMA10210 was not able to induce an inward current on Kenyon cells compared to LMA10211. Similarly, we did not find any significant effect of LMA10210 on cockroach ganglionic depolarization, whereas these three compounds were able to induce an effect on the central nervous system of the third instar M. domestica larvae. Our data suggested that these three compounds could bind to distinct cockroach nicotinic acetylcholine receptors.

  2. Discovery of new muscarinic acetylcholine receptor antagonists from Scopolia tangutica

    PubMed Central

    Du, Nana; Liu, Yanfang; Zhang, Xiuli; Wang, Jixia; Zhao, Jianqiang; He, Jian; Zhou, Han; Mei, Lijuan; Liang, Xinmiao

    2017-01-01

    Scopolia tangutica (S. tangutica) is a traditional Chinese medicinal plant used for antispasmodics, anesthesia, analgesia and sedation. Its pharmacological activities are mostly associated with the antagonistic activity at muscarinic acetylcholine receptors (mAchRs) of several known alkaloids such as atropine and scopolamine. With our recent identification of four hydroxycinnamic acid amides from S. tangutica, we hypothesized that this plant may contain previously unidentified alkaloids that may also contribute to its in vivo effect. Herein, we used a bioassay-guided multi-dimension separation strategy to discover novel mAchR antagonists from S. tangutica. The core of this approach is to use label-free cell phenotypic assay to first identify active fractions, and then to guide purification of active ligands. Besides four tropanes and six cinnamic acid amides that have been previously isolated from S. tangutica, we recently identified two new tropanes, one new cinnamic acid amide, and nine other compounds. Six tropane compounds purified from S. tangutica for the first time were confirmed to be competitive antagonists of muscarinic receptor 3 (M3), including the two new ones 8 and 12 with IC50 values of 1.97 μM and 4.47 μM, respectively. Furthermore, the cinnamic acid amide 17 displayed 15-fold selectivity for M1 over M3 receptors. These findings will be useful in designing lead compounds for mAchRs and elucidating mechanisms of action of S. tangutica. PMID:28387362

  3. Revisiting the endocytosis of the m2 muscarinic acetylcholine receptor.

    PubMed

    Ockenga, Wymke; Tikkanen, Ritva

    2015-05-12

    The agonist-induced endocytosis of the muscarinic acetylcholine receptor M2 is different from that of the other members of the muscarinic receptor family. The uptake of the M2 receptor involves the adapter proteins of the β-arrestin family and the small GTPase ADP-ribosylation factor 6. However, it has remained inconclusive if M2 endocytosis is dependent on clathrin or the large GTPase dynamin. We here show by means of knocking down the clathrin heavy chain that M2 uptake upon agonist stimulation requires clathrin. The expression of various dominant-negative dynamin-2 mutants and the use of chemical inhibitors of dynamin function revealed that dynamin expression and membrane localization as such appear to be necessary for M2 endocytosis, whereas dynamin GTPase activity is not required for this process. Based on the data from the present and from previous studies, we propose that M2 endocytosis takes place by means of an atypical clathrin-mediated pathway that may involve a specific subset of clathrin-coated pits/vesicles.

  4. Anesthetics Target Interfacial Transmembrane Sites in Nicotinic Acetylcholine Receptors

    PubMed Central

    Forman, Stuart A.; Chiara, David C.; Miller, Keith W.

    2014-01-01

    General anesthetics are a heterogeneous group of small amphiphilic ligands that interact weakly at multiple allosteric sites on many pentameric ligand gated ion channels (pLGICs), resulting in either inhibition, potentiation of channel activity, or both. Allosteric principles imply that modulator sites must change configuration and ligand affinity during receptor state transitions. Thus, general anesthetics and related compounds are useful both as state-dependent probes of receptor structure and as potentially selective modulators of pLGIC functions. This review focuses on general anesthetic sites in nicotinic acetylcholine receptors, which were among the first anesthetic-sensitive pLGIC experimental models studied, with particular focus on sites formed by transmembrane domain elements. Structural models place many of these sites at interfaces between two or more pLGIC transmembrane helices both within subunits and between adjacent subunits, and between transmembrane helices and either lipids (the lipid-protein interface) or water (i.e. the ion channel). A single general anesthetic may bind at multiple allosteric sites in pLGICs, producing a net effect of either inhibition (e.g. blocking the ion channel) or enhanced channel gating (e.g. inter-subunit sites). Other general anesthetic sites identified by photolabeling or crystallography are tentatively linked to functional effects, including intra-subunit helix bundle sites and the lipid-protein interface. PMID:25316107

  5. Revisiting the Endocytosis of the M2 Muscarinic Acetylcholine Receptor

    PubMed Central

    Ockenga, Wymke; Tikkanen, Ritva

    2015-01-01

    The agonist-induced endocytosis of the muscarinic acetylcholine receptor M2 is different from that of the other members of the muscarinic receptor family. The uptake of the M2 receptor involves the adapter proteins of the β-arrestin family and the small GTPase ADP-ribosylation factor 6. However, it has remained inconclusive if M2 endocytosis is dependent on clathrin or the large GTPase dynamin. We here show by means of knocking down the clathrin heavy chain that M2 uptake upon agonist stimulation requires clathrin. The expression of various dominant-negative dynamin-2 mutants and the use of chemical inhibitors of dynamin function revealed that dynamin expression and membrane localization as such appear to be necessary for M2 endocytosis, whereas dynamin GTPase activity is not required for this process. Based on the data from the present and from previous studies, we propose that M2 endocytosis takes place by means of an atypical clathrin-mediated pathway that may involve a specific subset of clathrin-coated pits/vesicles. PMID:25985102

  6. Novel neonicotinoid-agarose affinity column for Drosophila and Musca nicotinic acetylcholine receptors.

    PubMed

    Tomizawa, M; Latli, B; Casida, J E

    1996-10-01

    Neonicotinoids such as the insecticide imidacloprid (IMI) act as agonists at the insect nicotinic acetylcholine receptor (nAChR). Head membranes of Drosophila melanogaster and Musca domestica have a single high-affinity binding site for [3H]IMI with KD values of 1-2 nM and Bmax values of 560-850 fmol/mg of protein. Locusta and Periplaneta nAChRs isolated with an alpha-bungarotoxin (alpha-BGT)-agarose affinity column are known to be alpha-subunit homooligomers. This study uses 1-[N-(6-chloro-3-pyridylmethyl)-N-ethyl]amino-1-amino-2-nitroethene++ + (which inhibits [3H]IMI binding to Drosophila and Musca head membranes at 2-3 nM) to develop a neonicotinoid-agarose affinity column. The procedure-introduction of Triton-solubilized Drosophila or Musca head membranes into this neonicotinoid-based column, elution with IMI, and analysis by lithium dodecyl sulfate-polyacrylamicle gel electrophoresis-gives only three proteins (69, 66, and 61 kDa) tentatively assigned as putative subunits of the nAChR; the same three proteins are obtained with Musca using the alpha-BGT-agarose affinity column. Photoaffinity labeling of the Drosophila and Musca putative subunits from the neonicotinoid column with 125I-alpha-BGT-4-azidosalicylic acid gives a labeled derivative of 66-69 kDa. The yield is 2-5 micrograms of receptor protein from 1 g of Drosophila or Musca heads. Neonicotinoid affinity chromatography to isolate native Drosophila and Musca receptors will facilitate studies on the structure and function of insect nAChRs.

  7. Immunological relationship between acetylcholine receptor and thymus: a possible significance in myasthenia gravis.

    PubMed Central

    Aharonov, A; Tarrab-Hazdai, R; Abramsky, O; Fuchs, S

    1975-01-01

    A defined immunological cross-reaction was observed between acetylcholine receptor fraction from the electric eel, Electrophorus electricus, and two calf thymus fractions. The cross-reaction was demonstrated on the cellular level by means of the lymphocyte transformation technique, and on the humoral level, by means of the microcomplement fixation assay. In the human disease myasthenia gravis both acetylcholine receptor at the neuromuscular junction and the thymus are affected, probably by an autoimmune mechanism. The immunological cross-reaction between acetylcholine receptor and thymic components may explain the association between endplate and thymus disorders in myasthenia gravis. PMID:1055418

  8. Photoaffinity labeling of the Torpedo californica nicotinic acetylcholine receptor with an aryl azide derivative of phosphatidylserine

    SciTech Connect

    Blanton, M.P.; Wang, H.H. )

    1990-02-06

    A photoactivatable analogue of phosphatidylserine, {sup 125}I-labeled 4-azidosalicylic acid-phosphatidylserine ({sup 125}I ASA-PS), was used to label both native acetylcholine receptor (AchR)-rich membranes from Torpedo californica and AchR membranes affinity purified from Torpedo reconstituted into asolectin vesicles. The radioiodinated arylazido group attaches directly to the phospholipid head group and thus probes for regions of the AchR structure in contact with the negatively charged head group of phosphatidylserine. All four subunits of the AchR incorporated the label, with the {alpha} subunit incorporating approximately twice as much as each of the other subunits on a per mole basis. The regions of the AchR {alpha} subunit that incorporated {sup 125}I ASA-PS were mapped by Staphylococcus aureus V8 protease digestion. The majority of label incorporated into fragments representing a more complete digestion of the {alpha} subunit was localized to 11.7- and 10.1-kDa V8 cleavage fragments, both beginning at Asn-339 and of sufficient length to contain the hydrophobic region M4. An 18.7-kDa fragment beginning at Ser-173 and of sufficient length to contain the hydrophobic regions M1, M2, and M3 was also significantly labeled. In contrast, V8 cleavage fragments representing roughly a third of the amino-terminal portion of the {alpha} subunit incorporated little or no detectable amount of probe.

  9. Conservation of neural nicotinic acetylcholine receptors from Drosophila to vertebrate central nervous systems.

    PubMed Central

    Bossy, B; Ballivet, M; Spierer, P

    1988-01-01

    Nicotinic acetylcholine receptors (nAChR) are found both in vertebrate and insect central nervous systems. We have isolated a Drosophila gene by crosshybridization with a vertebrate probe. Structural conservation of domains of the deduced protein and of intron/exon boundaries indicate that the Drosophila gene encodes an nAChR alpha-like subunit (ALS). That the Drosophila gene product most resembles the neuronal set of vertebrate nAChRs alpha-subunits is also indicated by the failure of an ALS-beta-galactosidase fusion protein to bind alpha-bungarotoxin on blots in contrast to vertebrate endplate alpha-subunit constructions. The ALS encoding gene exceeds 54 kb in length and the transcript has a very long and unusual 5' leader. As we found previously for a gene whose product is also involved in cholinergic synapses, acetylcholinesterase, the leader encodes short open reading frames, which might be involved in translation control. We also note the presence of opa repeats in the gene, as has been found for various Drosophila genes expressed in the nervous system. Images PMID:2840281

  10. Multiple conductance classes of mouse nicotinic acetylcholine receptors expressed in Xenopus oocytes.

    PubMed Central

    Kullberg, R; Owens, J L; Camacho, P; Mandel, G; Brehm, P

    1990-01-01

    Acetylcholine receptor (AcChoR) subunit mRNAs transcribed from mouse BC3H-1 cDNAs were injected into Xenopus oocytes and the expressed AcChoR channels were examined by single channel recording. Injection of alpha-, beta-, gamma-, and delta-subunit mRNAs produced two predominant channel classes with conductances of approximately 50 and approximately 12 pS, while infrequent openings of approximately 25-pS channels were also observed. Injection of alpha-, beta-, and gamma-subunit mRNAs produced a single class of approximately 12-pS AcChoR channels, which resembled the smallest conductance channels present in alpha beta gamma omega-injected oocytes. Assembly of delta-less channels may thus explain the lowest conductance AcChoR channels in alpha beta gamma delta-injected oocytes and might also account for similar channels that have been observed in vertebrate skeletal muscle. Images PMID:2315303

  11. Physiological characterization of human muscle acetylcholine receptors from ALS patients

    PubMed Central

    Palma, Eleonora; Inghilleri, Maurizio; Conti, Luca; Deflorio, Cristina; Frasca, Vittorio; Manteca, Alessia; Pichiorri, Floriana; Roseti, Cristina; Torchia, Gregorio; Limatola, Cristina; Grassi, Francesca; Miledi, Ricardo

    2011-01-01

    Amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of motor neurons leading to muscle paralysis. Research in transgenic mice suggests that the muscle actively contributes to the disease onset, but such studies are difficult to pursue in humans and in vitro models would represent a good starting point. In this work we show that tiny amounts of muscle from ALS or from control denervated muscle, obtained by needle biopsy, are amenable to functional characterization by two different technical approaches: “microtransplantation” of muscle membranes into Xenopus oocytes and culture of myogenic satellite cells. Acetylcholine (ACh)-evoked currents and unitary events were characterized in oocytes and multinucleated myotubes. We found that ALS acetylcholine receptors (AChRs) retain their native physiological characteristics, being activated by ACh and nicotine and blocked by α-bungarotoxin (α-BuTX), d-tubocurarine (dTC), and galantamine. The reversal potential of ACh-evoked currents and the unitary channel behavior were also typical of normal muscle AChRs. Interestingly, in oocytes injected with muscle membranes derived from ALS patients, the AChRs showed a significant decrease in ACh affinity, compared with denervated controls. Finally, riluzole, the only drug currently used against ALS, reduced, in a dose-dependent manner, the ACh-evoked currents, indicating that its action remains to be fully characterized. The two methods described here will be important tools for elucidating the role of muscle in ALS pathogenesis and for developing drugs to counter the effects of this disease. PMID:22128328

  12. Effects of a monoclonal anti-acetylcholine receptor antibody on the avian end-plate.

    PubMed Central

    Maselli, R A; Nelson, D J; Richman, D P

    1989-01-01

    1. The effects of anti-acetylcholine receptor (AChR) monoclonal antibodies (mAbs) 370 and 132A on miniature end-plate potentials (MEPPs) and end-plate currents (EPCs) in the posterior latissimus dorsi muscle of adult chickens were investigated. 2. After incubation of the electrophysiological preparation with mAb 370 (5-50 micrograms/ml), which blocks both agonist (carbamylcholine) and alpha-bungarotoxin (alpha-BTX) binding and induces a hyperacute form of experimental autoimmune myasthenia gravis (EAMG), MEPP and EPC amplitudes were irreversibly reduced. 3. This effect was not associated with any significant change in the time constant describing EPC decay (tau EPC), current reversal potential, or the voltage dependence of tau EPC. The tau EPC at -80 mV was 5.9 +/- 0.6 ms before incubation with mAb 370 (50 micrograms/ml) and 6.0 +/- 0.9 ms afterwards. Current reversal potential was -3.9 +/- 0.4 mV before mAb incubation and -4.8 +/- 1.5 mV afterwards. The change in membrane potential required to produce an e-fold change in tau EPC was 128 +/- 2.3 mV before antibody incubation compared to 125 +/- 6.6 mV after incubation. 4. A second anti-AChR mAb, 132A (50 micrograms/ml), which is capable of inducing the classically described form of EAMG without blocking agonist or alpha-BTX binding, or inducing hyperacute EAMG, produced no significant change in MEPP amplitude, EPC amplitude, tau EPC or EPC reversal potentials. 5. The mAb 370 (50 micrograms/ml) induced a partially reversible decrease of the quantal content of the neurally evoked end-plate potential (EPP). This effect was not observed with mAb 132A, (+)tubocurarine (10(-7)-10(-5) g/ml) or an irrelevant anti-oestrogen receptor mAb. 6. These data suggest that the rapid onset of weakness observed in chicken hatchlings after the injection of mAb 370 (Gomez & Richman, 1983) can be attributed to a combined effect of a block of acetylcholine (ACh)-induced ion channel activity in the postsynaptic membrane and a reduction of

  13. Muscarinic acetylcholine receptors: location of the ligand binding site

    SciTech Connect

    Hulme, E.; Wheatley, M.; Curtis, C.; Birdsall, N.

    1987-05-01

    The key to understanding the pharmacological specificity of muscarinic acetylcholine receptors (mAChR's) is the location within the receptor sequence of the amino acid residues responsible for ligand binding. To approach this problem, they have purified mAChR's from rat brain to homogeneity by sequential ion-exchange chromatography, affinity chromatography and molecular weight fractionation. Following labelling of the binding site with an alkylating affinity label, /sup 3/H-propylbenzilycholine mustard aziridinium ion (/sup 3/H-PrBCM), the mAChR was digested with a lysine-specific endoproteinase, and a ladder of peptides of increasing molecular weight, each containing the glycosylated N-terminus, isolated by chromatography on wheat-germ agglutinin sepharose. The pattern of labelling showed that a residue in the peptides containing transmembrane helices 2 and/or 3 of the mAChR was alkylated. The linkage was cleaved by 1 M hydroxylamine, showing that /sup 3/H-PrBCM was attached to an acidic residue, whose properties strongly suggested it to be embedded in a hydrophobic intramembrane region of the mAChR. Examination of the cloned sequence of the mAChR reveals several candidate residues, the most likely of which is homologous to an aspartic acid residue thought to protonate the retinal Schiff's base in the congeneric protein rhodopsin.

  14. Circulating antibodies against nicotinic acetylcholine receptors in chagasic patients

    PubMed Central

    GOIN, J C; VENERA, G; BONINO, M BISCOGLIO DE JIMÉNEZ; STERIN-BORDA, L

    1997-01-01

    Human and experimental Chagas' disease causes peripheral nervous system damage involving neuromuscular transmission alterations at the neuromuscular junction. Additionally, autoantibodies directed to peripheral nerves and sarcolemmal proteins of skeletal muscle have been described. In this work, we analyse the ability of serum immunoglobulin factors associated with human chagasic infection to bind the affinity-purified nicotinic acetylcholine receptor (nAChR) from electric organs of Discopyge tschudii and to identify the receptor subunits involved in the interaction. The frequency of serum anti-nAChR reactivity assayed by dot-blot was higher in seropositive chagasic patients than in uninfected subjects. Purified IgG obtained from chagasic patients immunoprecipitated a significantly higher fraction of the solubilized nAChR than normal IgG. Furthermore, immunoblotting assays indicated that α and β are the main subunits involved in the interaction. Chagasic IgG was able to inhibit the binding of α-bungarotoxin to the receptor in a concentration-dependent manner, confirming the contribution of the α-subunit in the autoantibody-receptor interaction. The presence of anti-nAChR antibodies was detected in 73% of chagasic patients with impairment of neuromuscular transmission in conventional electromyographical studies, indicating a strong association between seropositive reactivity against nAChR and electromyographical abnormalities in chagasic patients. The chronic binding of these autoantibodies to the nAChR could induce a decrease in the population of functional nAChRs at the neuromuscular junction and consequently contribute to the electrophysiological neuromuscular alterations described in the course of chronic Chagas' disease. PMID:9367405

  15. Acetylcholine Receptor Organization in Membrane Domains in Muscle Cells

    PubMed Central

    Piguet, Joachim; Schreiter, Christoph; Segura, Jean-Manuel; Vogel, Horst; Hovius, Ruud

    2011-01-01

    Nicotinic acetylcholine receptors (nAChR) in muscle fibers are densely packed in the postsynaptic region at the neuromuscular junction. Rapsyn plays a central role in directing and clustering nAChR during cellular differentiation and neuromuscular junction formation; however, it has not been demonstrated whether rapsyn is the only cause of receptor immobilization. Here, we used single-molecule tracking methods to investigate nAChR mobility in plasma membranes of myoblast cells during their differentiation to myotubes in the presence and absence of rapsyn. We found that in myoblasts the majority of nAChR were immobile and that ∼20% of the receptors showed restricted diffusion in small domains of ∼50 nm. In myoblasts devoid of rapsyn, the fraction of mobile nAChR was considerably increased, accompanied by a 3-fold decrease in the immobile population of nAChR with respect to rapsyn-expressing cells. Half of the mobile receptors were confined to domains of ∼120 nm. Measurements performed in heterologously transfected HEK cells confirmed the direct immobilization of nAChR by rapsyn. However, irrespective of the presence of rapsyn, about one-third of nAChR were confined in 300-nm domains. Our results show (i) that rapsyn efficiently immobilizes nAChR independently of other postsynaptic scaffold components; (ii) nAChR is constrained in confined membrane domains independently of rapsyn; and (iii) in the presence of rapsyn, the size of these domains is strongly reduced. PMID:20978122

  16. Rabies virus interaction with various cell lines is independent of the acetylcholine receptor.

    PubMed

    Reagan, K J; Wunner, W H

    1985-01-01

    Rabies virus infects most cells in vitro. The presence of the nicotinic acetylcholine receptor on the plasma membrane of various cell lines is not an obligate factor for rabies virus susceptibility of those cells.

  17. In vitro proliferative responses and antibody titers specific to human acetylcholine receptor synthetic peptides in patients with myasthenia gravis and relation to HLA class II genes.

    PubMed Central

    Brocke, S; Brautbar, C; Steinman, L; Abramsky, O; Rothbard, J; Neumann, D; Fuchs, S; Mozes, E

    1988-01-01

    To investigate which parts of the acetylcholine receptor are involved in the initiation and development of myasthenia gravis (MG), peptides representing different sequences of the human acetylcholine receptor alpha-subunit were synthesized. These peptides were tested for their ability to stimulate T cells of myasthenic patients and healthy control patients in proliferation assays and to bind to sera antibodies. Three of eight peptides discriminated significantly between the two groups in the proliferation assay, as well as in their ability to bind to serum antibodies. HLA-DR3 and DR5 were associated with proliferative responses to specific AChR peptides in the group of myasthenics. Acetylcholine receptor epitopes that might play a specific role in myasthenia gravis thus were demonstrated. PMID:2461962

  18. Muscarinic acetylcholine receptors regulating cell cycle progression are expressed in human gingival keratinocytes.

    PubMed

    Arredondo, J; Hall, L L; Ndoye, A; Chernyavsky, A I; Jolkovsky, D L; Grando, S A

    2003-02-01

    We have previously reported the presence in human gingival keratinocytes (GKC) of choline acetyltransferase, the acetylcholine (ACh) synthesizing enzyme, acetylcholinesterase, the ACh degrading enzyme, and alpha 3, alpha 5, alpha 7, beta 2 as well as alpha 9 nicotinic ACh receptor subunits. To expand the knowledge about the role of ACh in oral biology, we investigated the presence of the muscarinic ACh receptor (mAChR) subtypes in GKC. RT-PCR demonstrated the presence of m2, m3, m4, and m5 mRNA transcripts. Synthesis of the respective proteins was verified by immunoblotting with the subtype-specific antibodies that revealed receptor bands at the expected molecular weights. The antibodies mapped mAChR subtypes in the epithelium of human attached gingiva and also visualized them on the cell membrane of cultured GKC. The whole cell radioligand binding assay revealed that GKC have specific binding sites for the muscarinic ligand [3H]quinuclidinyl benzilate, Bmax = 222.9 fmol/106 cells with a Kd of 62.95 pM. The downstream coupling of the mAChRs to regulation of cell cycle progression in GKC was studied using quantitative RT-PCR and immunoblotting assays. Incubation of GKC for 24 h with 10 micro m muscarine increased relative amounts of Ki-67, PCNA and p53 mRNAs and PCNA, cyclin D1, p21 and p53 proteins. These effects were abolished in the presence of 50 micro m atropine. The finding in GKC of mAChRs coupled to regulation of the cell cycle progression demonstrate further the structure/function of the non-neuronal cholinergic system operating in human oral epithelium. The results obtained in this study help clarify the role for keratinocyte ACh axis in the physiologic control of oral gingival homeostasis.

  19. Non-Neuronal Functions of the M2 Muscarinic Acetylcholine Receptor

    PubMed Central

    Ockenga, Wymke; Kühne, Sina; Bocksberger, Simone; Banning, Antje; Tikkanen, Ritva

    2013-01-01

    Acetylcholine is an important neurotransmitter whose effects are mediated by two classes of receptors. The nicotinic acetylcholine receptors are ion channels, whereas the muscarinic receptors belong to the large family of G protein coupled seven transmembrane helix receptors. Beyond its function in neuronal systems, it has become evident that acetylcholine also plays an important role in non-neuronal cells such as epithelial and immune cells. Furthermore, many cell types in the periphery are capable of synthesizing acetylcholine and express at least some of the receptors. In this review, we summarize the non-neuronal functions of the muscarinic acetylcholine receptors, especially those of the M2 muscarinic receptor in epithelial cells. We will review the mechanisms of signaling by the M2 receptor but also the cellular trafficking and ARF6 mediated endocytosis of this receptor, which play an important role in the regulation of signaling events. In addition, we provide an overview of the M2 receptor in human pathological conditions such as autoimmune diseases and cancer. PMID:24705159

  20. Subunit-selective role of the M3 transmembrane domain of the nicotinic acetylcholine receptor in channel gating.

    PubMed

    De Rosa, María José; Corradi, Jeremías; Bouzat, Cecilia

    2008-02-01

    The nicotinic acetylcholine receptor (AChR) can be either hetero-pentameric, composed of alpha and non-alpha subunits, or homo-pentameric, composed of alpha7 subunits. To explore the subunit-selective contributions of transmembrane domains to channel gating we analyzed single-channel activity of chimeric muscle AChRs. We exchanged M3 between alpha1 and epsilon or alpha7 subunits. The replacement of M3 in alpha1 by epsilonM3 significantly alters activation properties. Channel activity appears as bursts of openings whose durations are 20-fold longer than those of wild-type AChRs. In contrast, 7-fold briefer openings are observed in AChRs containing the reverse epsilon chimeric subunit. The duration of the open state decreases with the increase in the number of alpha1M3 segments, indicating additive contributions of M3 of all subunits to channel closing. Each alpha1M3 segment decreases the energy barrier of the closing process by approximately 0.8 kcal/mol. Partial chimeric subunits show that small stretches of the M3 segment contribute additively to the open duration. The replacement of alpha1 sequence by alpha7 in M3 leads to 3-fold briefer openings whereas in M1 it leads to 10-fold prolonged openings, revealing that the subunit-selective role is unique to each transmembrane segment.

  1. Synthetic peptides of neurotoxins and rabies virus glycoprotein behave as antagonists in a functional assay for the acetylcholine receptor.

    PubMed

    Donnelly-Roberts, D L; Lentz, T L

    1989-01-01

    Peptides of portions of loop 2 (the "toxic" loop) of snake venom curare-mimetic neurotoxins (alpha-bungarotoxin and king cobra toxin b) and of a structurally similar region of the rabies virus glycoprotein were synthesized. The effect of the peptides on carbachol-induced 22Na+ flux into BC3H-1 cells, which contain nicotinic acetylcholine receptors on their surfaces, was measured. Both the neurotoxin and glycoprotein peptides inhibited ion transport with IC50 values of 10(-4) M to 7 x 10(-7) M. The most effective peptides correspond to neurotoxin loop 2 and inhibited 22Na+ flux in the micromolar range comparable to the competitive antagonist d-tubocurarine. These findings show that neurotoxin loop 2 and the corresponding rabies virus glycoprotein segment interact with the agonist binding site of teh acetylcholine receptor and that short synthetic peptides representing portions of larger molecules by themselves can exert a biological effect on a large macromolecular complex like the acetylcholine receptor.

  2. Acetylcholine receptor channels are present in undifferentiated satellite cells but not in embryonic myoblasts in culture.

    PubMed

    Cossu, G; Eusebi, F; Grassi, F; Wanke, E

    1987-09-01

    The expression and the physiological properties of acetylcholine receptors (AChRs) of mononucleated myogenic cells, isolated from either embryonic or adult muscle of the mouse, have been investigated using the gigaohm seal patch-clamp technique in combination with immunocytochemistry (with an anti-myosin antibody) and alpha-bungarotoxin binding techniques. Undifferentiated (myosin-negative) embryonic myoblasts, grown either in mass culture or under clonal conditions, were found to be unresponsive to ACh and did not bind alpha-bungarotoxin. On the contrary, undifferentiated satellite cells (from adult muscle) exhibited channels activated by ACh and alpha-bungarotoxin binding sites similar to those observed in differentiated (myosin-positive) embryonic myoblasts and myotubes. Two classes of ACh-activated channels with different opening frequencies were identified. The major class of channels had a conductance of about 42 pS and mean open time of 3.1-8.2 msec. The minor class of channels had smaller conductance (about 17 pS) and similar open time. During differentiation, the conductance of the two channels did not change significantly, while channel lifetime became shorter in myotubes derived from satellite cells but not in myotubes derived from embryonic myoblasts. The relative proportion of small over large channels was significantly larger in embryonic than in adult myogenic cells.

  3. A mutational analysis of the acetylcholine receptor channel transmitter binding site.

    PubMed Central

    Akk, G; Zhou, M; Auerbach, A

    1999-01-01

    Mutagenesis and single-channel kinetic analysis were used to investigate the roles of four acetylcholine receptor channel (AChR) residues that are candidates for interacting directly with the agonist. The EC50 of the ACh dose-response curve was increased following alpha-subunit mutations Y93F and Y198F and epsilon-subunit mutations D175N and E184Q. Single-channel kinetic modeling indicates that the increase was caused mainly by a reduced gating equilibrium constant (Theta) in alphaY198F and epsilonD175N, by an increase in the equilibrium dissociation constant for ACh (KD) and a reduction in Theta in alphaY93F, and only by a reduction in KD in epsilonE184Q. This mutation altered the affinity of only one of the two binding sites and was the only mutation that reduced competition by extracellular K+. Additional mutations of epsilonE184 showed that K+ competition was unaltered in epsilonE184D and was virtually eliminated in epsilonE184K, but that neither of these mutations altered the intrinsic affinity for ACh. Thus there is an apparent electrostatic interaction between the epsilonE184 side chain and K+ ( approximately 1.7kBT), but not ACh+. The results are discussed in terms of multisite and induced-fit models of ligand binding to the AChR. PMID:9876135

  4. Role of calcium in the regulation of acetylcholine receptor synthese in cultured muscle cells*.

    PubMed

    Birnbaum, M; Reis, M A; Shainberg, A

    1980-05-01

    Embroyonic muscles differentiated in vitro were used to study the effects of intracellular Ca2+ ([Ca2+1]i) variations on the amount of acetylcholine receptors ([AChR]) in the cell membrane. 2. Increased Ca2+ concentration in the growth medium ([Ca2+]o) caused a marked elevation of AChR levels, apparently through de novo synthesis. 3. Agents known to increase [Ca2+]i and its accumulation in the sarcoplasmic reticulum (SR), such as ionophore A23187, sodium dantrolene (DaNa), or high [Mg2+]o all enhanced alpha-bungarotoxin (alpha-BGT) binding after 48 h of treatment. 4. Electrical stimulation or caffeine, both affectors of SR calcium release, brought about a decrease in [AChR] probably by suppressing its synthesis. 5. The effects of simultaneous treatment with two AChR-inducing agents, namely, high [Ca2+]o in the presence of tetrodotoxin (TTX) or high [Mg2+]o were not additive, thus suggesting action via a common saturable mediator. 6. Intermediate AChR levels obtained following simultaneous treatments with opposing effects, e.g., electrical stimulation in the presence of high [Ca2+]o or DaNa, suggest contradictory actions on a common mediator. 7. All these observations indicate a strong correlation between SR calcium levels and [AChR] on myotubes; while calcium accumulation in the Sr was followed by increased AChR synthesis, calcium release was accompanied by suppression of receptor synthesis.

  5. Nicotinic Acetylcholine Receptor (nAChR) Dependent Chorda Tympani Taste Nerve Responses to Nicotine, Ethanol and Acetylcholine.

    PubMed

    Ren, Zuo Jun; Mummalaneni, Shobha; Qian, Jie; Baumgarten, Clive M; DeSimone, John A; Lyall, Vijay

    2015-01-01

    Nicotine elicits bitter taste by activating TRPM5-dependent and TRPM5-independent but neuronal nAChR-dependent pathways. The nAChRs represent common targets at which acetylcholine, nicotine and ethanol functionally interact in the central nervous system. Here, we investigated if the nAChRs also represent a common pathway through which the bitter taste of nicotine, ethanol and acetylcholine is transduced. To this end, chorda tympani (CT) taste nerve responses were monitored in rats, wild-type mice and TRPM5 knockout (KO) mice following lingual stimulation with nicotine free base, ethanol, and acetylcholine, in the absence and presence of nAChR agonists and antagonists. The nAChR modulators: mecamylamine, dihydro-β-erythroidine, and CP-601932 (a partial agonist of the α3β4* nAChR), inhibited CT responses to nicotine, ethanol, and acetylcholine. CT responses to nicotine and ethanol were also inhibited by topical lingual application of 8-chlorophenylthio (CPT)-cAMP and loading taste cells with [Ca2+]i by topical lingual application of ionomycin + CaCl2. In contrast, CT responses to nicotine were enhanced when TRC [Ca2+]i was reduced by topical lingual application of BAPTA-AM. In patch-clamp experiments, only a subset of isolated rat fungiform taste cells exposed to nicotine responded with an increase in mecamylamine-sensitive inward currents. We conclude that nAChRs expressed in a subset of taste cells serve as common receptors for the detection of the TRPM5-independent bitter taste of nicotine, acetylcholine and ethanol.

  6. Nicotinic Acetylcholine Receptor (nAChR) Dependent Chorda Tympani Taste Nerve Responses to Nicotine, Ethanol and Acetylcholine

    PubMed Central

    Ren, Zuo Jun; Mummalaneni, Shobha; Qian, Jie; Baumgarten, Clive M.; DeSimone, John A.; Lyall, Vijay

    2015-01-01

    Nicotine elicits bitter taste by activating TRPM5-dependent and TRPM5-independent but neuronal nAChR-dependent pathways. The nAChRs represent common targets at which acetylcholine, nicotine and ethanol functionally interact in the central nervous system. Here, we investigated if the nAChRs also represent a common pathway through which the bitter taste of nicotine, ethanol and acetylcholine is transduced. To this end, chorda tympani (CT) taste nerve responses were monitored in rats, wild-type mice and TRPM5 knockout (KO) mice following lingual stimulation with nicotine free base, ethanol, and acetylcholine, in the absence and presence of nAChR agonists and antagonists. The nAChR modulators: mecamylamine, dihydro-β-erythroidine, and CP-601932 (a partial agonist of the α3β4* nAChR), inhibited CT responses to nicotine, ethanol, and acetylcholine. CT responses to nicotine and ethanol were also inhibited by topical lingual application of 8-chlorophenylthio (CPT)-cAMP and loading taste cells with [Ca2+]i by topical lingual application of ionomycin + CaCl2. In contrast, CT responses to nicotine were enhanced when TRC [Ca2+]i was reduced by topical lingual application of BAPTA-AM. In patch-clamp experiments, only a subset of isolated rat fungiform taste cells exposed to nicotine responded with an increase in mecamylamine-sensitive inward currents. We conclude that nAChRs expressed in a subset of taste cells serve as common receptors for the detection of the TRPM5-independent bitter taste of nicotine, acetylcholine and ethanol. PMID:26039516

  7. Regulation of hippocampal inhibitory circuits by nicotinic acetylcholine receptors

    PubMed Central

    Griguoli, Marilena; Cherubini, Enrico

    2012-01-01

    The hippocampal network comprises a large variety of locally connected GABAergic interneurons exerting a powerful control on network excitability and which are responsible for the oscillatory behaviour crucial for information processing. GABAergic interneurons receive an important cholinergic innervation from the medial septum-diagonal band complex of the basal forebrain and are endowed with a variety of muscarinic and nicotinic acetylcholine receptors (mAChRs and nAChRs) that regulate their activity. Deficits in the cholinergic system lead to the impairment of high cognitive functions, which are particularly relevant in neurodegenerative pathologies such as Alzheimer's and Parkinson's diseases as well as in schizophrenia. Here, we highlight some recent advances in the mechanisms by which cholinergic signalling via nAChRs regulates local inhibitory circuits in the hippocampus, early in postnatal life and in adulthood. We also discuss recent findings concerning the functional role of nAChRs in controlling short- and long-term modifications of synaptic efficacy. Insights into these processes may provide new targets for the therapeutic control of pathological conditions associated with cholinergic dysfunctions. PMID:22124144

  8. Oseltamivir blocks human neuronal nicotinic acetylcholine receptor-mediated currents.

    PubMed

    Muraki, Katsuhiko; Hatano, Noriyuki; Suzuki, Hiroka; Muraki, Yukiko; Iwajima, Yui; Maeda, Yasuhiro; Ono, Hideki

    2015-02-01

    The effects of oseltamivir, a neuraminidase inhibitor, were tested on the function of neuronal nicotinic acetylcholine receptors (nAChRs) in a neuroblastoma cell line IMR32 derived from human peripheral neurons and on recombinant human α3β4 nAChRs expressed in HEK cells. IMR32 cells predominately express α3β4 nAChRs. Nicotine (nic, 30 μm)-evoked currents recorded at -90 mV in IMR32 cells using the whole-cell patch clamp technique were reversibly blocked by oseltamivir in a concentration-dependent manner. In contrast, an active metabolite of oseltamivir, oseltamivir carboxylate (OC) at 30 μm had little effect on the nic-evoked currents. Oseltamivir also blocked nic-evoked currents derived from HEK cells with recombinant α3β4 nAChRs. This blockade was voltage-dependent with 10, 30 and 100 μm oseltamivir inhibiting ~50% at -100, -60 and -40 mV, respectively. Non-inactivating currents in IMR32 cells and in HEK cells with α3β4 nAChRs, which were evoked by an endogenous nicotinic agonist, ACh (5 μm), were reversibly blocked by oseltamivir. These data demonstrate that oseltamivir blocks nAChRs, presumably via binding to a site in the channel pore.

  9. Nicotinic acetylcholine receptor agonist attenuates ILC2-dependent airway hyperreactivity

    PubMed Central

    Galle-Treger, Lauriane; Suzuki, Yuzo; Patel, Nisheel; Sankaranarayanan, Ishwarya; Aron, Jennifer L.; Maazi, Hadi; Chen, Lin; Akbari, Omid

    2016-01-01

    Allergic asthma is a complex and chronic inflammatory disorder that is associated with airway hyperreactivity (AHR) and driven by Th2 cytokine secretion. Type 2 innate lymphoid cells (ILC2s) produce large amounts of Th2 cytokines and contribute to the development of AHR. Here, we show that ILC2s express the α7-nicotinic acetylcholine receptor (α7nAChR), which is thought to have an anti-inflammatory role in several inflammatory diseases. We show that engagement of a specific agonist with α7nAChR on ILC2s reduces ILC2 effector function and represses ILC2-dependent AHR, while decreasing expression of ILC2 key transcription factor GATA-3 and critical inflammatory modulator NF-κB, and reducing phosphorylation of upstream kinase IKKα/β. Additionally, the specific α7nAChR agonist reduces cytokine production and AHR in a humanized ILC2 mouse model. Collectively, our data suggest that α7nAChR expressed by ILC2s is a potential therapeutic target for the treatment of ILC2-mediated asthma. PMID:27752043

  10. Neuronal Nicotinic Acetylcholine Receptor Modulators Reduce Sugar Intake

    PubMed Central

    Shariff, Masroor; Quik, Maryka; Holgate, Joan; Morgan, Michael; Patkar, Omkar L.; Tam, Vincent; Belmer, Arnauld; Bartlett, Selena E.

    2016-01-01

    Excess sugar consumption has been shown to contribute directly to weight gain, thus contributing to the growing worldwide obesity epidemic. Interestingly, increased sugar consumption has been shown to repeatedly elevate dopamine levels in the nucleus accumbens (NAc), in the mesolimbic reward pathway of the brain similar to many drugs of abuse. We report that varenicline, an FDA-approved nicotinic acetylcholine receptor (nAChR) partial agonist that modulates dopamine in the mesolimbic reward pathway of the brain, significantly reduces sucrose consumption, especially in a long-term consumption paradigm. Similar results were observed with other nAChR drugs, namely mecamylamine and cytisine. Furthermore, we show that long-term sucrose consumption increases α4β2 * and decreases α6β2* nAChRs in the nucleus accumbens, a key brain region associated with reward. Taken together, our results suggest that nAChR drugs such as varenicline may represent a novel treatment strategy for reducing sugar consumption. PMID:27028298

  11. Regulation of muscarinic acetylcholine receptor-mediated synaptic responses by adenosine receptors in the rat hippocampus.

    PubMed Central

    Morton, R A; Davies, C H

    1997-01-01

    1. Intracellular current clamp recordings were made from CA1 pyramidal neurones in rat hippocampal slices. Experiments were performed in the presence of ionotropic glutamate receptor antagonists and gamma-aminobutyric acid (GABA) receptor antagonists to block all fast excitatory and inhibitory synaptic transmission. A single stimulus, delivered extracellularly in the stratum oriens, caused a reduction in spike frequency adaptation in response to a depolarizing current step delivered 2 s after the stimulus. A 2- to 10-fold increase in stimulus intensity evoked a slow excitatory postsynaptic potential (EPSP) which was associated with a small increase in input resistance. The peak amplitude of the EPSP occurred approximately 2.5 s after the stimulus and its magnitude (up to 30 mV) and duration (10-50 s) increased with increasing stimulus intensity. 2. The slow EPSP was unaffected by the metabotropic glutamate receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine ((+)-MCPG; 1000 microM) but was greatly enhanced by the acetylcholinesterase inhibitor physostigmine (1-5 microM). Both the slow EPSP and the stimulus-evoked reduction in spike frequency adaptation were inhibited by the muscarinic acetylcholine receptor (mAChR) antagonist atropine (1-5 microM). These results are consistent with these effects being mediated by mAChRs. 3. Both the mAChR-mediated EPSP (EPSPm) and the associated reduction in spike frequency adaptation were reversibly depressed (up to 97%) by either adenosine (100 microM) or its non-hydrolysable analogue 2-chloroadenosine (CADO; 0.1-5.0 microM). These effects were often accompanied by postsynaptic hyperpolarization (up to 8 mV) and a reduction in input resistance (up to 11%). The selective adenosine A1 receptor agonists 2-chloro-N6-cyclopentyladenosine (CCPA; 0.1-0.4 microM) and R(-)N6-(2-phenylisopropyl)-adenosine (R-PIA; 1 microM) both depressed the EPSPm. In contrast, the adenosine A2A receptor agonist 2-p-(2-carboxyethyl)-phenethylamino-5

  12. Structure-function relationships of curaremimetic neurotoxin loop 2 and of a structurally similar segment of rabies virus glycoprotein in their interaction with the nicotinic acetylcholine receptor

    SciTech Connect

    Lentz, T.L. )

    1991-11-12

    Peptides corresponding to portions of curaremimetic neurotoxin loop 2 and to a structurally similar segment of rabies virus glycoprotein were synthetically modified in order to gain information on structure-function relationships of neurotoxin loop 2 interactions with the acetylcholine receptor. Binding of synthetic peptides to the acetylcholine receptor of Torpedo electric organ membranes was assessed by measuring their ability to inhibit the binding of {sup 125}I-{alpha}-bungarotoxin to the receptor. The peptides showing the highest affinity for the receptor were a peptide corresponding to the sequence of loop 2 (residues 25-44) of Ophiophagus hannah (king cobra) toxin b and the structurally similar segment of CVS rabies virus glycoprotein. These affinities were comparable to those of d-tubocurarine and suberyldicholine. These results demonstrate the importance of loop 2 in the neurotoxin interaction with the receptor. N- and C-terminal deletions of the loop 2 peptides and substitution of residues invariant or highly conserved among neurotoxins were performed in order to determine the role of individual residues in binding. Residues 25-40 are the most crucial in the interaction with the acetylcholine receptor. Since this region of the glycoprotein contains residues corresponding to all of the functionally invariant neurotoxin residues, it may interact with the acetylcholine receptor through a mechanism similar to that of the neurotoxins.

  13. Phosphorylation and function of alpha4beta2 receptor.

    PubMed

    Bermudez, Isabel; Moroni, Mirko

    2006-01-01

    The neuronal nicotinic acetylcholine receptor (nAChR) alpha4 and beta2 subunits expressed in heterologous expression systems assemble into high- and low-affinity receptors (Zwart and Vijverberg, 1998; Buisson and Bertrand, 2001; Houlihan et al., 2001; Nelson et al., 2003), which reflects the assembly of two distinct subunit stoichiometries of alpha4beta2 receptor (Nelson et al., 2003). The high-affinity receptor ([alpha4]2[beta2]3) is about 100-fold more sensitive to ACh than the low-affinity receptor ([alpha4]3[beta2]2) (Zwart and Vijverberg, 1998; Buisson and Bertrand, 2001; Houlihan et al., 2001; Nelson et al., 2003). Recent evidence implicated 14-3-3 proteins as modulators of the relative abundance of nAChR subunits in the endoplasmic reticulum (ER), where ligand-gated ion channels assemble. The 14-3-3 proteins influence ER-to-plasma membrane trafficking of multimeric cell-surface proteins (O'Kelly et al., 2002). 14-3-3 proteins bind components of these multimeric proteins, and this interaction overrides dibasic COP1 retention signal to permit forward transport of the protein (O'Kelly et al., 2002). In the case of alpha4beta2 nAChRs, 14-3-3 binds the alpha4 subunit, and this association is dependent on phosphorylation of a serine residue within a protein kinase A(PKA) consensus sequence in the large cytoplasmic domain of the alpha4 subunit, which is also a binding motif recognized by 14-3-3 (Jeancloss et al., 2001; O'Kelly et al., 2002). The interplay among PKA, alpha4 subunits, and 14-3-3 proteins increases cell-surface expression of alpha4beta2 nAChRs by increasing steady-state levels of the alpha4 subunit available for assembly with beta2 subunits (Jeancloss et al., 2001). Because it is not known how 14-3-3-dependent changes in the steady-state levels of the alpha4 subunit might affect the functional type of alpha4beta2 receptors, we have investigated the effects of mutations of the 14-3-3 binding motif in the alpha4 subunit on alpha4beta2 nAChR function.

  14. Mechanism of nasal tolerance induced by a recombinant fragment of acetylcholine receptor for treatment of experimental myasthenia gravis.

    PubMed

    Im, S H; Barchan, D; Fuchs, S; Souroujon, M C

    2000-11-01

    Acetylcholine receptor (AChR) is the major autoantigen in myasthenia gravis (MG) and experimental autoimmune MG (EAMG). Here we analyze the mechanisms involved in suppression of ongoing EAMG in rats by nasal administration of a recombinant fragment from the human AChR alpha-subunit. We demonstrate that such a fragment, expressed without a fusion partner, confers nasal tolerance that can be adoptively transferred. Our observations suggest that the underlying mechanism of this nasal tolerance is active suppression involving a shift from a Th1 to a Th2/Th3-regulated AChR-specific response which may be mediated by down regulation of costimulatory factors.

  15. High-affinity binding of (/sup 3/H)acetylcholine to muscarinic cholinergic receptors

    SciTech Connect

    Kellar, K.J.; Martino, A.M.; Hall, D.P. Jr.; Schwartz, R.D.; Taylor, R.L.

    1985-06-01

    High-affinity binding of (/sup 3/H)acetylcholine to muscarinic cholinergic sites in rat CNS and peripheral tissues was measured in the presence of cytisin, which occupies nicotinic cholinergic receptors. The muscarinic sites were characterized with regard to binding kinetics, pharmacology, anatomical distribution, and regulation by guanyl nucleotides. These binding sites have characteristics of high-affinity muscarinic cholinergic receptors with a Kd of approximately 30 nM. Most of the muscarinic agonist and antagonist drugs tested have high affinity for the (/sup 3/H)acetylcholine binding site, but pirenzepine, an antagonist which is selective for M-1 receptors, has relatively low affinity. The ratio of high-affinity (/sup 3/H)acetylcholine binding sites to total muscarinic binding sites labeled by (/sup 3/H)quinuclidinyl benzilate varies from 9 to 90% in different tissues, with the highest ratios in the pons, medulla, and heart atrium. In the presence of guanyl nucleotides, (/sup 3/H) acetylcholine binding is decreased, but the extent of decrease varies from 40 to 90% in different tissues, with the largest decreases being found in the pons, medulla, cerebellum, and heart atrium. The results indicate that (/sup 3/H)acetylcholine binds to high-affinity M-1 and M-2 muscarinic receptors, and they suggest that most M-2 sites have high affinity for acetylcholine but that only a small fraction of M-1 sites have such high affinity.

  16. Optical studies of nicotinic acetylcholine receptor subtypes in the guinea-pig enteric nervous system.

    PubMed

    Obaid, A L; Nelson, M E; Lindstrom, J; Salzberg, B M

    2005-08-01

    Nicotinic transmission in the enteric nervous system (ENS) is extensive, but the role of individual nicotinic acetylcholine receptor (nAChR) subtypes in the functional connectivity of its plexuses has been elusive. Using monoclonal antibodies (mAbs) against neuronal alpha3-, alpha4-, alpha3/alpha5-, beta2-, beta4- and alpha7-subunits, combined with radioimmunoassays and immunocytochemistry, we demonstrate that guinea-pig enteric ganglia contain all of these nAChR-subunits with the exception of alpha4, and so, differ from mammalian brain. This information alone, however, is insufficient to establish the functional role of the identified nAChR-subtypes within the enteric networks and, ultimately, their specific contributions to gastrointestinal physiology. We have used voltage-sensitive dyes and a high-speed CCD camera, in conjunction with specific antagonists to various nAChRs, to elucidate some of the distinct contributions of the individual subtypes to the behaviour of enteric networks. In the guinea-pig, the submucous plexus has the extraordinary advantage that it is virtually two-dimensional, permitting optical recording, with single cell resolution, of the electrical activity of all of its neurones. In this plexus, the block of alpha3beta2-, alpha3beta4- and/or alpha7-nAChRs always results in a decrease in the magnitude of the synaptic response. However, the magnitude of the fast excitatory post-synaptic potentials (epsps) evoked by electrical stimulation of a neighbouring ganglion varies from cell to cell, reflecting the differential expression of subunits already observed using mAbs, as well as the strengths of the activated synaptic inputs. At the same time, we observe that submucous neurones have a substantial mecamylamine (Mec)-insensitive (non-nicotinic) component to their fast epsps, which may point to the presence of purinergic or serotonergic fast epsps in this system. In the myenteric plexus, on the other hand, the antagonist-induced changes in the

  17. Ligand binding to nicotinic acetylcholine receptor investigated by surface plasmon resonance.

    PubMed

    Kröger, D; Hucho, F; Vogel, H

    1999-08-01

    Ligand binding to the nicotinic acetylcholine receptor is studied by surface plasmon resonance. Biotinylated bungarotoxin, immobilized on a streptavidin-coated gold film, binds nicotinic acetylcholine receptor both in detergent-solubilized and in lipid vesicle-reconstituted form with high specificity. In the latter case, nonspecific binding to the sensor surface is significantly reduced by reconstituting the receptor into poly(ethylene glycol)-lipid-containing sterically stabilized vesicles. By preincubation of a bulk nicotinic acetylcholine receptor sample with the competing ligands carbamoylcholine and decamethonium bromide, the subsequent specific binding of the receptor to the surface-immobilized bungarotoxin is reduced, depending on the concentration of competing ligand. This competition assay allows the determination of the dissociation constants of the acetylcholine receptor-carbamoylcholine complex. A K(D) = 3.5 × 10(-)(6) M for the detergent-solubilized receptor and a K(D) = 1.4 × 10(-)(5) M for the lipid vesicle-reconstituted receptor are obtained. For decamethonium bromide, a K(D) = 4.5 × 10(-)(5) M is determined for the detergent-solubilized receptor. This approach is of general importance for investigating ligand-receptor interactions in case of small ligand molecules by mass-sensitive techniques.

  18. Developmental regulation of nicotinic acetylcholine receptors within midbrain dopamine neurons

    PubMed Central

    Azam, Layla; Chen, Yiling; Leslie, Frances M.

    2007-01-01

    We have combined anatomical and functional methodologies to provide a comprehensive analysis of the properties of nicotinic acetylcholine receptors (nAChRs) on developing dopamine (DA) neurons. Double-labeling in situ hybridization was used to examine the expression of nAChR subunit mRNAs within developing midbrain DA neurons. As brain maturation progressed there was a change in the pattern of subunit mRNA expression within DA neurons, such that α3 and α4 subunits declined and α6 mRNA increased. Although there were strong similarities in subunit mRNA expression in substantia nigra (SNc) and ventral tegmental area (VTA), there was higher expression of α4 mRNA in SNc than VTA at gestational day (G)15, and of α5, α6 and β3 mRNAs during postnatal development. Using a superfusion neurotransmitter release paradigm to functionally characterize nicotine-stimulated release of [3H]DA from striatal slices, the properties of the nAChRs on DA terminals were also found to change with age. Functional nAChRs were detected on striatal terminals at G18. There was a decrease in maximal release in the first postnatal week, followed by an increase in nicotine efficacy and potency during the second and third postnatal weeks. In the transition from adolescence (postnatal days (P) 30 and 40) to adulthood, there was a complex pattern of functional maturation of nAChRs in ventral, but not dorsal, striatum. In males, but not females, there were significant changes in both nicotine potency and efficacy during this developmental period. These findings suggest that nAChRs may play critical functional roles throughout DA neuronal maturation. PMID:17197101

  19. Two subsites in the binding domain of the acetylcholine receptor: an aromatic subsite and a proline subsite.

    PubMed

    Kachalsky, S G; Jensen, B S; Barchan, D; Fuchs, S

    1995-11-07

    The ligand binding site of the nicotinic acetylcholine receptor (AcChoR) is localized in the alpha-subunit within a domain containing the tandem Cys-192 and -193. By analyzing the binding-site region of AcChoR from animal species that are resistant to alpha-neurotoxins, we have previously shown that four residues in this region, at positions 187, 189, 194, and 197, differ between animals sensitive (e.g., mouse) and resistant (e.g., mongoose and snake) to alpha-bungarotoxin (alpha-BTX). In the present study, we performed site-directed mutagenesis on a fragment of the mongoose AcChoR alpha-subunit (residues 122-205) and exchanged residues 187, 189, 194, and 197, either alone or in combination, with those present in the mouse alpha-subunit sequence. Only the mongoose fragment in which all four residues were mutated to the mouse ones exhibited alpha-BTX binding similar to that of the mouse fragment. The mongoose double mutation in which Leu-194 and His-197 were replaced with proline residues, which are present at these positions in the mouse AcChoR and in all other toxin binders, bound alpha-BTX to approximately 60% of the level of binding exhibited by the mouse fragment. In addition, replacement of either Pro-194 or -197 in the mouse fragment with serine and histidine, respectively, markedly decreased alpha-BTX binding. All other mutations resulted in no or just a small increase in alpha-BTX binding. These results have led us to propose two subsites in the binding domain for alpha-BTX: the proline subsite, which includes Pro-194 and -197 and is critical for alpha-BTX binding, and the aromatic subsite, which includes amino acid residues 187 and 189 and determines the extent of alpha-BTX binding.

  20. Two subsites in the binding domain of the acetylcholine receptor: an aromatic subsite and a proline subsite.

    PubMed Central

    Kachalsky, S G; Jensen, B S; Barchan, D; Fuchs, S

    1995-01-01

    The ligand binding site of the nicotinic acetylcholine receptor (AcChoR) is localized in the alpha-subunit within a domain containing the tandem Cys-192 and -193. By analyzing the binding-site region of AcChoR from animal species that are resistant to alpha-neurotoxins, we have previously shown that four residues in this region, at positions 187, 189, 194, and 197, differ between animals sensitive (e.g., mouse) and resistant (e.g., mongoose and snake) to alpha-bungarotoxin (alpha-BTX). In the present study, we performed site-directed mutagenesis on a fragment of the mongoose AcChoR alpha-subunit (residues 122-205) and exchanged residues 187, 189, 194, and 197, either alone or in combination, with those present in the mouse alpha-subunit sequence. Only the mongoose fragment in which all four residues were mutated to the mouse ones exhibited alpha-BTX binding similar to that of the mouse fragment. The mongoose double mutation in which Leu-194 and His-197 were replaced with proline residues, which are present at these positions in the mouse AcChoR and in all other toxin binders, bound alpha-BTX to approximately 60% of the level of binding exhibited by the mouse fragment. In addition, replacement of either Pro-194 or -197 in the mouse fragment with serine and histidine, respectively, markedly decreased alpha-BTX binding. All other mutations resulted in no or just a small increase in alpha-BTX binding. These results have led us to propose two subsites in the binding domain for alpha-BTX: the proline subsite, which includes Pro-194 and -197 and is critical for alpha-BTX binding, and the aromatic subsite, which includes amino acid residues 187 and 189 and determines the extent of alpha-BTX binding. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7479887

  1. Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist.

    PubMed

    Haga, Kazuko; Kruse, Andrew C; Asada, Hidetsugu; Yurugi-Kobayashi, Takami; Shiroishi, Mitsunori; Zhang, Cheng; Weis, William I; Okada, Tetsuji; Kobilka, Brian K; Haga, Tatsuya; Kobayashi, Takuya

    2012-01-25

    The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.

  2. Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist

    SciTech Connect

    Haga, Kazuko; Kruse, Andrew C.; Asada, Hidetsugu; Yurugi-Kobayashi, Takami; Shiroishi, Mitsunori; Zhang, Cheng; Weis, William I.; Okada, Tetsuji; Kobilka, Brian K.; Haga, Tatsuya; Kobayashi, Takuya

    2012-03-15

    The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.

  3. Subunit profiling and functional characteristics of acetylcholine receptors in GT1-7 cells.

    PubMed

    Arai, Yuki; Ishii, Hirotaka; Kobayashi, Makito; Ozawa, Hitoshi

    2017-03-01

    GnRH neurons form a final common pathway for the central regulation of reproduction. Although the involvement of acetylcholine in GnRH secretion has been reported, direct effects of acetylcholine and expression profiles of acetylcholine receptors (AChRs) still remain to be studied. Using immortalized GnRH neurons (GT1-7 cells), we analyzed molecular expression and functionality of AChRs. Expression of the mRNAs were identified in the order α7 > β2 = β1 ≧ α4 ≧ α5 = β4 = δ > α3 for nicotinic acetylcholine receptor (nAChR) subunits and m4 > m2 for muscarinic acetylcholine receptor (mAChR) subtypes. Furthermore, this study revealed that α7 nAChRs contributed to Ca(2+) influx and GnRH release and that m2 and m4 mAChRs inhibited forskolin-induced cAMP production and isobutylmethylxanthine-induced GnRH secretion. These findings demonstrate the molecular profiles of AChRs, which directly contribute to GnRH secretion in GT1-7 cells, and provide one possible regulatory action of acetylcholine in GnRH neurons.

  4. Interaction of dinotefuran and its analogues with nicotinic acetylcholine receptors of cockroach nerve cords.

    PubMed

    Mori, Kazuki; Okumoto, Takashi; Kawahara, Nobuyuki; Ozoe, Yoshihisa

    2002-02-01

    To investigate the action of dinotefuran (MTI-446, 1-methyl-2-nitro-3-(tetrahydro-3-furylmethyl)guanidine), a recently developed insecticide, on insect nicotinic acetylcholine receptors (nAChRs), we determined the potencies of the compound and 15 analogues in inhibiting the specific binding of [3H]epibatidine (EPI), a nAChR agonist, and [3H]alpha-bungarotoxin (alpha-BGT), a competitive nAChR antagonist, to the nerve cord membranes of American cockroaches (Periplaneta americana). Racemic dinotefuran inhibited [3H]EPI binding with an IC50 of 890 nM and [3H]alpha-BGT binding with an IC50 of 36.1 microM. Scatchard analysis indicated that the dinotefuran inhibition of [3H]EPI binding was a competitive one. Slight structural modification caused a drastic reduction in potency; only four analogues were found to be equipotent to or more potent than dinotefuran. Chloropyridinyl and chlorothiazolyl neonicotinoid insecticides displayed two or three orders of magnitude higher potency than dinotefuran. There was a good correlation between the IC50 values of tested compounds obtained with [3H]EPI and those obtained with [3H]alpha-BGT. A better correlation was observed between 3-h knockdown activities (KD50) against German cockroaches (Blattella germanica) and IC50 values obtained from [3H]EPI assays than between 24-h lethal activities (LD50) and IC50 values. While the results indicate that dinotefuran and its analogues interact with the ACh-binding site in cockroach nAChRs, it remains to be elucidated why they displayed lower potencies than those expected based on their insecticidal activities.

  5. Glutamatergic contributions to nicotinic acetylcholine receptor agonist-evoked cholinergic transients in the prefrontal cortex.

    PubMed

    Parikh, Vinay; Man, Kingson; Decker, Michael W; Sarter, Martin

    2008-04-02

    Because modulation of cortical cholinergic neurotransmission has been hypothesized to represent a necessary mechanism mediating the beneficial cognitive effects of nicotine and nicotinic acetylcholine receptor (nAChR) subtype-selective agonists, we used choline-sensitive microelectrodes for the real-time measurement of ACh release in vivo, to characterize cholinergic transients evoked by nicotine and the alpha4beta2*-selective nAChR partial agonist 2-methyl-3-(2-(S)-pyrrolindinylmethoxy)pyridine dihydrochloride (ABT-089), a clinically effective cognition enhancer. In terms of cholinergic signal amplitudes, ABT-089 was significantly more potent than nicotine in evoking ACh cholinergic transients. Moreover, cholinergic signals evoked by ABT-089 were characterized by faster signal rise time and decay rate. The nAChR antagonist mecamylamine attenuated the cholinergic signals evoked by either compound. Cholinergic signals evoked by ABT-089 were more efficaciously attenuated by the relatively beta2*-selective nAChR antagonist dihydro-beta-erythroidine. The alpha7 antagonist methyllycaconitine did not affect choline signal amplitudes but partly attenuated the relatively slow decay rate of nicotine-evoked cholinergic signals. Furthermore, the AMPA receptor antagonist DNQX as well as the NMDA receptor antagonist APV more potently attenuated cholinergic signals evoked by ABT-089. Using glutamate-sensitive microelectrodes to measure glutamatergic transients, ABT-089 was more potent than nicotine in evoking glutamate release. Glutamatergic signals were highly sensitive to tetrodotoxin-induced blockade of voltage-regulated sodium channels. Together, the present evidence indicates that compared with nicotine, ABT-089 evokes more potent and sharper cholinergic transients in prefrontal cortex. Glutamatergic mechanisms necessarily mediate the cholinergic effects of nAChR agonists in the prefrontal cortex.

  6. Acetylcholine receptor pathway mutations explain various fetal akinesia deformation sequence disorders.

    PubMed

    Michalk, Anne; Stricker, Sigmar; Becker, Jutta; Rupps, Rosemarie; Pantzar, Tapio; Miertus, Jan; Botta, Giovanni; Naretto, Valeria G; Janetzki, Catrin; Yaqoob, Nausheen; Ott, Claus-Eric; Seelow, Dominik; Wieczorek, Dagmar; Fiebig, Britta; Wirth, Brunhilde; Hoopmann, Markus; Walther, Marisa; Körber, Friederike; Blankenburg, Markus; Mundlos, Stefan; Heller, Raoul; Hoffmann, Katrin

    2008-02-01

    Impaired fetal movement causes malformations, summarized as fetal akinesia deformation sequence (FADS), and is triggered by environmental and genetic factors. Acetylcholine receptor (AChR) components are suspects because mutations in the fetally expressed gamma subunit (CHRNG) of AChR were found in two FADS disorders, lethal multiple pterygium syndrome (LMPS) and Escobar syndrome. Other AChR subunits alpha1, beta1, and delta (CHRNA1, CHRNB1, CHRND) as well as receptor-associated protein of the synapse (RAPSN) previously revealed missense or compound nonsense-missense mutations in viable congenital myasthenic syndrome; lethality of homozygous null mutations was predicted but never shown. We provide the first report to our knowledge of homozygous nonsense mutations in CHRNA1 and CHRND and show that they were lethal, whereas novel recessive missense mutations in RAPSN caused a severe but not necessarily lethal phenotype. To elucidate disease-associated malformations such as frequent abortions, fetal edema, cystic hygroma, or cardiac defects, we studied Chrna1, Chrnb1, Chrnd, Chrng, and Rapsn in mouse embryos and found expression in skeletal muscles but also in early somite development. This indicates that early developmental defects might be due to somite expression in addition to solely muscle-specific effects. We conclude that complete or severe functional disruption of fetal AChR causes lethal multiple pterygium syndrome whereas milder alterations result in fetal hypokinesia with inborn contractures or a myasthenic syndrome later in life.

  7. Modelling and simulation of ion channels: applications to the nicotinic acetylcholine receptor.

    PubMed

    Sansom, M S; Adcock, C; Smith, G R

    1998-01-01

    Molecular dynamics simulations with experimentally derived restraints have been used to develop atomic models of M2 helix bundles forming the pore-lining domains of the nicotinic acetylcholine receptor and related ligand-gated ion channels. M2 helix bundles have been used in microscopic simulations of the dynamics and energetics of water and ions within an ion channel. Translational and rotational motion of water are restricted within the pore, and water dipoles are aligned relative to the pore axis by the surrounding helix dipoles. Potential energy profiles for translation of a Na+ ion along the pore suggest that the protein and water components of the interaction energy exert an opposing effect on the ion, resulting in a relatively flat profile which favors cation permeation. Empirical conductance calculations based on a pore radius profile suggest that the M2 helix model is consistent with a single channel conductance of ca. 50 pS. Continuum electrostatics calculations indicate that a ring of glutamate residues at the cytoplasmic mouth of the alpha 7 nicotinic receptor M2 helix bundle may not be fully ionized. A simplified model of the remainder of the channel protein when added to the M2 helix bundle plays a significant role in enhancing the ion selectivity of the channel.

  8. Substituted 2-Aminopyrimidines Selective for α7-Nicotinic Acetylcholine Receptor Activation and Association with Acetylcholine Binding Proteins.

    PubMed

    Kaczanowska, Katarzyna; Camacho Hernandez, Gisela Andrea; Bendiks, Larissa; Kohs, Larissa; Cornejo-Bravo, Jose Manuel; Harel, Michal; Finn, M G; Taylor, Palmer

    2017-03-15

    Through studies with ligand binding to the acetylcholine binding protein (AChBP), we previously identified a series of 4,6-substituted 2-aminopyrimidines that associate with this soluble surrogate of the nicotinic acetylcholine receptor (nAChR) in a cooperative fashion, not seen for classical nicotinic agonists and antagonists. To examine receptor interactions of this structural family on ligand-gated ion channels, we employed HEK cells transfected with cDNAs encoding three requisite receptor subtypes: α7-nAChR, α4β2-nAChR, and a serotonin receptor (5-HT3AR), along with a fluorescent reporter. Initial screening of a series of over 50 newly characterized 2-aminopyrimidines with affinity for AChBP showed only two to be agonists on the α7-nAChR below 10 μM concentration. Their unique structural features were incorporated into design of a second subset of 2-aminopyrimidines yielding several congeners that elicited α7 activation with EC50 values of 70 nM and Kd values for AChBP in a similar range. Several compounds within this series exhibit specificity for the α7-nAChR, showing no activation or antagonism of α4β2-nAChR or 5-HT3AR at concentrations up to 10 μM, while others were weaker antagonists (or partial agonists) on these receptors. Analysis following cocrystallization of four ligand complexes with AChBP show binding at the subunit interface, but with an orientation or binding pose that differs from classical nicotinic agonists and antagonists and from the previously analyzed set of 2-aminopyrimidines that displayed distinct cooperative interactions with AChBP. Orientations of aromatic side chains of these complexes are distinctive, suggesting new modes of binding at the agonist-antagonist site and perhaps an allosteric action for heteromeric nAChRs.

  9. Theoretical investigation of interaction between the set of ligands and α7 nicotinic acetylcholine receptor

    NASA Astrophysics Data System (ADS)

    Glukhova, O. E.; Prytkova, T. R.; Shmygin, D. S.

    2016-03-01

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

  10. Unique pharmacology of heteromeric α7β2 nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes.

    PubMed

    Zwart, Ruud; Strotton, Merrick; Ching, Jennifer; Astles, Peter C; Sher, Emanuele

    2014-03-05

    α7β2 is a novel type of nicotinic acetylcholine receptor shown to be uniquely expressed in cholinergic neurons of the basal forebrain and in hippocampal interneurons. We have compared the pharmacological properties of recombinant homomeric α7 and heteromeric α7β2 nicotinic acetylcholine receptors in order to reveal the pharmacological consequences of β2 subunit incorporation into the pentamer. The non-selective agonist epibatidine did not distinguish α7β2 from α7 nicotinic acetylcholine receptors, but three other non-selective agonists (nicotine, cytisine and varenicline) were less efficacious on α7β2 than on α7. A more dramatic change in efficacy was seen with eight different selective α7 agonists. Because of their very low intrinsic efficacy, some compounds became very efficacious functional antagonists at α7β2 receptors. Three α4β2 nicotinic receptor selective agonists that were not active on α7, were also inactive on α7β2, and dihydro-β-erythroidine, an α4β2 receptor-preferring antagonist, inhibited α7 and α7β2 in a similar manner. These results reveal significant effects of β2 incorporation in determining the relative efficacy of several non-selective and α7 selective agonists, and also show that incorporation of β2 subunits does not cause a shift to a more “β2-like” pharmacology of α7 nicotinic acetylcholine receptors.

  11. Auto/paracrine control of inflammatory cytokines by acetylcholine in macrophage-like U937 cells through nicotinic receptors.

    PubMed

    Chernyavsky, Alexander I; Arredondo, Juan; Skok, Maryna; Grando, Sergei A

    2010-03-01

    Although acetylcholine (ACh) is well known for its neurotransmitter function, recent studies have indicated that it also functions as an immune cytokine that prevents macrophage activation through a 'cholinergic (nicotinic) anti-inflammatory pathway'. In this study, we used the macrophage-like U937 cells to elucidate the mechanisms of the physiologic control of cytokine production by auto/paracrine ACh through the nicotinic class of ACh receptors (nAChRs) expressed in these cells. Stimulation of cells with lipopolysaccharide up-regulated expression of alpha1, alpha4, alpha5, alpha7, alpha10, beta1 and beta3 subunits, down-regulated alpha6 and beta2 subunits, and did not alter the relative quantity of alpha9 and beta4 mRNAs. Distinct nAChR subtypes showed differential regulation of the production of pro- and anti-inflammatory cytokines. While inhibition of the expression of the TNF-alpha gene was mediated predominantly by the alpha-bungarotoxin sensitive nAChRs, that of the IL-6 and IL-18 genes-by the mecamylamine-sensitive nAChRs. Both the Mec- and alphaBtx-sensitive nAChRs regulated expression of the IL-1beta gene equally efficiently. Upregulation of IL-10 production by auto/paracrine ACh was mediated predominantly through alpha7 nAChR. These findings offer a new insight on how nicotinic agonists control inflammation, thus laying a groundwork for the development of novel immunomodulatory therapies based on the nAChR subtype selectivity of nicotinic agonists.

  12. Recent Duplication and Functional Divergence in Parasitic Nematode Levamisole-Sensitive Acetylcholine Receptors

    PubMed Central

    Duguet, Thomas B.; Charvet, Claude L.; Forrester, Sean G.; Wever, Claudia M.; Dent, Joseph A.; Neveu, Cedric; Beech, Robin N.

    2016-01-01

    Helminth parasites rely on fast-synaptic transmission in their neuromusculature to experience the outside world and respond to it. Acetylcholine plays a pivotal role in this and its receptors are targeted by a wide variety of both natural and synthetic compounds used in human health and for the control of parasitic disease. The model, Caenorhabditis elegans is characterized by a large number of acetylcholine receptor subunit genes, a feature shared across the nematodes. This dynamic family is characterized by both gene duplication and loss between species. The pentameric levamisole-sensitive acetylcholine receptor has been characterized from C. elegans, comprised of five different subunits. More recently, cognate receptors have been reconstituted from multiple parasitic nematodes that are found to vary in subunit composition. In order to understand the implications of receptor composition change and the origins of potentially novel drug targets, we investigated a specific example of subunit duplication based on analysis of genome data for 25 species from the 50 helminth genome initiative. We found multiple independent duplications of the unc-29, acetylcholine receptor subunit, where codon substitution rate analysis identified positive, directional selection acting on amino acid positions associated with subunit assembly. Characterization of four gene copies from a model parasitic nematode, Haemonchus contortus, demonstrated that each copy has acquired unique functional characteristics based on phenotype rescue of transgenic C. elegans and electrophysiology of receptors reconstituted in Xenopus oocytes. We found evidence that a specific incompatibility has evolved for two subunits co-expressed in muscle. We demonstrated that functional divergence of acetylcholine receptors, driven by directional selection, can occur more rapidly than previously thought and may be mediated by alteration of receptor assembly. This phenomenon is common among the clade V parasitic

  13. Cellular approaches to the interaction between cannabinoid receptor ligands and nicotinic acetylcholine receptors.

    PubMed

    Oz, Murat; Al Kury, Lina; Keun-Hang, Susan Yang; Mahgoub, Mohamed; Galadari, Sehamuddin

    2014-05-15

    Cannabinoids are among the earliest known drugs to humanity. Cannabis plant contains various phytochemicals that bind to cannabinoid receptors. In addition, synthetic and endogenously produced cannabinoids (endocannabinoids) constitute other classes of cannabinoid receptor ligands. Although many pharmacological effects of these cannabinoids are mediated by the activation of cannabinoid receptors, recent studies indicate that cannabinoids also modulate the functions of various integral membrane proteins including ion channels, receptors, neurotransmitter transporters, and enzymes by mechanism(s) not involving the activation of known cannabinoid receptors. Currently, the mechanisms of these effects were not fully understood. However, it is likely that direct actions of cannabinoids are closely linked to their lipophilic structures. This report will focus on the actions of cannabinoids on nicotinic acetylcholine receptors and will examine the results of recent studies in this field. In addition some mechanistic approaches will be provided. The results discussed in this review indicate that, besides cannabinoid receptors, further molecular targets for cannabinoids exist and that these targets may represent important novel sites to alter neuronal excitability.

  14. Functional Characterization of a Novel Class of Morantel-Sensitive Acetylcholine Receptors in Nematodes

    PubMed Central

    Courtot, Elise; Charvet, Claude L.; Beech, Robin N.; Harmache, Abdallah; Wolstenholme, Adrian J.; Holden-Dye, Lindy; O’Connor, Vincent; Peineau, Nicolas; Woods, Debra J.; Neveu, Cedric

    2015-01-01

    Acetylcholine receptors are pentameric ligand–gated channels involved in excitatory neuro-transmission in both vertebrates and invertebrates. In nematodes, they represent major targets for cholinergic agonist or antagonist anthelmintic drugs. Despite the large diversity of acetylcholine-receptor subunit genes present in nematodes, only a few receptor subtypes have been characterized so far. Interestingly, parasitic nematodes affecting human or animal health possess two closely related members of this gene family, acr-26 and acr-27 that are essentially absent in free-living or plant parasitic species. Using the pathogenic parasitic nematode of ruminants, Haemonchus contortus, as a model, we found that Hco-ACR-26 and Hco-ACR-27 are co-expressed in body muscle cells. We demonstrated that co-expression of Hco-ACR-26 and Hco-ACR-27 in Xenopus laevis oocytes led to the functional expression of an acetylcholine-receptor highly sensitive to the anthelmintics morantel and pyrantel. Importantly we also reported that ACR-26 and ACR-27, from the distantly related parasitic nematode of horses, Parascaris equorum, also formed a functional acetylcholine-receptor highly sensitive to these two drugs. In Caenorhabditis elegans, a free-living model nematode, we demonstrated that heterologous expression of the H. contortus and P. equorum receptors drastically increased its sensitivity to morantel and pyrantel, mirroring the pharmacological properties observed in Xenopus oocytes. Our results are the first to describe significant molecular determinants of a novel class of nematode body wall muscle AChR. PMID:26625142

  15. Muscarinic acetylcholine receptor X-ray structures: potential implications for drug development.

    PubMed

    Kruse, Andrew C; Hu, Jianxin; Kobilka, Brian K; Wess, Jürgen

    2014-06-01

    Muscarinic acetylcholine receptor antagonists are widely used as bronchodilating drugs in pulmonary medicine. The therapeutic efficacy of these agents depends on the blockade of M3 muscarinic receptors expressed on airway smooth muscle cells. All muscarinic antagonists currently used as bronchodilating agents show high affinity for all five muscarinic receptor subtypes, thus increasing the likelihood of unwanted side effects. Recent X-ray crystallographic studies have provided detailed structural information about the nature of the orthosteric muscarinic binding site (the conventional acetylcholine binding site) and an 'outer' receptor cavity that can bind allosteric (non-orthosteric) drugs. These new findings should guide the development of selective M3 receptor blockers that have little or no effect on other muscarinic receptor subtypes.

  16. Development of a photoactivatable allosteric ligand for the m1 muscarinic acetylcholine receptor.

    PubMed

    Davie, Briana J; Sexton, Patrick M; Capuano, Ben; Christopoulos, Arthur; Scammells, Peter J

    2014-10-15

    The field of G protein-coupled receptor drug discovery has benefited greatly from the structural and functional insights afforded by photoactivatable ligands. One G protein-coupled receptor subfamily for which photoactivatable ligands have been developed is the muscarinic acetylcholine receptor family, though, to date, all such ligands have been designed to target the orthosteric (endogenous ligand) binding site of these receptors. Herein we report the synthesis and pharmacological investigation of a novel photoaffinity label, MIPS1455 (4), designed to bind irreversibly to an allosteric site of the M1 muscarinic acetylcholine receptor; a target of therapeutic interest for the treatment of cognitive deficits. MIPS1455 may be a valuable molecular tool for further investigating allosteric interactions at this receptor.

  17. Inhibition of human α7 nicotinic acetylcholine receptors by cyclic monoterpene carveol.

    PubMed

    Lozon, Yosra; Sultan, Ahmed; Lansdell, Stuart J; Prytkova, Tatiana; Sadek, Bassem; Yang, Keun-Hang Susan; Howarth, Frank Christopher; Millar, Neil S; Oz, Murat

    2016-04-05

    Cyclic monoterpenes are a group of phytochemicals with antinociceptive, local anesthetic, and anti-inflammatory actions. Effects of cyclic monoterpenes including vanilin, pulegone, eugenole, carvone, carvacrol, carveol, thymol, thymoquinone, menthone, and limonene were investigated on the functional properties of the cloned α7 subunit of the human nicotinic acetylcholine receptor expressed in Xenopus oocytes. Monoterpenes inhibited the α7 nicotinic acetylcholine receptor in the order carveol>thymoquinone>carvacrol>menthone>thymol>limonene>eugenole>pulegone≥carvone≥vanilin. Among the monoterpenes, carveol showed the highest potency on acetylcholine-induced responses, with IC50 of 8.3µM. Carveol-induced inhibition was independent of the membrane potential and could not be reversed by increasing the concentration of acetylcholine. In line with functional experiments, docking studies indicated that cyclic monoterpenes such as carveol may interact with an allosteric site located in the α7 transmembrane domain. Our results indicate that cyclic monoterpenes inhibit the function of human α7 nicotinic acetylcholine receptors, with varying potencies.

  18. The nicotinic acetylcholine receptor: Binding of nitroxide analogs of a local anesthetic and a photoactivatable analog of phosphatidylserine

    SciTech Connect

    Blanton, M.P.

    1989-01-01

    Electron spin resonance was used to contrast the accessibility of tertiary and quaternary amine local anesthetics to their high affinity binding site in the desensitized Torpedo californica acetylcholine receptor (AchR). Preincubation of AchR-rich membranes with agonist resulted in a substantial reduction in the initial association of the quaternary amine local anesthetic C6SLMEI with the receptor. The time-dependent reduction in association follows a biphasic exponential function having rate constants of 0.19 min{sup {minus}1} and 0.03 min{sup {minus}1}. In contrast, agonist preincubation did not produce a comparable decrease in the association of C6SL, a tertiary amine analog, with the AchR. The results are modeled in two ways: (1) A charge gate near the channel mouth in the desensitized receptor limits access of the permanently charged cationic local anesthetic (C6SLMEI), but not for the uncharged form of the tertiary amine anesthetic C6SL. (2) A hydrophobic pathway, possibly through a corridor in the annular lipid surrounding receptor subunits, allows the uncharged form of C6SL to reach the high affinity binding site in the AchR. A photoactivatable analog of phosphatidylserine {sup 125}I 4-azido salicylic acid-phosphatidylserine ({sup 125}I ASA-PS) was use to label both Torpedo californica acetylcholine receptor-rich membranes and reconstituted AchR membranes. All four subunits of the AchR were found to incorporate label, with the {alpha} subunit incorporating approximately twice as much as each of the other subunits on a per mole basis. The regions of the AchR {alpha} subunit that incorporate {sup 125}I ASA-PS were mapped by Staphylococcus aureus V8 protease digestion. Eighty-one per cent of the incorporated label was localized to 11.7 and 10.1 kdal V8 cleavage fragments.

  19. Ligand-binding properties of an unusual nicotinic acetylcholine receptor subtype on isolated outer hair cells from guinea pig cochlea.

    PubMed

    Lawoko, G; Järlebark, L; Heilbronn, E

    1995-07-28

    Acetylcholine receptors on isolated guinea pig cochlear outer hair cells (OHC) were characterized by radioligand binding. Equilibrium binding of [125I]alpha-bungarotoxin revealed a KD of 62 +/- 2 nM, Bmax = 7.2 +/- 1.8 x 10(7) binding sites/OHC, and a slowly reversible dissociation rate constant, kappa-1 = 2.2 +/- 0.01 x 10(-4) min-1. L-[3H]Nicotine bound reversibly (estimated KD approximately 230 nM and Bmax approximately 5 x 10(7)) with kinetic rate constants of association kappa-1 = 6.2 +/- 0.06 x 10(4) min-1 nM-1 and dissociation kappa-1 = 0.23 +/- 0.003 min-1. [3H]Strychnine bound to OHC with a KD of 35 +/- 6 nM and Bmax = 2.6 +/- 0.5 x 10(7), and binding increased 3-4 fold after membrane depolarization with 56.2 mM [K+], suggesting additional binding sites. Binding, seen only at > nM concentrations, of [3H]3-quinuclidinyl benzilate (KD = 11.5 +/- 5 nM; Bmax = 2.5 +/- 0.6 x 10(6)) was competitively inhibited by the muscarinic antagonists atropine and 4-DAMP (IC50 of 6.1 +/- 0.5 and 6.5 +/- 0.4 nM). The OHC receptor is thus an atypical nicotinic acetylcholine receptor subtype with unusual pharmacological properties.

  20. Development of the electromotor system of Torpedo marmorata: distribution of extracellular matrix and cytoskeletal components during acetylcholine receptor focalization.

    PubMed

    Richardson, G P; Fiedler, W; Fox, G Q

    1987-03-01

    A combination of direct fluorescence and indirect immunofluorescence microscopy has been used to compare the distribution of the acetylcholine receptor with the distribution of major cytoskeletal and extracellular matrix components during electrocyte differentiation in the electric organs of Torpedo marmorata. Laminin, fibronectin and extracellular matrix proteoglycan are always more extensively distributed around the differentiating cell than the acetylcholine receptor-rich patch that forms on the ventral surface of the cell. The distribution of acetylcholinesterase within the ventral surface of the differentiating electrocyte closely resembles the distribution of the acetylcholine receptor. Areas of apparently high acetylcholine receptor density within the ventrally forming acetylcholine receptor-rich patch are always areas of apparently high extracellular matrix proteoglycan density but are not always areas of high laminin or fibronectin density. Desmin levels appear to increase at the onset of differentiation and desmin initially accumulates in the ventral pole of each myotube as it begins to form an electrocyte. During differentiation F-actin-positive filament bundles are observed that extend from the nuclei down to the ventrally forming acetylcholine receptor-rich patch. Most filament bundles terminate in the acetylcholine receptor-rich region of the cell membrane. Electron-microscopic autoradiography suggests that the filament bundles attach to the membrane at sites where small acetylcholine receptor clusters are found. The results of this study suggest that, out of the four extracellular matrix components studied, only the distribution of acetylcholinesterase (which may be both matrix- and membrane-bound at this stage) closely parallels that of the acetylcholine receptor, and that F-actin filament bundles terminate in a region of the cell that is becoming an area of high acetylcholine receptor density.

  1. Nootropic alpha7 nicotinic receptor allosteric modulator derived from GABAA receptor modulators.

    PubMed

    Ng, Herman J; Whittemore, Edward R; Tran, Minhtam B; Hogenkamp, Derk J; Broide, Ron S; Johnstone, Timothy B; Zheng, Lijun; Stevens, Karen E; Gee, Kelvin W

    2007-05-08

    Activation of brain alpha7 nicotinic acetylcholine receptors (alpha7 nAChRs) has broad therapeutic potential in CNS diseases related to cognitive dysfunction, including Alzheimer's disease and schizophrenia. In contrast to direct agonist activation, positive allosteric modulation of alpha7 nAChRs would deliver the clinically validated benefits of allosterism to these indications. We have generated a selective alpha7 nAChR-positive allosteric modulator (PAM) from a library of GABAA receptor PAMs. Compound 6 (N-(4-chlorophenyl)-alpha-[[(4-chloro-phenyl)amino]methylene]-3-methyl-5-isoxazoleacet-amide) evokes robust positive modulation of agonist-induced currents at alpha7 nAChRs, while preserving the rapid native characteristics of desensitization, and has little to no efficacy at other ligand-gated ion channels. In rodent models, it corrects sensory-gating deficits and improves working memory, effects consistent with cognitive enhancement. Compound 6 represents a chemotype for allosteric activation of alpha7 nAChRs, with therapeutic potential in CNS diseases with cognitive dysfunction.

  2. Use of intact rat brain cells as a model to study regulation of muscarinic acetylcholine receptors

    SciTech Connect

    Lee, J.H.; El-Fakahany, E.E.

    1985-08-12

    Intact rat brain cells were dissociated and used to study the regulation of muscarinic acetylcholine receptors upon exposure to muscarinic receptor agonists. Incubation of cells with carbamylcholine resulted in a time-dependent decrease in subsequent (/sup 3/H)N-methylscopolamine specific binding, an effect which reached a steady state after 3 hr at 37/sup 0/C. This effect of carbamylcholine was dependent on the concentration of the agonist in the incubation medium and was due to a reduction in the maximal binding capacity of the receptor with no decrease in the affinity of the remaining receptors. This preparation might be useful in future studies to elucidate the mechanisms underlying the regulation of muscarinic acetylcholine receptors in the central nervous system. 20 references, 3 tables.

  3. Binding of /sup 3/H-acetylcholine to cholinergic receptors in bovine cerebral arteries

    SciTech Connect

    Shimohama, S.; Tsukahara, T.; Taniguchi, T.; Fujiwara, M.

    1985-11-18

    Cholinergic receptor sites in bovine cerebral arteries were analyzed using radioligand binding techniques with the cholinergic agonist, /sup 3/H-acetylcholine (ACh), as the ligand. Specific binding of /sup 3/H-ACh to membrane preparations of bovine cerebral arteries was saturable, of two binding sites, with dissociation constant (K/sub D/) values of 0.32 and 23.7 nM, and maximum binding capacity (Bmax) values of 67 and 252 fmol/mg protein, respectively. Specific binding of /sup 3/H-ACh was displaced effectively by muscarinic cholinergic agents and less effectively by nicotinic cholinergic agents. IC/sub 50/ values of cholinergic drugs for /sup 3/H-ACh binding were as follows: atropine, 38.5 nM; ACh, 59.8 nM; oxotremorine, 293 nM; scopolamine 474 nM; carbamylcholine, 990 nM. IC/sub 50/ values of nicotinic cholinergic agents such as nicotine, cytisine and ..cap alpha..-bungarotoxin exceeded 50 ..mu..M. Choline acetyltransferase activity was 1.09 nmol/mg protein/hour in the cerebral arteries. These findings suggest that the cholinergic nerves innervate the bovine cerebral arteries and that there are at least two classes of ACh binding sites of different affinities on muscarinic reporters in these arteries. 18 references, 2 figures, 2 tables.

  4. Suppression of ongoing experimental myasthenia by oral treatment with an acetylcholine receptor recombinant fragment.

    PubMed

    Im, S H; Barchan, D; Fuchs, S; Souroujon, M C

    1999-12-01

    Myasthenia gravis (MG) is an autoimmune disorder in which the nicotinic acetylcholine receptor (AChR) is the major autoantigen. In an attempt to develop an antigen-specific therapy for MG, we administered a nonmyasthenogenic recombinant fragment of AChR orally to rats. This fragment, corresponding to the extracellular domain of the human AChR alpha-subunit (Halpha1-205), protected rats from subsequently induced experimental autoimmune myasthenia gravis (EAMG) and suppressed ongoing EAMG when treatment was initiated during either the acute or chronic phases of disease. Prevention and suppression of EAMG were accompanied by a significant decrease in AChR-specific humoral and cellular responses. The underlying mechanism for the Halpha1-205-induced oral tolerance seems to be active suppression, mediated by a shift from a T-helper 1 (Th1) to a Th2/Th3 response. This shift was assessed by changes in the cytokine profile, a deviation of anti-AChR IgG isotypes from IgG2 to IgG1, and a suppressed AChR-specific delayed-type hypersensitivity response. Our results in experimental myasthenia suggest that oral administration of AChR-specific recombinant fragments may be considered for antigen-specific immunotherapy of myasthenia gravis.

  5. Identification of epitopes within a highly immunogenic region of acetylcholine receptor by a phage epitope library.

    PubMed

    Barchan, D; Balass, M; Souroujon, M C; Katchalski-Katzir, E; Fuchs, S

    1995-11-01

    We have employed a hexapeptide phage-epitope library to identify epitopes for a mAb (mAb 5.14), which is directed to a determinant within a highly immunogenic, cytoplasmic region of the alpha-subunit of acetylcholine receptor (AChR). We have selected two different peptide-presenting phages (SWDDIR-phage and LWILTR-phage) which interact specifically with mAb 5.14. This interaction is specifically inhibited by AChR and by synthetic peptides corresponding to the hexapeptides presented by the selected phages. Although mAb 5.14 binds to AChR in its native as well as its denatured form, the selected hexapeptides do not exist as such in the AChR molecule. However, three amino acid sequence homologies with these hexapeptides were shown to be present in the cytoplasmic region of Torpedo AChR. By extending the selected hexapeptides, at one or both ends, with amino acid residues flanking the hexapeptides in the phage, we obtained mimotopes with an up to two order of magnitude higher affinity to the Ab. These extended peptides were able to efficiently block the binding of mAb 5.14 to both peptide-presenting phages, and to AChR.

  6. Molecular characterisation of nicotinic acetylcholine receptor subunits from the cat flea, Ctenocephalides felis (Siphonaptera: Pulicidae).

    PubMed

    Bass, Chris; Lansdell, Stuart J; Millar, Neil S; Schroeder, Iris; Turberg, Andreas; Field, Linda M; Williamson, Martin S

    2006-01-01

    As part of a program to monitor the susceptibility of cat flea populations to the insecticide imidacloprid we have examined the cat flea nicotinic acetylcholine receptor, the target site protein of the neonicotinoid group of insecticides. Seven nAChR subunits (six alpha-type and one beta-type) were identified in cat flea using a degenerate PCR-based strategy. Five of these were expressed in vitro by creating chimeras containing the N-terminal ligand-binding domain of the cat flea subunits and the C-terminal region of the Drosophila Dalpha2 (SAD) subunit. Two of the five chimeric subunits, Cfalpha1/Dalpha2 and Cfalpha3/Dalpha2, when co-expressed with rat beta2 in Drosophila S2 cells, showed high-affinity binding of both epibatidine (Kd=1.6+/-0.6 and 0.13+/-0.06nM, respectively), and imidacloprid (Ki=142+/-34 and 28.7+/-2.4nM, respectively). It is likely therefore that Cfalpha1 and Cfalpha3 contribute to nAChR populations in vivo that are sensitive to imidacloprid. The identification of cat flea nAChR subunits that have a high affinity for imidacloprid presents candidate genes in which to look for resistance-associated mutations if target-site resistance to imidacloprid arises in domestic pet flea populations.

  7. Structure-function relationships of curaremimetic neurotoxin loop 2 and of a structurally similar segment of rabies virus glycoprotein in their interaction with the nicotinic acetylcholine receptor.

    PubMed

    Lentz, T L

    1991-11-12

    Peptides corresponding to portions of curaremimetic neurotoxin loop 2 and to a structurally similar segment of rabies virus glycoprotein were synthetically modified in order to gain information on structure-function relationships of neurotoxin loop 2 interactions with the acetylcholine receptor. Binding of synthetic peptides to the acetylcholine receptor of Torpedo electric organ membranes was assessed by measuring their ability to inhibit the binding of 125I-alpha-bungarotoxin to the receptor. The peptides showing the highest affinity for the receptor were a peptide corresponding to the sequence of loop 2 (residues 25-44) of Ophiophagus hannah (king cobra) toxin b (IC50 = 5.7 x 10(-6) M) and the structurally similar segment (residues 173-203) of CVS rabies virus glycoprotein (IC50 = 2.6 x 10(-6) M). These affinities were comparable to those of d-tubocurarine (IC50 = 3.4 x 10(-6) M) and suberyldicholine (IC50 = 2.5 x 10(-6) M). These results demonstrate the importance of loop 2 in the neurotoxin interaction with the receptor. N- and C-terminal deletions of the loop 2 peptides and substitution of residues invariant or highly conserved among neurotoxins were performed in order to determine the role of individual residues in binding. Residues 25-40 are the most crucial in the interaction with the acetylcholine receptor. Modifications involving Lys-27, Trp-29, Phe-33, Arg-37, and Gly-38 reduced affinity of binding. R37D and F33T modifications reduced the affinity of alpha-bungarotoxin residues 28-40 by an order of magnitude. Arg-37 may correspond to the positively charged quaternary ammonium group and Phe-33 to the hydrophobic acetyl methyl group of acetylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)

  8. Inhibition of nicotinic acetylcholine receptors, a novel facet in the pleiotropic activities of snake venom phospholipases A2.

    PubMed

    Vulfius, Catherine A; Kasheverov, Igor E; Starkov, Vladislav G; Osipov, Alexey V; Andreeva, Tatyana V; Filkin, Sergey Yu; Gorbacheva, Elena V; Astashev, Maxim E; Tsetlin, Victor I; Utkin, Yuri N

    2014-01-01

    Phospholipases A2 represent the most abundant family of snake venom proteins. They manifest an array of biological activities, which is constantly expanding. We have recently shown that a protein bitanarin, isolated from the venom of the puff adder Bitis arietans and possessing high phospholipolytic activity, interacts with different types of nicotinic acetylcholine receptors and with the acetylcholine-binding protein. To check if this property is characteristic to all venom phospholipases A2, we have studied the capability of these enzymes from other snakes to block the responses of Lymnaea stagnalis neurons to acetylcholine or cytisine and to inhibit α-bungarotoxin binding to nicotinic acetylcholine receptors and acetylcholine-binding proteins. Here we present the evidence that phospholipases A2 from venoms of vipers Vipera ursinii and V. nikolskii, cobra Naja kaouthia, and krait Bungarus fasciatus from different snake families suppress the acetylcholine- or cytisine-elicited currents in L. stagnalis neurons and compete with α-bungarotoxin for binding to muscle- and neuronal α7-types of nicotinic acetylcholine receptor, as well as to acetylcholine-binding proteins. As the phospholipase A2 content in venoms is quite high, under some conditions the activity found may contribute to the deleterious venom effects. The results obtained suggest that the ability to interact with nicotinic acetylcholine receptors may be a general property of snake venom phospholipases A2, which add a new target to the numerous activities of these enzymes.

  9. Effects of the α subunit on imidacloprid sensitivity of recombinant nicotinic acetylcholine receptors

    PubMed Central

    Matsuda, K; Buckingham, S D; Freeman, J C; Squire, M D; Baylis, H A; Sattelle, D B

    1998-01-01

    Imidacloprid is a new insecticide with selective toxicity for insects over vertebrates. Recombinant (α4β2) chicken neuronal nicotinic acetylcholine receptors (AChRs) and a hybrid nicotinic AChR formed by co-expression of a Drosophila melanogaster neuronal α subunit (SAD) with the chicken β2 subunit were heterologously expressed in Xenopus oocytes by nuclear injection of cDNAs. The agonist actions of imidacloprid and other nicotinic AChR ligands ((+)-epibatidine, (−)-nicotine and acetylcholine) were compared on both recombinant nicotinic AChRs by use of two-electrode, voltage-clamp electrophysiology. Imidacloprid alone of the 4 agonists behaved as a partial agonist on the α4β2 receptor; (+)-epibatidine, (−)-nicotine and acetylcholine were all full, or near full, agonists. Imidacloprid was also a partial agonist of the hybrid Drosophila SAD chicken β2 receptor, as was (−)-nicotine, whereas (+)-epibatidine and acetylcholine were full agonists. The EC50 of imidacloprid was decreased by replacing the chicken α4 subunit with the Drosophila SAD α subunit. This α subunit substitution also resulted in an increase in the EC50 for (+)-epibatidine, (−)-nicotine and acetylcholine. Thus, the Drosophila (SAD) α subunit contributes to the greater apparent affinity of imidacloprid for recombinant insect/vertebrate nicotinic AChRs. Imidacloprid acted as a weak antagonist of ACh-mediated responses mediated by SADβ2 hybrid receptors and as a weak potentiator of ACh responses mediated by α4β2 receptors. This suggests that imidacloprid has complex effects upon these recombinant receptors, determined at least in part by the α subunit. PMID:9504393

  10. INHIBITORY EFFECTS OF VOLATILE ORGANIC COMPOUNDS ON NEURONAL NICOTINIC ACETYLCHOLINE RECEPTORS.

    EPA Science Inventory

    INHIBITORY EFFECTS OF VOLATILE ORGANIC COMPOUNDS ON NEURONAL NICOTINIC ACETYLCHOLINE RECEPTORS.
    A.S. Bale*; P.J. Bushnell; C.A. Meacham; T.J. Shafer
    Neurotoxicology Division, NHEERL, ORD, US Environmental Protection Agency, Research Triangle Park, NC, USA
    Toluene (TOL...

  11. Effect of a nicotinic acetylcholine receptor agonists and antagonists on motor function in mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nicotinic acetylcholine receptors (nAChR) are ligand-gated cation channels found throughout the body, and serve to mediate diverse physiological functions. Muscle-type nAChR located in the motor endplate region of muscle fibers play an integral role in muscle contraction and thus motor function. The...

  12. The Nicotinic Acetylcholine Receptor: The Founding Father of the Pentameric Ligand-gated Ion Channel Superfamily*

    PubMed Central

    Changeux, Jean-Pierre

    2012-01-01

    A critical event in the history of biological chemistry was the chemical identification of the first neurotransmitter receptor, the nicotinic acetylcholine receptor. Disciplines as diverse as electrophysiology, pharmacology, and biochemistry joined together in a unified and rational manner with the common goal of successfully identifying the molecular device that converts a chemical signal into an electrical one in the nervous system. The nicotinic receptor has become the founding father of a broad family of pentameric membrane receptors, paving the way for their identification, including that of the GABAA receptors. PMID:23038257

  13. Proteins with epitopes of the acetylcholine receptor in epithelial cell cultures of thymomas in myasthenia gravis.

    PubMed Central

    Marx, A.; Kirchner, T.; Hoppe, F.; O'Connor, R.; Schalke, B.; Tzartos, S.; Müller-Hermelink, H. K.

    1989-01-01

    Thymomas from 12 patients with myasthenia gravis (MG) were investigated for the presence of epitopes of the alpha-subunit of the nicotinic acetylcholine receptor (AchR) using monoclonal antibodies (MAb) reacting against the AchR. In all but two of the tumors epitopes corresponding to antigenic determinants located on the cytoplasmic side of the AchR were identified. From eight thymomas cell lines were established that have been kept in culture for up to 6 months. The cultured cells expressed the same AchR-epitopes as did the primary tumors. During early passages the percentage of epithelial cells positive for the AchR epitopes approximately mirrored the percentage of positive cells in the original tumors. With passaging the relative number of positive cells usually declined but in some cultures an increase was observed. Three cell lines that showed extensive staining with an MAb against the AchR were radiolabeled to characterize the antigen. From protein extracts of these three cell lines proteins of 45 kd and 156 kd molecular weight (MW) were precipitated. These proteins are different from other proteins described in the context of both thymomas and MG. The negative reactivity with MAb against other epitopes of the alpha-subunit, especially against the main immunogenic region (MIR), speaks in favor of membrane-associated proteins of only limited crossreactivity to the AchR. A previous study found an almost exclusive occurrence of these AchR-epitopes in thymomas associated with MG, but not in other thymomas of similar histologic type. The expression of the proteins described here could therefore play a role in the triggering of the autoimmune process against the AchR of the motor, endplate in MG patients. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:2468286

  14. Docking of 6-chloropyridazin-3-yl derivatives active on nicotinic acetylcholine receptors into molluscan acetylcholine binding protein (AChBP).

    PubMed

    Artali, Roberto; Bombieri, Gabriella; Meneghetti, Fiorella

    2005-04-01

    The crystal structure of Acetylcholine Binding Protein (AChBP), homolog of the ligand binding domain of nAChR, has been used as model for computational investigations on the ligand-receptor interactions of derivatives of 6-chloropyridazine substituted at C3 with 3,8-diazabicyclo[3.2.1]octane, 2,5-diazabicyclo[2.2.1]heptane and with piperazine and homopiperazine, substituted or not at N4. The ligand-receptor complexes have been analyzed by docking techniques using the binding site of HEPES complexed with AChBP as template. The good relationship between the observed binding affinity and the calculated docking energy confirms that this model provides a good starting point for understanding the binding domain of neuronal nicotinic receptors. An analysis of the possible factors significant for the ligand recognition has evidenced, besides the cation-pi interaction, the distance between the chlorine atom of the pyridazinyl group and the carbonylic oxygen of Leu B112 as an important parameter in the modulation of the binding energy.

  15. Binding of N-methylscopolamine to the extracellular domain of muscarinic acetylcholine receptors

    PubMed Central

    Jakubík, Jan; Randáková, Alena; Zimčík, Pavel; El-Fakahany, Esam E.; Doležal, Vladimír

    2017-01-01

    Interaction of orthosteric ligands with extracellular domain was described at several aminergic G protein-coupled receptors, including muscarinic acetylcholine receptors. The orthosteric antagonists quinuclidinyl benzilate (QNB) and N-methylscopolamine (NMS) bind to the binding pocket of the muscarinic acetylcholine receptor formed by transmembrane α-helices. We show that high concentrations of either QNB or NMS slow down dissociation of their radiolabeled species from all five subtypes of muscarinic acetylcholine receptors, suggesting allosteric binding. The affinity of NMS at the allosteric site is in the micromolar range for all receptor subtypes. Using molecular modelling of the M2 receptor we found that E172 and E175 in the second extracellular loop and N419 in the third extracellular loop are involved in allosteric binding of NMS. Mutation of these amino acids to alanine decreased affinity of NMS for the allosteric binding site confirming results of molecular modelling. The allosteric binding site of NMS overlaps with the binding site of some allosteric, ectopic and bitopic ligands. Understanding of interactions of NMS at the allosteric binding site is essential for correct analysis of binding and action of these ligands. PMID:28091608

  16. Binding of N-methylscopolamine to the extracellular domain of muscarinic acetylcholine receptors

    NASA Astrophysics Data System (ADS)

    Jakubík, Jan; Randáková, Alena; Zimčík, Pavel; El-Fakahany, Esam E.; Doležal, Vladimír

    2017-01-01

    Interaction of orthosteric ligands with extracellular domain was described at several aminergic G protein-coupled receptors, including muscarinic acetylcholine receptors. The orthosteric antagonists quinuclidinyl benzilate (QNB) and N-methylscopolamine (NMS) bind to the binding pocket of the muscarinic acetylcholine receptor formed by transmembrane α-helices. We show that high concentrations of either QNB or NMS slow down dissociation of their radiolabeled species from all five subtypes of muscarinic acetylcholine receptors, suggesting allosteric binding. The affinity of NMS at the allosteric site is in the micromolar range for all receptor subtypes. Using molecular modelling of the M2 receptor we found that E172 and E175 in the second extracellular loop and N419 in the third extracellular loop are involved in allosteric binding of NMS. Mutation of these amino acids to alanine decreased affinity of NMS for the allosteric binding site confirming results of molecular modelling. The allosteric binding site of NMS overlaps with the binding site of some allosteric, ectopic and bitopic ligands. Understanding of interactions of NMS at the allosteric binding site is essential for correct analysis of binding and action of these ligands.

  17. Sigma receptor ligand N,N'-di-(ortho-tolyl)guanidine inhibits release of acetylcholine in the guinea pig ileum.

    PubMed

    Cambell, B G; Keana, J F; Weber, E

    1991-11-26

    The inhibition of stimulated contractions of the guinea pig ileum longitudinal muscle/myenteric plexus preparation by sigma receptor ligands has been previously described. In this study, the stimulated release of [3H]acetylcholine from cholinergic nerve terminals in this same preparation was monitored in the presence and absence of sigma receptor ligands. N,N'-Di-(orthotolyl)guanidine (DTG) and other compounds selective for the sigma receptor inhibited stimulated [3H]acetylcholine release. These results suggest that their inhibition of stimulated contractions in this preparation was mediated by inhibition of acetylcholine release.

  18. The nicotinic acetylcholine receptor CHRNA5/A3/B4 gene cluster: Dual role in nicotine addiction and lung cancer

    PubMed Central

    Improgo, Ma. Reina D.; Scofield, Michael D.; Tapper, Andrew R.; Gardner, Paul D.

    2010-01-01

    More than 1 billion people around the world smoke, with 10 million cigarettes sold every minute. Cigarettes contain thousands of harmful chemicals including the psychoactive compound, nicotine. Nicotine addiction is initiated by the binding of nicotine to nicotinic acetylcholine receptors, ligand-gated cation channels activated by the endogenous neurotransmitter, acetylcholine. These receptors serve as prototypes for all ligand-gated ion channels and have been extensively studied in an attempt to elucidate their role in nicotine addiction. Many of these studies have focused on heteromeric nicotinic acetylcholine receptors containing α4 and β2 subunits and homomeric nicotinic acetylcholine receptors containing the α7 subunit, two of the most abundant subtypes expressed in the brain. Recently however, a series of linkage analyses, candidate-gene analyses and genome-wide association studies have brought attention to three other members of the nicotinic acetylcholine receptor family: the α5, α3 and β4 subunits. The genes encoding these subunits lie in a genomic cluster that contains variants associated with increased risk for several diseases including nicotine dependence and lung cancer. The underlying mechanisms for these associations have not yet been elucidated but decades of research on the nicotinic receptor gene family as well as emerging data provide insight on how these receptors may function in pathological states. Here, we review this body of work, focusing on the clustered nicotinic acetylcholine receptor genes and evaluating their role in nicotine addiction and lung cancer. PMID:20685379

  19. Muscarinic acetylcholine receptor-interacting proteins (mAChRIPs): targeting the receptorsome.

    PubMed

    Borroto-Escuela, D O; Agnati, Luigi F; Fuxe, Kjell; Ciruela, F

    2012-01-01

    Muscarinic acetylcholine receptors comprise a large family of G protein-coupled receptors that are involved in the regulation of many important functions of the central and peripheral nervous system. To achieve such a large range of physiological effects, these receptors interact with a large array of accessory proteins including scaffold molecules, ion channels and enzymes that operate as molecular transducers of muscarinic function in addition to the canonical heterotrimeric G proteins. Interestingly, as demonstrated for others G protein-coupled receptors, this type of receptor is also able to oligomerise, a fact that has been shown to play a critical role in their subcellular distribution, trafficking, and fine tuning of cholinergic signalling. On the other hand, the specificity of these receptor interactions may be largely determined by the occurrence of precise protein-interacting motifs, posttranslational modifications, and the differential tissue distribution and stoichiometry of the receptor-interacting proteins. Thus, the exhaustive cataloguing and documentation of muscarinic acetylcholine receptor-interacting proteins and the grasp of their specific function will explain key physiological differences in muscarinic-mediated cholinergic transmission. Overall, a better comprehension of the muscarinic receptor interactome will have a significant impact on the cholinergic pharmacology and thus provide previously unrealised opportunities to achieve greater specificity in muscarinic-related drug discovery and diagnostics.

  20. Expression of muscarinic acetylcholine and dopamine receptor mRNAs in rat basal ganglia

    SciTech Connect

    Weiner, D.M. Howard Hughes Medical Inst., Bethesda, MD ); Levey, A.I. Johns Hopkins Univ., Baltimore, MD ); Brann, M.R. )

    1990-09-01

    Within the basal ganglia, acetylcholine and dopamine play a central role in the extrapyramidal control of motor function. The physiologic effects of these neurotransmitters are mediated by a diversity of receptor subtypes, several of which have now been cloned. Muscarinic acetylcholine receptors are encoded by five genes (m1-m5), and of the two known dopamine receptor subtypes (D1 and D2) the D2 receptor gene has been characterized. To gain insight into the physiological roles of each of these receptor subtypes, the authors prepared oligodeoxynucleotide probes to localize receptor subtype mRNAs within the rat striatum and substantia nigra by in situ hybridization histochemistry. Within the striatum, three muscarinic (m1, m2, m4) receptor mRNAs and the D2 receptor mRNA were detected. The m1 mRNA was expressed in most neurons; the m2 mRNA, in neurons which were both very large and rare; and the m4 and D2 mRNAs, in 40-50% of the neurons, one-third of which express both mRNAs. Within the substantia nigra, pars compacta, only the m5 and D2 mRNAs were detected, and most neurons expressed both mRNAs. These data provide anatomical evidence for the identity of the receptor subtypes which mediate the diverse effects of muscarinic and dopaminergic drugs on basal ganglia function.

  1. Abundance, distribution, mobility and oligomeric state of M2 muscarinic acetylcholine receptors in live cardiac muscle

    PubMed Central

    Nenasheva, Tatiana A.; Neary, Marianne; Mashanov, Gregory I.; Birdsall, Nigel J.M.; Breckenridge, Ross A.; Molloy, Justin E.

    2013-01-01

    M2 muscarinic acetylcholine receptors modulate cardiac rhythm via regulation of the inward potassium current. To increase our understanding of M2 receptor physiology we used Total Internal Reflection Fluorescence Microscopy to visualize individual receptors at the plasma membrane of transformed CHOM2 cells, a cardiac cell line (HL-1), primary cardiomyocytes and tissue slices from pre- and post-natal mice. Receptor expression levels between individual cells in dissociated cardiomyocytes and heart slices were highly variable and only 10% of murine cardiomyocytes expressed muscarinic receptors. M2 receptors were evenly distributed across individual cells and their density in freshly isolated embryonic cardiomyocytes was ~ 1 μm− 2, increasing at birth (to ~ 3 μm− 2) and decreasing back to ~ 1 μm− 2 after birth. M2 receptors were primarily monomeric but formed reversible dimers. They diffused freely at the plasma membrane, moving approximately 4-times faster in heart slices than in cultured cardiomyocytes. Knowledge of receptor density and mobility has allowed receptor collision rate to be modeled by Monte Carlo simulations. Our estimated encounter rate of 5–10 collisions per second, may explain the latency between acetylcholine application and GIRK channel opening. PMID:23357106

  2. Diversity of nicotinic acetylcholine receptors in rat hippocampal neurons. I. Pharmacological and functional evidence for distinct structural subtypes.

    PubMed

    Alkondon, M; Albuquerque, E X

    1993-06-01

    Nicotinic acetylcholine receptors present on cultured hippocampal neurons from fetal rats were characterized by means of whole-cell patch-clamp technique, using a number of structurally divergent agonists and highly selective antagonists. Based upon the decay kinetics of the currents elicited by 3 mM acetylcholine (ACh) and their sensitivities to agonists and antagonists, the neurons were shown to exhibit four current types, IA, IB, II and III. Rapidly decaying currents (type IA) that were blocked by alpha-bungarotoxin (10 nM), kappa-bungarotoxin (10 nM) and methyllycaconitine (MLA, 1 nM) were the most frequent and were found in 83% of the neurons tested. Type II currents (found in 5% of the neurons) were blocked by dihydro-beta-erythroidine (10 nM), and by high concentrations of MLA and kappa-bungarotoxin (100 nM each) but not by alpha-bungarotoxin (100 nM). Type III currents (elicited in 2% of the neurons) decayed slowly and were blocked by (+/-)-mecamylamine (1 microM) but not by alpha-bungarotoxin, kappa-bungarotoxin or MLA (each at 100 nM). Some of the cells (10% of the neurons) had mixed responses (named type IB), which were only partially blocked by MLA (1 nM) or dihydro-beta-erythroidine (10 nM) alone and were completely blocked by combination of the two agents. The order of potency of agonists in activating the currents was the following: for type IA, (+)-anatoxin-a > 1,1-dimethyl-4-phenyl-piperazinium > (-)-nicotine > cystisine > ACh > carbachol > (+)-nicotine > arecoline > suberyldicholine; for type II, ACh > (+)-anatoxin-a > (-)-nicotine > 1,1-dimethyl-4-phenyl-piperazinium > carbachol > cytisine > (+)-nicotine > suberyldicholine > arecoline. Certain agonists were particularly useful in discriminating among the various types of currents: ACh, carbachol, (-)-nicotine and suberyldicholine for type II, and cytisine for type III currents. The EC50 of ACh was approximately 130 microM for type IA and approximately 2 microM for type II currents. A marked

  3. Nicotine inhibits Fc epsilon RI-induced cysteinyl leukotrienes and cytokine production without affecting mast cell degranulation through alpha 7/alpha 9/alpha 10-nicotinic receptors.

    PubMed

    Mishra, Neerad C; Rir-sima-ah, Jules; Boyd, R Thomas; Singh, Shashi P; Gundavarapu, Sravanthi; Langley, Raymond J; Razani-Boroujerdi, Seddigheh; Sopori, Mohan L

    2010-07-01

    Smokers are less likely to develop some inflammatory and allergic diseases. In Brown-Norway rats, nicotine inhibits several parameters of allergic asthma, including the production of Th2 cytokines and the cysteinyl leukotriene LTC(4). Cysteinyl leukotrienes are primarily produced by mast cells, and these cells play a central role in allergic asthma. Mast cells express a high-affinity receptor for IgE (FcepsilonRI). Following its cross-linking, cells degranulate and release preformed inflammatory mediators (early phase) and synthesize and secrete cytokines/chemokines and leukotrienes (late phase). The mechanism by which nicotine modulates mast cell activation is unclear. Using alpha-bungarotoxin binding and quantitative PCR and PCR product sequencing, we showed that the rat mast/basophil cell line RBL-2H3 expresses nicotinic acetylcholine receptors (nAChRs) alpha7, alpha9, and alpha10; exposure to exceedingly low concentrations of nicotine (nanomolar), but not the biologically inactive metabolite cotinine, for > or = 8 h suppressed the late phase (leukotriene/cytokine production) but not degranulation (histamine and hexosaminidase release). These effects were unrelated to those of nicotine on intracellular free calcium concentration but were causally associated with the inhibition of cytosolic phospholipase A(2) activity and the PI3K/ERK/NF-kappaB pathway, including phosphorylation of Akt and ERK and nuclear translocation of NF-kappaB. The suppressive effect of nicotine on the late-phase response was blocked by the alpha7/alpha9-nAChR antagonists methyllycaconitine and alpha-bungarotoxin, as well as by small interfering RNA knockdown of alpha7-, alpha9-, or alpha10-nAChRs, suggesting a functional interaction between alpha7-, alpha9-, and alpha10-nAChRs that might explain the response of RBL cells to nanomolar concentrations of nicotine. This "hybrid" receptor might serve as a target for novel antiallergic/antiasthmatic therapies.

  4. Expression of five acetylcholine receptor subunit genes in Brugia malayi adult worms

    PubMed Central

    Li, Ben-Wen; Rush, Amy C.; Weil, Gary J.

    2015-01-01

    Acetylcholine receptors (AChRs) are required for body movement in parasitic nematodes and are targets of “classical” anthelmintic drugs such as levamisole and pyrantel and of newer drugs such as tribendimidine and derquantel. While neurotransmission explains the effects of these drugs on nematode movement, their effects on parasite reproduction are unexplained. The levamisole AChR type (L-AChRs) in Caenorhabditis elegans is comprised of five subunits: Cel-UNC-29, Cel-UNC-38, Cel-UNC-63, Cel-LEV-1 and Cel-LEV-8. The genome of the filarial parasite Brugia malayi contains nine AChRs subunits including orthologues of Cel-unc-29, Cel-unc-38, and Cel-unc-63. We performed in situ hybridization with RNA probes to localize the expression of five AChR genes (Bm1_35890-Bma-unc-29, Bm1_20330-Bma-unc-38, Bm1_38195-Bma-unc-63, Bm1_48815-Bma-acr-26 and Bm1_40515-Bma-acr-12) in B. malayi adult worms. Four of these genes had similar expression patterns with signals in body muscle, developing embryos, spermatogonia, uterine wall adjacent to stretched microfilariae, wall of Vas deferens, and lateral cord. Three L-AChR subunit genes (Bma-unc-29, Bma-unc-38 and Bma-unc-63) were expressed in body muscle, which is a known target of levamisole. Bma-acr-12 was co-expressed with these levamisole subunit genes in muscle, and this suggests that its protein product may form receptors with other alpha subunits. Bma-acr-26 was expressed in male muscle but not in female muscle. Strong expression signals of these genes in early embryos and gametes in uterus and testis suggest that AChRs may have a role in nervous system development of embryogenesis and spermatogenesis. This would be consistent with embryotoxic effects of drugs that target these receptors in filarial worms. Our data show that the expression of these receptor genes is tightly regulated with regard to localization in adult worms and developmental stage in embryos and gametes. These results may help to explain the broad effects

  5. Use of Monoclonal Antibodies to Study the Structure and Function of Nicotinic Acetylcholine Receptors on Electric Organ and Muscle and to Determine the Structure of Nicotinic Acetylcholine Receptors on Neurons

    DTIC Science & Technology

    1988-03-16

    observed when the sections were coincubated in 400 nrL cold mAb 270. Adjacent Nissl -stained sections were used to identify labeled structures...affinity reagent for the acetylcholine receptor binding site. J Biol Chem 259:11662-11665. 7. Whiting PJ, JM Lindstrom. 1986 . Purification and...characterization of a nicotinic acetylcholine receptor from chick brain. Biochemistry 2502082-2093. 8. Whiting PJ, JM Lindstrom. 1986 . Pharmacological

  6. Immunohistochemical localization of monoclonal antibodies to the nicotinic acetylcholine receptor in chick midbrain.

    PubMed Central

    Swanson, L W; Lindstrom, J; Tzartos, S; Schmued, L C; O'Leary, D D; Cowan, W M

    1983-01-01

    We used the indirect immunofluorescence method to determine the crossreactivity of a library of 57 monoclonal antibodies (mAbs) against each of the subunits of the nicotinic acetylcholine receptor (nAcChoR) isolated from Torpedo and Electrophorus electric organs or from fetal calf and human muscle, with specific neural elements in the midbrain of the chick. Out of 17 mAbs that recognized motor end plates on chick muscle, 14 produced a similar pattern of labeling in the midbrain: the neuronal perikarya and dendrites in the lateral spiriform nucleus (SpL) were intensely labeled, and there was moderate labeling of fibers in certain of the deeper layers of the optic tectum, which disappeared after the SpL was destroyed electrolytically. Two lines of evidence suggest that the mAbs may be crossreacting with nAcChoRs in the midbrain. First, all of the mAbs that stained the SpL also stained neuromuscular junctions in skeletal muscle, whereas none of the 40 mAbs that failed to stain end plates crossreacted with the SpL; second, in vitro immunological studies and blocking experiments on tissue sections (in which unlabeled mAbs were used to block the staining of a directly fluorescein-treated mAb) indicated the presence of mAbs specific for unique antigenic determinants on all four of the subunits (alpha, beta, gamma, and delta) from Torpedo nAcChoR in chick midbrain and muscle. On the other hand, the distribution of mAb staining in the optic tectum does not closely parallel that of either acetylcholinesterase staining or of 125I-labeled alpha-bungarotoxin binding; no toxin binding has been observed autoradiographically in the SpL, but the nucleus does contain moderately dense acetylcholinesterase staining. Take together, our observations suggest that there may be a cholinergic input to the SpL and that the projection fibers from the SpL to the optic tectum (which are also stained with an antiserum to [Leu]enkephalin) may contain presynaptic nAcChoRs. It is clear, however

  7. Nicotinic acetylcholine receptors: upregulation, age-related effects and associations with drug use

    PubMed Central

    Melroy-Greif, W. E.; Stitzel, J. A.; Ehringer, M. A.

    2016-01-01

    Nicotinic acetylcholine receptors are ligand-gated ion channels that exogenously bind nicotine. Nicotine produces rewarding effects by interacting with these receptors in the brain’s reward system. Unlike other receptors, chronic stimulation by an agonist induces an upregulation of receptor number that is not due to increased gene expression in adults; while upregulation also occurs during development and adolescence there have been some opposing findings regarding a change in corresponding gene expression. These receptors have also been well studied with regard to human genetic associations and, based on evidence suggesting shared genetic liabilities between substance use disorders, numerous studies have pointed to a role for this system in comorbid drug use. This review will focus on upregulation of these receptors in adulthood, adolescence and development, as well as the findings from human genetic association studies which point to different roles for these receptors in risk for initiation and continuation of drug use. PMID:26351737

  8. The structure of the third intracellular loop of the muscarinic acetylcholine receptor M2 subtype.

    PubMed

    Ichiyama, Susumu; Oka, Yoshiaki; Haga, Kazuko; Kojima, Shuichi; Tateishi, Yukihiro; Shirakawa, Masahiro; Haga, Tatsuya

    2006-01-09

    We have examined whether the long third intracellular loop (i3) of the muscarinic acetylcholine receptor M2 subtype has a rigid structure. Circular dichroism (CD) and nuclear magnetic resonance spectra of M2i3 expressed in and purified from Escherichia coli indicated that M2i3 consists mostly of random coil. In addition, the differential CD spectrum between the M2 and M2deltai3 receptors, the latter of which lacks most of i3 except N- and C-terminal ends, gave no indication of secondary structure. These results suggest that the central part of i3 of the M2 receptor has a flexible structure.

  9. Crystal Structures of the M1 and M4 Muscarinic Acetylcholine Receptors

    PubMed Central

    Thal, David M.; Sun, Bingfa; Feng, Dan; Nawaratne, Vindhya; Leach, Katie; Felder, Christian C.; Bures, Mark G.; Evans, David A.; Weis, William I.; Bachhawat, Priti; Kobilka, Tong Sun; Sexton, Patrick M.; Kobilka, Brian K.; Christopoulos, Arthur

    2016-01-01

    Summary Muscarinic M1–M5 acetylcholine receptors are G protein-coupled receptors (GPCRs) that regulate many vital functions of the central and peripheral nervous systems. In particular, the M1 and M4 receptor subtypes have emerged as attractive drug targets for treatments of neurological disorders, such as Alzheimer's disease and schizophrenia, but the high conservation of the acetylcholine-binding pocket has spurred current research into targeting allosteric sites on these receptors. Here, we report the first crystal structures of the M1 and M4 muscarinic receptors bound to the inverse agonist, tiotropium. Comparison of these structures to each other, as well as the previously reported M2 and M3 receptor structures, reveals differences in the orthosteric and allosteric binding sites that contribute to a role in drug selectivity at this important receptor family. We also report identification of a cluster of residues that form a network linking the orthosteric and allosteric sites of the M4 receptor, which provides new insight into how allosteric modulation may be transmitted between the two spatially distinct domains. PMID:26958838

  10. Subnanosecond polarized fluorescence photobleaching: rotational diffusion of acetylcholine receptors on developing muscle cells.

    PubMed Central

    Yuan, Y; Axelrod, D

    1995-01-01

    Polarized fluorescence recovery after photobleaching (PFRAP) is a technique for measuring the rate of rotational motion of biomolecules on living, nondeoxygenated cells with characteristic times previously ranging from milliseconds to many seconds. Although very broad, that time range excludes the possibility of quantitatively observing freely rotating membrane protein monomers that typically should have a characteristic decay time of only several microseconds. This report describes an extension of the PFRAP technique to a much shorter time scale. With this new system, PFRAP experiments can be conducted with sample time as short as 0.4 microseconds and detection of possible characteristic times of less than 2 microseconds. The system is tested on rhodamine-alpha-bungarotoxin-labeled acetylcholine receptors (AChRs) on myotubes grown in primary cultures of embryonic rat muscle, in both endogenously clustered and nonclustered regions of AChR distribution. It is found that approximately 40% of the AChRs in nonclustered regions undergoes rotational diffusion fast enough to possibly arise from unrestricted monomer Brownian motion. The AChRs in clusters, on the other hand, are almost immobile. The effects of rat embryonic brain extract (which contains AChR aggregating factors) on the myotube AChR were also examined by the fast PFRAP system. Brain extract is known to abolish the presence of endogenous clusters and to induce the formation of new clusters. It is found here that rotational diffusion of AChR in the extract-induced clusters is as slow as that in endogenous clusters on untreated cells but that rotational diffusion in the nonclustered regions of extract-treated myotubes remains rapid. Images FIGURE 3 PMID:8527682

  11. Differential Contribution of Subunit Interfaces to α9α10 Nicotinic Acetylcholine Receptor Function.

    PubMed

    Boffi, Juan Carlos; Marcovich, Irina; Gill-Thind, JasKiran K; Corradi, Jeremías; Collins, Toby; Lipovsek, María Marcela; Moglie, Marcelo; Plazas, Paola V; Craig, Patricio O; Millar, Neil S; Bouzat, Cecilia; Elgoyhen, Ana Belén

    2017-03-01

    Nicotinic acetylcholine receptors can be assembled from either homomeric or heteromeric pentameric subunit combinations. At the interface of the extracellular domains of adjacent subunits lies the acetylcholine binding site, composed of a principal component provided by one subunit and a complementary component of the adjacent subunit. Compared with neuronal nicotinic acetylcholine cholinergic receptors (nAChRs) assembled from α and β subunits, the α9α10 receptor is an atypical member of the family. It is a heteromeric receptor composed only of α subunits. Whereas mammalian α9 subunits can form functional homomeric α9 receptors, α10 subunits do not generate functional channels when expressed heterologously. Hence, it has been proposed that α10 might serve as a structural subunit, much like a β subunit of heteromeric nAChRs, providing only complementary components to the agonist binding site. Here, we have made use of site-directed mutagenesis to examine the contribution of subunit interface domains to α9α10 receptors by a combination of electrophysiological and radioligand binding studies. Characterization of receptors containing Y190T mutations revealed unexpectedly that both α9 and α10 subunits equally contribute to the principal components of the α9α10 nAChR. In addition, we have shown that the introduction of a W55T mutation impairs receptor binding and function in the rat α9 subunit but not in the α10 subunit, indicating that the contribution of α9 and α10 subunits to complementary components of the ligand-binding site is nonequivalent. We conclude that this asymmetry, which is supported by molecular docking studies, results from adaptive amino acid changes acquired only during the evolution of mammalian α10 subunits.

  12. Differential Contribution of Subunit Interfaces to α9α10 Nicotinic Acetylcholine Receptor Function

    PubMed Central

    Boffi, Juan Carlos; Marcovich, Irina; Gill-Thind, JasKiran K.; Corradi, Jeremías; Collins, Toby; Lipovsek, María Marcela; Moglie, Marcelo; Plazas, Paola V.; Craig, Patricio O.; Millar, Neil S.; Bouzat, Cecilia

    2017-01-01

    Nicotinic acetylcholine receptors can be assembled from either homomeric or heteromeric pentameric subunit combinations. At the interface of the extracellular domains of adjacent subunits lies the acetylcholine binding site, composed of a principal component provided by one subunit and a complementary component of the adjacent subunit. Compared with neuronal nicotinic acetylcholine cholinergic receptors (nAChRs) assembled from α and β subunits, the α9α10 receptor is an atypical member of the family. It is a heteromeric receptor composed only of α subunits. Whereas mammalian α9 subunits can form functional homomeric α9 receptors, α10 subunits do not generate functional channels when expressed heterologously. Hence, it has been proposed that α10 might serve as a structural subunit, much like a β subunit of heteromeric nAChRs, providing only complementary components to the agonist binding site. Here, we have made use of site-directed mutagenesis to examine the contribution of subunit interface domains to α9α10 receptors by a combination of electrophysiological and radioligand binding studies. Characterization of receptors containing Y190T mutations revealed unexpectedly that both α9 and α10 subunits equally contribute to the principal components of the α9α10 nAChR. In addition, we have shown that the introduction of a W55T mutation impairs receptor binding and function in the rat α9 subunit but not in the α10 subunit, indicating that the contribution of α9 and α10 subunits to complementary components of the ligand-binding site is nonequivalent. We conclude that this asymmetry, which is supported by molecular docking studies, results from adaptive amino acid changes acquired only during the evolution of mammalian α10 subunits. PMID:28069778

  13. Synaptic modulation of excitatory synaptic transmission by nicotinic acetylcholine receptors in spinal ventral horn neurons.

    PubMed

    Mine, N; Taniguchi, W; Nishio, N; Izumi, N; Miyazaki, N; Yamada, H; Nakatsuka, T; Yoshida, M

    2015-04-02

    Nicotinic acetylcholine receptors (nAChRs) are distributed widely in the central nervous system and play important roles in higher brain functions, including learning, memory, and recognition. However, functions of the cholinergic system in spinal motoneurons remain poorly understood. In this study, we investigated the actions of presynaptic and postsynaptic nAChRs in spinal ventral horn neurons by performing whole-cell patch-clamp recordings on lumbar slices from male rats. The application of nicotine or acetylcholine generated slow inward currents and increased the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs). Slow inward currents by acetylcholine or nicotine were not inhibited by tetrodotoxin (TTX) or glutamate receptor antagonists. In the presence of TTX, the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) were also increased by acetylcholine or nicotine. A selective α4β2 nicotinic receptor antagonist, dihydro-β-erythroidine hydrobromide (DhβE), significantly decreased nicotine-induced inward currents without affecting the enhancement of sEPSCs and mEPSCs. In addition, a selective α7 nicotinic receptor antagonist, methyllycaconitine, did not affect either nicotine-induced inward currents or the enhancement of sEPSCs and mEPSCs. These results suggest that α4β2 AChRs are localized at postsynaptic sites in the spinal ventral horn, non-α4β2 and non-α7 nAChRs are located presynaptically, and nAChRs enhance excitatory synaptic transmission in the spinal ventral horn.

  14. In vivo Therapy with Monoclonal Anti-I-A Antibody Suppresses Immune Responses to Acetylcholine Receptor

    NASA Astrophysics Data System (ADS)

    Waldor, Matthew K.; Sriram, Subramaniam; McDevitt, Hugh O.; Steinman, Lawrence

    1983-05-01

    A monoclonal antibody to I-A gene products of the immune response gene complex attenuates both humoral and cellular responses to acetylcholine receptor and appears to suppress clinical manifestations of experimental autoimmune myasthenia gravis. This demonstrates that use of antibodies against immune response gene products that are associated with susceptibility to disease may be feasible for therapy in autoimmune conditions such as myasthenia gravis.

  15. Nicotine at concentrations found in cigarette smokers activates and desensitizes nicotinic acetylcholine receptors in CA1 interneurons of rat hippocampus.

    PubMed

    Alkondon, M; Pereira, E F; Almeida, L E; Randall, W R; Albuquerque, E X

    2000-10-01

    Behavioral effects of cigarette smoking are attributed to the interactions of nicotine with brain nicotinic acetylcholine receptors (nAChRs). However, the mechanisms by which nAChR function in developing and mature brain is affected by a smoker's level of nicotine (50-500 nM) remain unclear. Thus, the objective of this study was to determine the concentration- and time-dependent effects of nicotine on alpha7 and alpha4beta2 nAChRs, the two major brain subtypes, natively expressed in CA1 interneurons of rat hippocampal slices. Only at concentrations > or =5 microM did nicotine (applied for 6-60 s) elicit action potentials or measurable whole-cell currents (EC(50)=158 microM) in stratum radiatum interneurons that express alpha7 nAChRs. Continuous exposure for 10-15 min of the neurons to nicotine (0.5-2.5 microM) inhibited alpha7 nAChR-mediated currents (IC(50)=640 nM) evoked by choline (10 mM). Nicotine (> or =0.125 microM) applied to the neurons for 1-5 min induced slowly desensitizing whole-cell currents (EC(50)=3.2 microM) in stratum lacunosum moleculare interneurons; this effect was mediated by alpha4beta2 nAChRs. Also via activation of alpha4beta2 nAChRs, nicotine (0.125-0.5 microM) increased the frequency and amplitude of GABAergic postsynaptic currents (PSCs) in stratum radiatum interneurons. However, exposure of the neurons for 10-15 min to nicotine (0.25-0.5 microM) resulted in desensitization of alpha4beta2 nAChRs. It is suggested that nanomolar concentrations of nicotine after acute intake suppress inhibitory inputs to pyramidal cells through a disinhibitory mechanism involving activation of alpha4beta2 nAChRs and desensitization of alpha7 nAChRs, and after chronic intake leads to up-regulation of both receptor subtypes via desensitization. These findings have direct implications to the actions of nicotine in cigarette smokers.

  16. Effects of dichlorobenzene on acetylcholine receptors in human neuroblastoma SH-SY5Y cells.

    PubMed

    Yan, Ren-Ming; Chiung, Yin-Mei; Pan, Chien-Yuan; Liu, Jenn-Hwa; Liu, Pei-Shan

    2008-11-20

    para-Dichlorobenzene (DCB), a deodorant and an industrial chemical, is a highly volatile compound and is known to be an indoor air contaminant. Because of its widespread use and volatility, the toxicity of DCB presents a concern to industrial workers and public. Some toxic aspects of DCB have already been focused but its effects on neuronal signal transduction have been hitherto unknown. The effects of DCB on the cytosolic calcium homeostasis are investigated in human neuroblastoma SH-SY5Y cells in this study. DCB, above 200 microM, was found to induce a rise in cytosolic calcium concentration that could not be counteracted by nicotinic acetylcholine receptor (nAChR) and muscarinic acetylcholine receptor (mAChR) antagonists but was partially inhibited by thapsigargin. To understand the actions of DCB on the acetylcholine receptors, we investigated its effects on the changes of cytosolic calcium concentration following nicotinic AChR stimulation with epibatidine and muscarinic AChR stimulation with methacholine in human neuroblastoma SH-SY5Y cells. DCB inhibited the cytosolic calcium concentration rise induced by epibatidine and methacholine with respective IC(50)s of 34 and 294 microM. The inhibitions of DCB were not the same as thapsigargin's inhibition. In the electrophysiological observations, DCB blocked the influx currents induced by epibatidine. Our findings suggest that DCB interferes with the functional activities of AChR, including its coupling influx currents and cytosolic calcium elevations.

  17. Acetylcholine receptor pathway in lung cancer: New twists to an old story

    PubMed Central

    Niu, Xiao-Min; Lu, Shun

    2014-01-01

    Genome-wide association studies revealed that allelic variation in the α5-α3-β4 nicotine acetylcholine receptor (nAChR) cluster on chromosome 15q24-15q25.1 was associated with lung cancer risk. nAChRs are membrane ligand-gated cation channels whose activation is triggered by the binding of the endogenous neurotransmitter acetylcholine (ACh) or other biologic compounds including nicotine. nAChRs have been found on lung cancer cells, underscoring the idea that the non-neuronal nAChR pathway has important implications for lung cancer. Several studies focusing on the treatment with nAChR antagonists with improved selectivity might trigger novel strategies for the intervention and prevention of lung cancer. Here we review the genetic risk factors for lung cancer in the nAChR gene cluster, the roles of nicotine receptors, and the molecular mechanisms of acetylcholine receptor pathways to lead to more opportunities for intervention and prevention of lung cancer. PMID:25302169

  18. Mutations in Dalpha1 or Dbeta2 nicotinic acetylcholine receptor subunits can confer resistance to neonicotinoids in Drosophila melanogaster.

    PubMed

    Perry, Trent; Heckel, David G; McKenzie, John A; Batterham, Philip

    2008-05-01

    Resistance to insecticides by modification of their molecular targets is a serious problem in chemical control of many arthropod pests. Neonicotinoids target the nicotinic acetylcholine receptor (nAChR) of arthropods. The spectrum of possible resistance-conferring mutations of this receptor is poorly understood. Prediction of resistance is complicated by the existence of multiple genes encoding the different subunits of this essential component of neurotransmission. We focused on the cluster of three Drosophila melanogaster nAChR subunit genes at cytological region 96A. EMS mutagenesis and selection for resistance to nitenpyram was performed on hybrids carrying a deficiency for this chromosomal region. Two complementation groups were defined for the four strains isolated. Molecular characterisation of the mutations found lesions in two nAChR subunit genes, Dalpha1 (encoding an alpha-type subunit) and Dbeta2 (beta-type). Mutations conferring resistance in beta-type receptors have not previously been reported, but we found several lesions in the Dbeta2 sequence, including locations distant from the predicted neonicotinoid-binding site. This study illustrates that mutations in a single-receptor subunit can confer nitenpyram resistance. Moreover, some of the mutations may protect the insect against nitenpyram by interfering with subunit assembly or channel activation, rather than affecting binding affinities of neonicotinoids to the channel.

  19. Barium permeability of neuronal nicotinic receptor alpha 7 expressed in Xenopus oocytes.

    PubMed Central

    Sands, S B; Costa, A C; Patrick, J W

    1993-01-01

    The rat alpha 7 neuronal nicotinic acetylcholine receptor was expressed and studied in Xenopus oocytes. The magnitude and reversal potential of instantaneous whole cell currents were examined in solutions containing varying concentrations of either calcium or barium, and in the presence or absence of the intracellular calcium chelator BAPTA. In external barium, application of nicotine elicits an inwardly rectifying response; in calcium the response is larger and has a linear IV relation. Pretreatment of oocytes with BAPTA-AM could not prevent activation of calcium-dependent chloride channels in external Ringer containing calcium. Using an extended GHK equation, the permeability ratio PBa/PNa of the alpha 7 receptor was determined to be about 17. Our results suggest that alpha 7 nicotinic receptors are highly permeable to divalent cations. PMID:8312496

  20. Characterization of a series of anabaseine-derived compounds reveals that the 3-(4)-dimethylaminocinnamylidine derivative is a selective agonist at neuronal nicotinic alpha 7/125I-alpha-bungarotoxin receptor subtypes.

    PubMed

    de Fiebre, C M; Meyer, E M; Henry, J C; Muraskin, S I; Kem, W R; Papke, R L

    1995-01-01

    Investigation of the naturally occurring, nicotinic agonist anabaseine and novel derivatives has shown that these compounds have cytoprotective and memory-enhancing effects. The hypothesis that these arise at least in part through actions on brain nicotinic receptors was evaluated by examining the ability of these compounds to displace the binding of nicotinic ligands and to affect the function of the alpha 4 beta 2 and alpha 7 receptor subtypes expressed in Xenopus oocytes. The derivative 3-(4)-dimethylaminocinnamylidine anabaseine (DMAC) was found to be a selective alpha 7 receptor agonist; it was more potent than nicotine, acetylcholine, anabaseine, and other derivatives at activating the alpha 7 receptor subtype, while displaying little agonist activity at alpha 4 beta 2 and other receptor subtypes. Compared with anabaseine and the other derivatives, DMAC was the most potent at displacing 125I-alpha-bungarotoxin binding (putative alpha 7) and the least potent at displacing [3H]cytisine binding (putative alpha 4 beta 2) to brain membranes. Independently of agonist activities, all of the novel compounds displayed secondary inhibitory activity at both receptor subtypes. At the alpha 4 beta 2 receptor subtype, inhibition by the 3-(2,4)-dimethoxybenzylidene derivative was enhanced by coapplication of acetylcholine, suggesting a noncompetitive form of inhibition. Anabaseine and nicotine prolonged the time course of activation of alpha 4 beta 2 receptors, compared with acetylcholine, suggesting sequential channel-blocking activity. As selective agonists, anabaseine derivatives such as DMAC may be useful for elucidating the function of alpha 7 nicotinic receptors, including their potential role(s) in the cytoprotective and memory-enhancing effects of nicotinic agents.

  1. Two types of muscarinic acetylcholine receptors in Drosophila and other arthropods.

    PubMed

    Collin, Caitlin; Hauser, Frank; Gonzalez de Valdivia, Ernesto; de Valdivia, Ernesto Gonzalez; Li, Shizhong; Reisenberger, Julia; Carlsen, Eva M M; Khan, Zaid; Hansen, Niels O; Puhm, Florian; Søndergaard, Leif; Niemiec, Justyna; Heninger, Magdalena; Ren, Guilin R; Grimmelikhuijzen, Cornelis J P

    2013-09-01

    Muscarinic acetylcholine receptors (mAChRs) play a central role in the mammalian nervous system. These receptors are G protein-coupled receptors (GPCRs), which are activated by the agonists acetylcholine and muscarine, and blocked by a variety of antagonists. Mammals have five mAChRs (m1-m5). In this study, we cloned two structurally related GPCRs from the fruit fly Drosophila melanogaster, which, after expression in Chinese hamster ovary cells, proved to be muscarinic acetylcholine receptors. One mAChR (the A-type; encoded by gene CG4356) is activated by acetylcholine (EC50, 5 × 10(-8) M) and muscarine (EC50, 6 × 10(-8) M) and blocked by the classical mAChR antagonists atropine, scopolamine, and 3-quinuclidinyl-benzilate (QNB), while the other (the B-type; encoded by gene CG7918) is also activated by acetylcholine, but has a 1,000-fold lower sensitivity to muscarine, and is not blocked by the antagonists. A- and B-type mAChRs were also cloned and functionally characterized from the red flour beetle Tribolium castaneum. Recently, Haga et al. (Nature 2012, 482: 547-551) published the crystal structure of the human m2 mAChR, revealing 14 amino acid residues forming the binding pocket for QNB. These residues are identical between the human m2 and the D. melanogaster and T. castaneum A-type mAChRs, while many of them are different between the human m2 and the B-type receptors. Using bioinformatics, one orthologue of the A-type and one of the B-type mAChRs could also be found in all other arthropods with a sequenced genome. Protostomes, such as arthropods, and deuterostomes, such as mammals and other vertebrates, belong to two evolutionarily distinct lineages of animal evolution that split about 700 million years ago. We found that animals that originated before this split, such as cnidarians (Hydra), had two A-type mAChRs. From these data we propose a model for the evolution of mAChRs.

  2. α7 nicotinic acetylcholine receptors: a therapeutic target in the structure era.

    PubMed

    Taly, Antoine; Charon, Sebastien

    2012-05-01

    The nicotinic acetylcholine receptors (nAChR) are ligand-gated ion channels involved in cognitive processes and are associated with brain disorders which makes them interesting drug targets. This article presents a general overview of the receptor to introduce the α7 nAChR as a drug target. The advances in understanding of the structure/function properties of the nAChR produced during the last decade are detailed as they are crucial for rational drug design. The allosteric properties of the nAChR will also be described because they also have important consequences for drug design.

  3. Incorporation of acetylcholine receptors and Cl- channels in Xenopus oocytes injected with Torpedo electroplaque membranes.

    PubMed Central

    Marsal, J; Tigyi, G; Miledi, R

    1995-01-01

    A method was developed to transplant assembled nicotinic acetylcholine receptors (AcChoRs) and Cl- channels from the electric organ of Torpedo to the membrane of Xenopus oocytes. Membrane vesicles from Torpedo electroplaques were injected into the oocytes and, within a few hours, the oocyte membrane acquired AcChoRs and Cl- channels. The mechanism of expression of these receptors and channels is very different from that which follows the injection of mRNA, since the appearance of receptors after membrane injection does not require de novo protein synthesis or N-glycosylation. This, and other controls, indicate that the foreign receptor-bearing membranes fuse with the oocyte membrane and cause the appearance of functional receptors and channels. All this makes the Xenopus oocyte an even more powerful tool for studies of the structure and function of membrane proteins. PMID:7761478

  4. Nicotine-morphine interactions at α4β2, α7 and α3(⁎) nicotinic acetylcholine receptors.

    PubMed

    Talka, Reeta; Salminen, Outi; Whiteaker, Paul; Lukas, Ronald J; Tuominen, Raimo K

    2013-02-15

    Nicotine and opioids share several behavioral and rewarding properties. Although both opioids and nicotine have their own specific mechanism of action, there is empirical and experimental evidence of interactions between these drugs. We studied receptor-level interactions of nicotine and morphine at α4β2, α7 and α3(⁎) nicotinic acetylcholine receptors. [(3)H]epibatidine displacement was used to determine if morphine binds competitively to nicotinic acetylcholine receptors. Functional interactions of morphine and nicotine were studied with calcium fluorometry and (86)Rb(+) efflux assays. Morphine displaced [(3)H]epibatidine from nicotinic agonist binding sites in all cell lines studied. The Ki values for morphine were 13.2μM in SH-EP1-hα4β2 cells, 0.16μM and 126μM in SH-SY5Y cells and 43.7μM in SH-EP1-hα7 cells. In SH-EP1-hα4β2 cells expressing α4β2 nicotinic acetylcholine receptors, morphine acted as a partial agonist of (86)Rb(+) efflux comparable to cytisine (with EC50 values of 53.3μM for morphine and 5.38μM for cytisine). The effect of morphine was attenuated concentration-dependently by the nicotinic antagonist mecamylamine. In the SH-SY5Y cell line expressing several subtypes of nicotinic acetylcholine receptors morphine had an inhibitory effect on nicotine induced (86)Rb(+) ion efflux mediated by α3(⁎) nicotinic acetylcholine receptors. These results suggest that morphine acts as a partial agonist at α4β2 nicotinic acetylcholine receptors and as a weak antagonist at α3(⁎) nicotinic acetylcholine receptors.

  5. Monkey adrenal chromaffin cells express α6β4* nicotinic acetylcholine receptors.

    PubMed

    Hernández-Vivanco, Alicia; Hone, Arik J; Scadden, Mick L; Carmona-Hidalgo, Beatriz; McIntosh, J Michael; Albillos, Almudena

    2014-01-01

    Nicotinic acetylcholine receptors (nAChRs) that contain α6 and β4 subunits have been demonstrated functionally in human adrenal chromaffin cells, rat dorsal root ganglion neurons, and on noradrenergic terminals in the hippocampus of adolescent mice. In human adrenal chromaffin cells, α6β4* nAChRs (the asterisk denotes the possible presence of additional subunits) are the predominant subtype whereas in rodents, the predominant nAChR is the α3β4* subtype. Here we present molecular and pharmacological evidence that chromaffin cells from monkey (Macaca mulatta) also express α6β4* receptors. PCR was used to show the presence of transcripts for α6 and β4 subunits and pharmacological characterization was performed using patch-clamp electrophysiology in combination with α-conotoxins that target the α6β4* subtype. Acetylcholine-evoked currents were sensitive to inhibition by BuIA[T5A,P6O] and MII[H9A,L15A]; α-conotoxins that inhibit α6-containing nAChRs. Two additional agonists were used to probe for the expression of α7 and β2-containing nAChRs. Cells with currents evoked by acetylcholine were relatively unresponsive to the α7-selctive agonist choline but responded to the agonist 5-I-A-85380. These studies provide further insights into the properties of natively expressed α6β4* nAChRs.

  6. Differential roles for disulfide bonds in the structural integrity and biological activity of kappa-Bungarotoxin, a neuronal nicotinic acetylcholine receptor antagonist.

    PubMed

    Grant, G A; Luetje, C W; Summers, R; Xu, X L

    1998-09-01

    kappa-Bungarotoxin, a kappa-neurotoxin derived from the venom of the banded Krait, Bungarus multicinctus, is a homodimeric protein composed of subunits of 66 amino acid residues containing five disulfide bonds. kappa-Bungarotoxin is a potent, selective, and slowly reversible antagonist of alpha3 beta2 neuronal nicotinic acetylcholine receptors. kappa-Bungarotoxin is structurally related to the alpha-neurotoxins, such as alpha-bungarotoxin derived from the same snake, which are monomeric in solution and which effectively antagonize muscle type receptors (alpha1 beta1 gamma delta) and the homopentameric neuronal type receptors (alpha7, alpha8, and alpha9). Like the kappa-neurotoxins, the long alpha-neurotoxins contain the same five conserved disulfide bonds, while the short alpha-neurotoxins only contain four of the five. Systematic removal of single disulfide bonds in kappa-bungarotoxin by site-specific mutagenesis reveals a differential role for each of the disulfide bonds. Removal of either of the two disulfides connecting elements of the carboxy terminal loop of this toxin (Cys 46-Cys 58 and Cys 59-Cys 64) interferes with the ability of the toxin to fold. In contrast, removal of each of the other three disulfides does not interfere with the general folding of the toxin and yields molecules with biological activity. In fact, when either C3-C21 or C14-C42 are removed individually, no loss in biological activity is seen. However, removing both produces a polypeptide chain which fails to fold properly. Removal of the C27-C31 disulfide only reduces the activity of the toxin 46.6-fold. This disulfide may play a role in specific interaction of the toxin with specific neuronal receptors.

  7. Investigation of the presence and antinociceptive function of muscarinic acetylcholine receptors in the African naked mole-rat (Heterocephalus glaber).

    PubMed

    Jørgensen, Kristine B; Krogh-Jensen, Karen; Pickering, Darryl S; Kanui, Titus I; Abelson, Klas S P

    2016-01-01

    The present study investigated the cholinergic system in the African naked mole-rat (Heterocephalus glaber) with focus on the muscarinic acetylcholine receptor subtypes M1 and M4. The protein sequences for the subtypes m 1-5 of the naked mole-rat were compared to that of the house mouse (Mus musculus) using basic local alignment search tool (BLAST). The presence and function of M1 and M4 was investigated in vivo, using the formalin test with the muscarinic receptor agonists xanomeline and VU0152100. Spinal cord tissue from the naked mole-rat was used for receptor saturation binding studies with [(3)H]-N-methylscopolamine. The BLAST test revealed 95 % protein sequence homology showing the naked mole-rat to have the genetic potential to express all five muscarinic acetylcholine receptor subtypes. A significant reduction in pain behavior was demonstrated after administration of 8.4 mg/kg in the formalin test. Administration of 50 mg/kg VU0152100 resulted in a non-significant tendency towards antinociception. The antinociceptive effects were reversed by the muscarinic acetylcholine receptor antagonist atropine. Binding studies indicated presence of muscarinic acetylcholine receptors with a radioligand affinity comparable to that reported in mice. In conclusion, muscarinic acetylcholine receptor subtypes are present in the naked mole-rat and contribute to antinociception in the naked mole-rat.

  8. Correlation of phospholipid structure with functional effects on the nicotinic acetylcholine receptor. A modulatory role for phosphatidic acid.

    PubMed Central

    Bhushan, A; McNamee, M G

    1993-01-01

    Fourier transform infrared spectroscopy is used to characterize specific interactions between negatively charged lipids, such as phosphatidic acid, and the purified nicotinic acetylcholine receptor from Torpedo californica. The specific interaction of phosphatidic acid with acetylcholine receptor is demonstrated by the receptor-induced perturbation of the lipid ionization state, which is monitored using Fourier transform infrared bands arising from the phosphate head group. The acetylcholine receptor shifts the pKa of phosphatidic acid molecules adjacent to the receptor to a lower value by almost 2 pH units from 8.5 to 6.6. Decreased pH also leads to changes in ion channel function and to changes in the secondary structure of the acetylcholine receptor in membranes containing ionizable phospholipids. Phospholipase D restores functional activity of acetylcholine receptor reconstituted in an unfavorable environment containing phosphatidylcholine by generating phosphatidic acid. Lipids such as phosphatidic acid may serve as allosteric effectors for membrane protein function and the lipid-protein interface could be a site for activity-dependent changes that lead to modulation of synaptic efficacy. PMID:8471723

  9. Changes in acetylcholine content, release and muscarinic receptors in rat hippocampus under cold stress

    SciTech Connect

    Fatranska, M.; Budai, D.; Gulya, K; Kvetnansky, R.

    1989-01-01

    The aim was to study the mechanism of the previously established decrease in acetylcholine (ACh) concentration in the rat hippocampus under cold stress. Male rats were exposed for 14 days to cold (5/degree/C) or kept (controls) at room temperature (24/degree/C). Acetylcholine content, release and muscarinic receptor binding were investigated in the hippocampus. Cold exposure resulted in a decrease of ACh concentration in the dorsal hippocampus. Moreover, the potassium-evoked release of ACh from hippocampal slices was increased and an increase of maximal binding capacity of (/sup 3/H)(-) quinuclidinyl benzilate in the dorsal hippocampus of cold exposed animals was also observed. Thus the decrease of hippocampal ACh concentration under cold exposure is probably due to its increased release. On balance then, our results demonstrate that cold stress in the rat induces significant activation of the hippocampal cholinergic system.

  10. Study of the Peripheral Nerve Fibers Myelin Structure Changes during Activation of Schwann Cell Acetylcholine Receptors

    PubMed Central

    Verdiyan, Ekaterina E.; Allakhverdiev, Elvin S.; Maksimov, Georgy V.

    2016-01-01

    In the present paper we consider a new type of mechanism by which neurotransmitter acetylcholine (ACh) regulates the properties of peripheral nerve fibers myelin. Our data show the importance of the relationship between the changes in the number of Schwann cell (SC) acetylcholine receptors (AChRs) and the axon excitation (different intervals between action potentials (APs)). Using Raman spectroscopy, an effect of activation of SC AChRs on the myelin membrane fluidity was investigated. It was found, that ACh stimulates an increase in lipid ordering degree of the myelin lipids, thus providing evidence for specific role of the “axon-SC” interactions at the axon excitation. It was proposed, that during the axon excitation, the SC membrane K+- depolarization and the Ca2+—influx led to phospholipase activation or exocytosis of intracellular membrane vesicles and myelin structure reorganization. PMID:27455410

  11. Dispersal and reformation of acetylcholine receptor clusters of cultured rat myotubes treated with inhibitors of energy metabolism.

    PubMed

    Bloch, R J

    1979-09-01

    The effects of energy metabolism inhibitors on the distribution of acetylcholine receptors (AChRs) in the surface membranes of non-innervated, cultured rat myotubes were studied by visualizing the AChRs with monotetramethylrhodamine-alpha-bungarotoxin. Incubation of myotubes with inhibitors of energy metabolism causes a large decrease in the fraction of myotubes displaying clusters of AChR. This decrease is reversible, and is dependent on temperature, the concentration of inhibitor, and the duration of treatment. Cluster dispersal is probably not the result of secondary effects on Ca++ or cyclic nucleotide metabolism, membrane potential, cytoskeletal elements, or protein synthesis. Sequential observations of identified cells treated with sodium azide showed that clusters appear to disperse by movements of receptors within the sarcolemma without accompanying changes in cell shape. AChR clusters dispersed by pretreating cells with sodium azide rapidly reform upon removal of the inhibitor. Reclustering involves the formation of small aggregates of AChR, which act as foci for further aggregation and which appear to be precursors of large AChR clusters. Small AChR aggregates also appear to be precursors of clusters which form on myotubes never exposed to azide. Reclustering after azide treatment does not necessarily occur at the same sites occupied by clusters before dispersal, nor does it employ only receptors which had previously been in clusters. Cluster reformation can be blocked by cycloheximide, colchicine, and drugs which alter the intracellular cation composition.

  12. The Role of α7 Nicotinic Acetylcholine Receptor in Modulation of Heart Rate Dynamics in Endotoxemic Rats

    PubMed Central

    Mazloom, Roham; Eftekhari, Golnar; Rahimi, Maryam; Khori, Vahid; Hajizadeh, Sohrab; Dehpour, Ahmad R.; Mani, Ali R.

    2013-01-01

    Previous reports have indicated that artificial stimulation of the vagus nerve reduces systemic inflammation in experimental models of sepsis. This phenomenon is a part of a broader cholinergic anti-inflammatory pathway which activates the vagus nerve to modulate inflammation through activation of alpha7 nicotinic acetylcholine receptors (α7nACHR). Heart rate variability represents the complex interplay between autonomic nervous system and cardiac pacemaker cells. Reduced heart rate variability and increased cardiac cycle regularity is a hallmark of clinical conditions that are associated with systemic inflammation (e.g. endotoxemia and sepsis). The present study was aimed to assess the role of α7nACHR in modulation of heart rate dynamics during systemic inflammation. Systemic inflammation was induced by injection of endotoxin (lipopolysaccharide) in rats. Electrocardiogram and body temperature were recorded in conscious animals using a telemetric system. Linear and non-linear indices of heart rate variability (e.g. sample entropy and fractal-like temporal structure) were assessed. RT-PCR and immunohistochemistry studies showed that α7nACHR is expressed in rat atrium and is mainly localized at the endothelial layer. Systemic administration of an α7nACHR antagonist (methyllycaconitine) did not show a significant effect on body temperature or heart rate dynamics in naïve rats. However, α7nACHR blockade could further reduce heart rate variability and elicit a febrile response in endotoxemic rats. Pre-treatment of endotoxemic animals with an α7nACHR agonist (PHA-543613) was unable to modulate heart rate dynamics in endotoxemic rats but could prevent the effect of endotoxin on body temperature within 24 h experiment. Neither methyllycaconitine nor PHA-543613 could affect cardiac beating variability of isolated perfused hearts taken from control or endotoxemic rats. Based on our observations we suggest a tonic role for nicotinic acetylcholine receptors in

  13. Muscarinic acetylcholine receptors in the nucleus accumbens core and shell contribute to cocaine priming-induced reinstatement of drug seeking

    PubMed Central

    Yee, Judy; Famous, Katie R.; Hopkins, Thomas J.; McMullen, Michael C.; Pierce, R. Christopher; Schmidt, Heath D.

    2011-01-01

    Muscarinic acetylcholine receptors in the nucleus accumbens play an important role in mediating the reinforcing effects of cocaine. However, there is a paucity of data regarding the role of accumbal muscarinic acetylcholine receptors in the reinstatement of cocaine-seeking behavior. The goal of these experiments was to assess the role of muscarinic acetylcholine receptors in the nucleus accumbens core and shell in cocaine and sucrose priming-induced reinstatement. Rats were initially trained to self-administer cocaine or sucrose on a fixed-ratio schedule of reinforcement. Lever-pressing behavior was then extinguished and followed by a subsequent reinstatement phase during which operant responding was induced by either a systemic injection of cocaine in cocaine-experienced rats or non-contingent delivery of sucrose pellets in subjects with a history of sucrose self-administration. Results indicated that systemic administration of the muscarinic acetylcholine receptor antagonist scopolamine (5.0 mg/kg, i.p.) dose-dependently attenuated cocaine, but not sucrose, reinstatement. Furthermore, administration of scopolamine (36.0 μg) directly into the nucleus accumbens shell or core attenuated cocaine-priming induced reinstatement. In contrast, infusion of scopolamine (36.0 μg) directly into the accumbens core, but not shell, attenuated sucrose reinstatement, which suggests that muscarinic acetylcholine receptors in these two subregions of the nucleus accumbens have differential roles in sucrose seeking. Taken together, these results indicate that cocaine-priming induced reinstatement is mediated, in part, by increased signaling through muscarinic acetylcholine receptors in the shell subregion of the nucleus accumbens. Muscarinic acetylcholine receptors in the core of the accumbens, in contrast, appear to play a more general (i.e. not cocaine specific) role in motivated behaviors. PMID:21034738

  14. Mammalian Nicotinic Acetylcholine Receptors: From Structure to Function

    PubMed Central

    Albuquerque, Edson X.; Pereira, Edna F. R.; Alkondon, Manickavasagom; Rogers, Scott W.

    2009-01-01

    The classical studies of nicotine by Langley at the turn of the 20th century introduced the concept of a “receptive substance,” from which the idea of a “receptor” came to light. Subsequent studies aided by the Torpedo electric organ, a rich source of muscle-type nicotinic receptors (nAChRs), and the discovery of α-bungarotoxin, a snake toxin that binds pseudo-irreversibly to the muscle nAChR, resulted in the muscle nAChR being the best characterized ligand-gated ion channel hitherto. With the advancement of functional and genetic studies in the late 1980s, the existence of nAChRs in the mammalian brain was confirmed and the realization that the numerous nAChR subtypes contribute to the psychoactive properties of nicotine and other drugs of abuse and to the neuropathology of various diseases, including Alzheimer’s, Parkinson’s, and schizophrenia, has since emerged. This review provides a comprehensive overview of these findings and the more recent revelations of the impact that the rich diversity in function and expression of this receptor family has on neuronal and nonneuronal cells throughout the body. Despite these numerous developments, our understanding of the contributions of specific neuronal nAChR subtypes to the many facets of physiology throughout the body remains in its infancy. PMID:19126755

  15. Common molecular mechanisms in field- and agrin-induced acetylcholine receptor clustering.

    PubMed

    Sabrina, F; Stollberg, J

    1997-04-01

    1. The aggregation of acetylcholine receptors at the developing neuromuscular junction is critical to the development and function of this synapse. In vitro studies have shown that receptor aggregation can be induced by the finding of agrin to the muscle cell surface and by the electric field-induced concentration of a (nonreceptor) molecule at the cathodal cell pole. 2. We report here on the interaction between agrin binding and electric fields with respect to the distribution of receptors and agrin binding sites. 3. (a) Pretreatment of cells with agrin completely blocks the development of field-induced receptor clusters. (b) Field-induced aggregation of receptors precedes the field-induced aggregation of agrin binding sites by approximately 30 min. (c) Electric fields prevent agrin-induced receptor clustering despite the presence of agrin binding sites and freely diffusing receptors. 4. These results indicate that another membrane component-but not the agrin binding site and not the receptor-is required for agrin-induced receptor clustering. They also suggest that electric fields and agrin cause receptor clustering via common molecular mechanisms.

  16. Subcellular localization of creatine kinase in Torpedo electrocytes: association with acetylcholine receptor-rich membranes

    PubMed Central

    1985-01-01

    Creatine kinase (CK, EC 2.7.3.2) has recently been identified as the intermediate isoelectric point species (pl 6.5-6.8) of the Mr 40,000- 43,000 nonreceptor, peripheral v-proteins in Torpedo marmorata acetylcholine receptor-rich membranes (Barrantes, F. J., G. Mieskes, and T. Wallimann, 1983, Proc. Natl. Acad. Sci. USA, 80: 5440-5444). In the present study, this finding is substantiated at the cellular and subcellular level of the T. marmorata electric organ by immunofluorescence and by protein A-gold labeling of either ultrathin cryosections of electrocytes or purified receptor-membrane vesicles that use subunit-specific anti-chicken creatine kinase antibodies. The muscle form of the kinase, on the one hand, is present throughout the entire T. marmorata electrocyte except in the nuclei. The brain form of the kinase, on the other hand, is predominantly located on the ventral, innervated face of the electrocyte, where it is closely associated with both surfaces of the postsynaptic membrane, and secondarily in the synaptic vesicles at the presynaptic terminal. Labeling of the noninnervated dorsal membrane is observed at the invaginated sac system. In the case of purified acetylcholine receptor-rich membranes, antibodies specific for chicken B-CK label only one face of the isolated vesicles. No immunoreaction is observed with anti-chicken M-CK antibodies. A discussion follows on the possible implications of these localizations of creatine kinase in connection with the function of the acetylcholine receptor at the postsynaptic membrane, the Na/K ATPase at the dorsal electrocyte membrane, and the ATP-dependent transmitter release at the nerve ending. PMID:3884630

  17. Pharmacological profile of zacopride and new quaternarized fluorobenzamide analogues on mammalian α7 nicotinic acetylcholine receptor.

    PubMed

    Bourdin, Céline M; Lebreton, Jacques; Mathé-Allainmat, Monique; Thany, Steeve H

    2015-08-15

    From quaternarization of quinuclidine enantiomers of 2-fluoro benzamide LMA10203 in dichloromethane, the corresponding N-chloromethyl derivatives LMA10227 and LMA10228 were obtained. Here, we compared the agonist action of known zacopride and its 2-fluoro benzamide analogues, LMA10203, LMA10227 and LMA10228 against mammalian homomeric α7 nicotinic acetylcholine receptor expressed in Xenopus oocytes. We found that LMA10203 was a partial agonist of α7 receptor with a pEC50 value of 4.25 ± 0.06 μM whereas LMA10227 and LMA10228 were poorly active on α7 homomeric nicotinic receptor. LMA10227 and LMA10228 were identified as antagonists of acetylcholine-induced currents with IC50 values of 28.4 μM and 39.3 μM whereas LMA10203 and zacopride possessed IC50 values of 8.07 μM and 7.04 μM, respectively. Moreover, despite their IC50 values, LMA10227 was the most potent inhibitor of nicotine-induced current amplitudes (65.7 ± 2.1% inhibition). LMA10203 and LMA10228 had the same inhibitory effects (26.5 ± 7.5% and 33.2 ± 4.1%, respectively), whereas zacopride had no significant inhibitory effect (4.37 ± 4%) on nicotine-induced responses. Our results revealed different pharmacological properties between the four compounds on acetylcholine and nicotine currents. The mode of action of benzamide compounds may need to be reinterpreted with respect to the potential role of α7 receptor.

  18. Reconstitution of Purified Acetylcholine Receptors with Functional Ion Channels in Planar Lipid Bilayers

    NASA Astrophysics Data System (ADS)

    Nelson, N.; Anholt, R.; Lindstrom, J.; Montal, M.

    1980-05-01

    Acetylcholine receptor, solubilized and purified from Torpedo californica electric organ under conditions that preserve the activity of its ion channel, was reconstituted into vesicles of soybean lipid by the cholate-dialysis technique. The reconstituted vesicles were then spread into monolayers at an air-water interface and planar bilayers were subsequently formed by apposition of two monolayers. Addition of carbamoylcholine caused an increase in membrane conductance that was transient and relaxed spontaneously to the base level (i.e., became desensitized). The response to carbamoylcholine was dose dependent and competitively inhibited by curare. Fluctuations of membrane conductance corresponding to the opening and closing of receptor channels were observed. Fluctuation analysis indicated a single-channel conductance of 16± 3 pS (in 0.1 M NaCl) with a mean channel open time estimated to be 35± 5 ms. Thus, purified acetylcholine receptor reconstituted into lipid bilayers exhibited the pharmacological specificity, activation, and desensitization properties expected of this receptor in native membranes.

  19. Clebopride enhances contractility of the guinea pig stomach by blocking peripheral D2 dopamine receptor and alpha-2 adrenoceptor

    SciTech Connect

    Takeda, K.; Taniyama, K.; Kuno, T.; Sano, I.; Ishikawa, T.; Ohmura, I.; Tanaka, C. )

    1991-05-01

    The mechanism of action of clebopride on the motility of guinea pig stomach was examined by the receptor binding assay for bovine brain membrane and by measuring gastric contractility and the release of acetylcholine from the stomach. The receptor binding assay revealed that clebopride bound to the D2 dopamine receptor with a high affinity and to the alpha-2 adrenoceptor and 5-HT2 serotonin receptor with relatively lower affinity, and not to D1 dopamine, alpha-1 adrenergic, muscarinic acetylcholine, H1 histamine, or opioid receptor. In strips of the stomach, clebopride at 10{sup {minus} 8} M to 10{sup {minus} 5} M enhanced the electrical transmural stimulation-evoked contraction and the release of acetylcholine. This enhancement was attributed to the blockade of the D2 dopamine receptor and alpha-2 adrenoceptor because: (1) Maximum responses obtained with specific D2 dopamine receptor antagonist, domperidone, and with specific alpha-2 adrenoceptor antagonist, yohimbine, were smaller than that with clebopride, and the sum of the effects of these two specific receptor antagonists is approximately equal to the effect of clebopride. (2) The facilitatory effect of clebopride was partially eliminated by pretreatment of the sample with domperidone or yohimbine, and the facilitatory effect of clebopride was not observed in preparations treated with the combination of domperidone and yohimbine. Clebopride also antagonized the inhibitory effects of dopamine and clonidine on the electrical transmural stimulation-evoked responses. These results indicate that clebopride acts on post ganglionic cholinergic neurons at D2 and alpha-2 receptors in this preparation to enhance enteric nervous system stimulated motility.

  20. Evidence from the anti-idiotypic network that the acetylcholine receptor is a rabies virus receptor.

    PubMed

    Hanham, C A; Zhao, F; Tignor, G H

    1993-01-01

    We have developed idiotype-anti-idiotype monoclonal antibodies that provide evidence for rabies virus binding to the acetylcholine receptor (AChR). Hybridoma cell lines 7.12 and 7.25 resulted after fusion of NS-1 myeloma cells with spleen cells from a BALB/c mouse immunized with rabies virus strain CVS. Antibody 7.12 reacted with viral glycoprotein and neutralized virus infectivity in vivo. It also neutralized infectivity in vitro when PC12 cells, which express neuronal AChR, but not CER cells or neuroblastoma cells (clone N18), which have no AChR, were used. Antibody 7.25 reacted with nucleocapsid protein. Anti-idiotypic monoclonal antibody B9 was produced from fusion of NS-1 cells with spleen cells from a mouse immunized with 7.12 Fab. In an enzyme-linked immunosorbent assay and immunoprecipitation, B9 reacted with 7.12, polyclonal rabies virus immune dog serum, and purified AChR. The binding of B9 to 7.12 and immune dog serum was inhibited by AChR. B9 also inhibited the binding of 7.12 to rabies virus both in vitro and in vivo. Indirect immunofluorescence revealed that B9 reacted at neuromuscular junctions of mouse tissue. B9 also reacted in indirect immunofluorescence with distinct neurons in mouse and monkey brain tissue as well as with PC12 cells. B9 staining of neuronal elements in brain tissue of rabies virus-infected mice was greatly reduced. Rabies virus inhibited the binding of B9 to PC12 cells. Mice immunized with B9 developed low-titer rabies virus-neutralizing antibody. These mice were protected from lethal intramuscular rabies virus challenge. In contrast, anti-idiotypic antibody raised against nucleocapsid antibody 7.25 did not react with AChR.

  1. Role in the selectivity of neonicotinoids of insect-specific basic residues in loop D of the nicotinic acetylcholine receptor agonist binding site.

    PubMed

    Shimomura, Masaru; Yokota, Maiko; Ihara, Makoto; Akamatsu, Miki; Sattelle, David B; Matsuda, Kazuhiko

    2006-10-01

    The insecticide imidacloprid and structurally related neonicotinoids act selectively on insect nicotinic acetylcholine receptors (nAChRs). To investigate the mechanism of neonicotinoid selectivity, we have examined the effects of mutations to basic amino acid residues in loop D of the nAChR acetylcholine (ACh) binding site on the interactions with imidacloprid. The receptors investigated are the recombinant chicken alpha4beta2 nAChR and Drosophila melanogaster Dalpha2/chicken beta2 hybrid nAChR expressed in Xenopus laevis oocytes. Although mutations of Thr77 in loop D of the beta2 subunit resulted in a barely detectable effect on the imidacloprid concentration-response curve for the alpha4beta2 nAChR, T77R;E79V double mutations shifted the curve dramatically to higher affinity binding of imidacloprid. Likewise, T77K;E79R and T77N;E79R double mutations in the Dalpha2beta2 nAChR also resulted in a shift to a higher affinity for imidacloprid, which exceeded that observed for a single mutation of Thr77 to basic residues. By contrast, these double mutations scarcely influenced the ACh concentration-response curve, suggesting selective interactions with imidacloprid of the newly introduced basic residues. Computational, homology models of the agonist binding domain of the wild-type and mutant alpha4beta2 and Dalpha2beta2 nAChRs with imidacloprid bound were generated based on the crystal structures of acetylcholine binding proteins of Lymnaea stagnalis and Aplysia californica. The models indicate that the nitro group of imidacloprid interacts directly with the introduced basic residues at position 77, whereas those at position 79 either prevent or permit such interactions depending on their electrostatic properties, thereby explaining the observed functional changes resulting from site-directed mutagenesis.

  2. Implication of a multisubunit Ets-related transcription factor in synaptic expression of the nicotinic acetylcholine receptor.

    PubMed Central

    Schaeffer, L; Duclert, N; Huchet-Dymanus, M; Changeux, J P

    1998-01-01

    In adult muscle, transcription of the nicotinic acetylcholine receptor (AChR) is restricted to the nuclei located at the neuromuscular junction. The N-box, a new promoter element, was identified recently and shown to contribute to this compartmentalized synaptic expression of the AChR delta- and epsilon-subunits. We demonstrate that the N-box mediates transcriptional activation in cultured myotubes and identify the transcription factor that binds to the N-box as a heterooligomer in myotubes and adult muscle. The GABP (GA-binding protein) alpha-subunit belongs to the Ets family of transcription factors, whereas the beta-subunit shares homology with IkappaB and Drosophila Notch protein. GABP binding specificity to mutated N-box in vitro strictly parallels the sequence requirement for beta-galactosidase targeting to the endplate in vivo. In situ hybridization studies reveal that the mRNAs of both GABP subunits are abundant in mouse diaphragm, with preferential expression of the alpha-subunit at motor endplates. In addition, heregulin increases GABPalpha protein levels and regulates phosphorylation of both subunits in cultured chick myotubes. Finally, dominant-negative mutants of either GABPalpha or GABPbeta block heregulin-elicited transcriptional activation of the AChR delta and epsilon genes. These findings establish the expected connection with a presynaptic trophic factor whose release contributes to the accumulation of AChR subunit mRNAs at the motor endplate. PMID:9606190

  3. Molecular dynamics simulation of the M2 helices within the nicotinic acetylcholine receptor transmembrane domain: structure and collective motions.

    PubMed

    Hung, Andrew; Tai, Kaihsu; Sansom, Mark S P

    2005-05-01

    Multiple nanosecond duration molecular dynamics simulations were performed on the transmembrane region of the Torpedo nicotinic acetylcholine receptor embedded within a bilayer mimetic octane slab. The M2 helices and M2-M3 loop regions were free to move, whereas the outer (M1, M3, M4) helix bundle was backbone restrained. The M2 helices largely retain their hydrogen-bonding pattern throughout the simulation, with some distortions in the helical end and loop regions. All of the M2 helices exhibit bending motions, with the hinge point in the vicinity of the central hydrophobic gate region (corresponding to residues alphaL251 and alphaV255). The bending motions of the M2 helices lead to a degree of dynamic narrowing of the pore in the region of the proposed hydrophobic gate. Calculations of Born energy profiles for various structures along the simulation trajectory suggest that the conformations of the M2 bundle sampled correspond to a closed conformation of the channel. Principal components analyses of each of the M2 helices, and of the five-helix M2 bundle, reveal concerted motions that may be relevant to channel function. Normal mode analyses using the anisotropic network model reveal collective motions similar to those identified by principal components analyses.

  4. Muscarinic acetylcholine receptor-mediated stimulation of retinal ganglion cell photoreceptors.

    PubMed

    Sodhi, Puneet; Hartwick, Andrew T E

    2016-09-01

    Melanopsin-dependent phototransduction in intrinsically photosensitive retinal ganglion cells (ipRGCs) involves a Gq-coupled phospholipase C (PLC) signaling cascade. Acetylcholine, released in the mammalian retina by starburst amacrine cells, can also activate Gq-PLC pathways through certain muscarinic acetylcholine receptors (mAChRs). Using multielectrode array recordings of rat retinas, we demonstrate that robust spiking responses can be evoked in neonatal and adult ipRGCs after bath application of the muscarinic agonist carbachol. The stimulatory action of carbachol on ipRGCs was a direct effect, as confirmed through calcium imaging experiments on isolated ipRGCs in purified cultures. Using flickering (6 Hz) yellow light stimuli at irradiances below the threshold for melanopsin activation, spiking responses could be elicited in ipRGCs that were suppressed by mAChR antagonism. Therefore, this work identified a novel melanopsin-independent pathway for stimulating sustained spiking in ganglion cell photoreceptors. This mAChR-mediated pathway could enhance ipRGC spiking responses in conditions known to evoke retinal acetylcholine release, such as those involving flickering or moving visual stimuli. Furthermore, this work identifies a pharmacological approach for light-independent ipRGC stimulation that could be targeted by mAChR agonists.

  5. Neuronal nicotinic acetylcholine receptor activation modulates gamma-aminobutyric acid release from CA1 neurons of rat hippocampal slices.

    PubMed

    Alkondon, M; Pereira, E F; Barbosa, C T; Albuquerque, E X

    1997-12-01

    In the present study we investigated electrophysiologically the nicotinic responses of pyramidal neurons and interneurons visualized by infrared-assisted videomicroscopy and fluorescence in the CA1 field of hippocampal slices obtained from 8- to 24-day-old rats. Application of nicotinic agonists to CA1 neurons evoked at least four types of nicotinic responses. Of major interest was the ability of these agonists to induce the release of gamma-aminobutyric acid (GABA) from interneurons. Slowly decaying ACh whole-cell currents and GABA-mediated postsynaptic currents could be recorded from pyramidal neurons and interneurons, whereas fast-decaying nicotinic currents and fast current transients were recorded only from interneurons. Nicotinic responses were sensitive to blockade by d-tubocurarine (10 microM), which indicated that they were mediated by nicotinic acetylcholine receptors (nAChRs). The slowly decaying currents, the postsynaptic currents and the fast current transients were insensitive to blockade by the alpha-7 nAChR-specific antagonist methyllycaconitine (up to 1 microM) or alpha-bungarotoxin (100 nM). On the other hand, the slowly decaying nicotinic currents recorded from the interneurons were blocked by the alpha4beta2 nAChR-specific antagonist dihydro-beta-erythroidine, and the fast-desensitizing nicotinic currents were evoked by the alpha-7 nAChR-specific agonist choline. In experimental conditions similar to those used to record nicotinic responses from neurons in slice (i. e., in the absence of tetrodotoxin), we observed that nicotinic agonists can also induce the release of GABA from hippocampal neurons in culture. In summary, these results provide direct evidence for more than one subtype of functional nAChR in CA1 neurons and suggest that activation of nAChRs present in GABAergic interneurons can evoke inhibitory activity in CA1 pyramidal neurons, thereby modulating processing of information in the hippocampus.

  6. Number of junctional acetylcholine receptors: control by neural and muscular influences in the rat.

    PubMed Central

    Andreose, J S; Fumagalli, G; Lømo, T

    1995-01-01

    1. The number of acetylcholine receptors (AChRs) per neuromuscular junction in soleus muscles of adult rats was estimated from counts of 125I-alpha-bungarotoxin binding sites. The muscles were either denervated, denervated and electrically stimulated, paralysed by botulinum toxin (BoTX), or paralysed by tetrodotoxin (TTX). 2. After denervation, the number of junctional AChRs was normal after 18 days and then fell to 54 and 35% of normal after 33 and 57 days, respectively. 3. Direct high frequency muscle stimulation (100 Hz) maintained a normal number of junctional AChRs for at least 2 months when the stimulation started on the day of denervation. When the stimulation was started progressively later, the effect of the stimulation on AChR number disappeared within about a week. The disappearance was gradual and appeared to affect all the muscle fibres equally. 4. Stimulation at 100 Hz, starting on the day of denervation and stopping after 18 days, did not prevent the endplates from losing AChRs during the subsequent 15 days without stimulation. Thus 100 Hz stimulation and innervation are not equivalent in their effects on junctional AChR number. 5. Direct low frequency muscle stimulation from the day of denervation did not maintain a normal number of junctional AChRs, as the number of AChRs fell to 70 and 62% of normal after 33 days of stimulation at 20 and 10 Hz, respectively. 6. Endplates paralysed by BoTX or TTX for 33 days lost about as many junctional AChRs (54 and 55%) as endplates denervated for 33 days (46%). Direct stimulation at 100 Hz during the last 15 days of BoTX treatment reduced but did not prevent this AChR loss (36% loss at 33 days). 7. The results show that when motor nerve terminals in rat soleus muscles are removed by axotomy, they leave a 'trace' which, in conjunction with appropriate muscle stimulation, can maintain a normal number of AChRs in the postsynaptic region. In non-stimulated muscles the trace responsible for this maintenance

  7. Selective actions of Lynx proteins on different nicotinic acetylcholine receptors in the locust, Locusta migratoria manilensis.

    PubMed

    Wang, Xin; Bao, Haibo; Sun, Huahua; Zhang, Yixi; Fang, Jichao; Liu, Qinghong; Liu, Zewen

    2015-08-01

    Nicotinic acetylcholine receptors (nAChRs) are major neurotransmitter receptors and targets of neonicotinoid insecticides in the insect nervous system. The full function of nAChRs is often dependent on associated proteins, such as chaperones, regulators and modulators. Here, three Lynx (Ly-6/neurotoxin) proteins, Loc-lynx1, Loc-lynx2 and Loc-lynx3, were identified in the locust, Locusta migratoria manilensis. Co-expression with Lynx resulted in a dramatic increase in agonist-evoked macroscopic currents on nAChRs Locα1/β2 and Locα2/β2 in Xenopus oocytes, but no changes in agonist sensitivity. Loc-lynx1 and Loc-lynx3 only modulated nAChRs Locα1/β2 while Loc-lynx2 modulated Locα2/β2 specifically. Meanwhile, Loc-lynx1 induced a more significant increase in currents evoked by imidacloprid and epibatidine than Loc-lynx3, and the effects of Loc-lynx1 on imidacloprid and epibatidine were significantly higher than those on acetylcholine. Among three lynx proteins, only Loc-lynx1 significantly increased [(3) H]epibatidine binding on Locα1/β2. The results indicated that Loc-lynx1 had different modulation patterns in nAChRs compared to Loc-lynx2 and Loc-lynx3. Taken together, these findings indicated that three Lynx proteins were nAChR modulators and had selective activities in different nAChRs. Lynx proteins might display their selectivities from three aspects: nAChR subtypes, various agonists and different modulation patterns. Insect Lynx (Ly-6/neurotoxin) proteins act as the allosteric modulators on insect nicotinic acetylcholine receptors (nAChRs), the important targets of insecticides. We found that insect lynx proteins showed their selectivities from at least three aspects: nAChR subtypes, various agonists and different modulation patterns.

  8. Pharmacologic specificity of alpha-2 adrenergic receptor subtypes

    SciTech Connect

    Petrash, A.; Bylund, D.

    1986-03-01

    The authors have defined alpha-2 adrenergic receptor subtypes in human and rat tissues using prazosin as a subtype selective drug. Prazosin has a lower affinity (250 nM) at alpha-2A receptor and a higher affinity (5 nM) at alpha-2B receptors. In order to determine if other adrenergic drugs are selective for one or the other subtypes, the authors performed (/sup 3/H)yohimbine inhibition experiments with various adrenergic drugs in tissues containing alpha-2A, alpha-2B or both subtypes. Oxymetazoline, WB4101 and yohimbine were found to be 80-, 20- and 10-fold more potent at alpha-2A receptors than at alpha-2B receptors. Phentolamine, adazoxan, (+)- and (-)-mianserin, clonidine, (+)-butaclamol, (-)- and (+)-norepinephrine, epinephrine, dopamine and thioridazine were found to have equal affinities for the two subtypes. These results further validate the subdivision of alpha-2 adrenergic receptors into alpha-2A and alpha-2B subtypes.

  9. A fluorinated quinuclidine benzamide named LMA 10203 acts as an agonist of insect nicotinic acetylcholine receptors.

    PubMed

    Mathé-Allainmat, Monique; Bodereau-Dubois, Béatrice; Lapied, Bruno; Lebreton, Jacques; Thany, Steeve H

    2012-06-01

    In the present study, we take advantage of the fact that cockroach dorsal unpaired median neurons express different nicotinic acetylcholine receptor subtypes to demonstrate that simple quinuclidine benzamides such as the 2-fluorinated benzamide LMA 10203, could act as an agonist of cockroach α-bungarotoxin-insensitive nicotinic acetylcholine receptor subtype, called nAChR2. Indeed, 1 mM LMA 10203 induced ionic currents which were partially blocked by 0.5 μM α-bungarotoxin and methyllycaconitine and completely blocked by 5 μM mecamylamine. Moreover, the current-voltage curve revealed that the ionic current induced by LMA 10203 increased from -30 mV to +20 mV confirming that it acted as an agonist of α-bungarotoxin-insensitive nAChR2. In addition, 1 mM LMA 10203 induced a depolarization of the sixth abdominal ganglion and this neuroexcitatory activity was completely blocked by 5 μM mecamylamine. These data suggest that nAChR2 was also expressed at the postsynaptic level on the synapse between the cercal afferent nerve and the giant interneurons. Interestingly, despite LMA 10203 being an agonist of cockroach nicotinic receptors, it had a poor insecticidal activity. We conclude that LMA 10203 could be used as an interesting compound to identify specific insect nAChR subtypes.

  10. Three austin family compounds from Penicillium brasilianum exhibit selective blocking action on cockroach nicotinic acetylcholine receptors.

    PubMed

    Kataoka, Saori; Furutani, Shogo; Hirata, Koichi; Hayashi, Hideo; Matsuda, Kazuhiko

    2011-01-01

    Austin (AT) and its derivatives (dehydroaustin (DAT) and acetoxydehydroaustin (ADAT)) produced by Penicillium brasilianum MG-11 exhibit toxicity to insects, yet their targets are unknown. Here, we used whole-cell patch-clamp electrophysiology to investigate the action of AT family compounds on cockroach acetylcholine (ACh), γ-aminobutyric acid (GABA) and l-glutamate receptors expressed in the American cockroach (Periplaneta americana) neuron. U-tube application of AT or its derivatives did not induce any current amplitudes, suggesting that they did not act as agonist of these three receptors. In the second step of experiments, they were bath-applied for 1min before co-application with the corresponding ligand. We found that AT and its derivatives had no effect on GABA and l-glutamate-induced currents, whereas they significantly reduced ACh- and epibatidine-induced currents, showing that these compounds acted as selective antagonists of nicotinic acetylcholine receptors (nAChRs) expressed in the cockroach neuron. Of the compounds, DAT showed the highest blocking potency for nAChRs, differentially attenuating the peak and slowly desensitizing current amplitude of ACh-induced responses with pIC(50) (=-logIC(50) (M)) values of 6.11 and 5.91, respectively. DAT reduced the maximum normalized response to ACh without a significant shift in EC(50), suggesting that the blocking action is not competitive with ACh.

  11. An ER-resident membrane protein complex regulates nicotinic acetylcholine receptor subunit composition at the synapse

    PubMed Central

    Almedom, Ruta B; Liewald, Jana F; Hernando, Guillermina; Schultheis, Christian; Rayes, Diego; Pan, Jie; Schedletzky, Thorsten; Hutter, Harald; Bouzat, Cecilia; Gottschalk, Alexander

    2009-01-01

    Nicotinic acetylcholine receptors (nAChRs) are homo- or heteropentameric ligand-gated ion channels mediating excitatory neurotransmission and muscle activation. Regulation of nAChR subunit assembly and transfer of correctly assembled pentamers to the cell surface is only partially understood. Here, we characterize an ER transmembrane (TM) protein complex that influences nAChR cell-surface expression and functional properties in Caenorhabditis elegans muscle. Loss of either type I TM protein, NRA-2 or NRA-4 (nicotinic receptor associated), affects two different types of muscle nAChRs and causes in vivo resistance to cholinergic agonists. Sensitivity to subtype-specific agonists of these nAChRs is altered differently, as demonstrated by whole-cell voltage-clamp of dissected adult muscle, when applying exogenous agonists or after photo-evoked, channelrhodopsin-2 (ChR2) mediated acetylcholine (ACh) release, as well as in single-channel recordings in cultured embryonic muscle. These data suggest that nAChRs desensitize faster in nra-2 mutants. Cell-surface expression of different subunits of the ‘levamisole-sensitive' nAChR (L-AChR) is differentially affected in the absence of NRA-2 or NRA-4, suggesting that they control nAChR subunit composition or allow only certain receptor assemblies to leave the ER. PMID:19609303

  12. Vector-averaged gravity does not alter acetylcholine receptor single channel properties

    NASA Technical Reports Server (NTRS)

    Reitstetter, R.; Gruener, R.

    1994-01-01

    To examine the physiological sensitivity of membrane receptors to altered gravity, we examined the single channel properties of the acetylcholine receptor (AChR), in co-cultures of Xenopus myocytes and neurons, to vector-averaged gravity in the clinostat. This experimental paradigm produces an environment in which, from the cell's perspective, the gravitational vector is "nulled" by continuous averaging. In that respect, the clinostat simulates one aspect of space microgravity where the gravity force is greatly reduced. After clinorotation, the AChR channel mean open-time and conductance were statistically not different from control values but showed a rotation-dependent trend that suggests a process of cellular adaptation to clinorotation. These findings therefore suggest that the ACHR channel function may not be affected in the microgravity of space despite changes in the receptor's cellular organization.

  13. α7-nicotinic acetylcholine receptor agonists for cognitive enhancement in schizophrenia.

    PubMed

    Freedman, Robert

    2014-01-01

    α7-Nicotinic acetylcholine receptors have emerged as a potential therapeutic target for the treatment of neurocognitive dysfunctions in schizophrenia that are often resistant to existing antipsychotic drugs. Molecular evidence for involvement in schizophrenia of CHRNA7, the gene for the receptor subunit, in the neurobiology of deficits in attention is a critical rationale for the clinical study of α7-nicotinic receptor agonists to improve neurocognition. Initial clinical trials show enhancement of inhibitory neuron function related to sensory gating and increased attention and working memory, as well as improvement in negative symptoms such as anhedonia and alogia. Further development of this therapeutic strategy requires assessment of interactions with patients' heavy cigarette smoking and the relationship of this mechanism to the therapeutic effects of clozapine and olanzapine, both highly effective therapeutics with significant side effects.

  14. Desensitization of human muscarinic acetylcholine receptor m2 subtypes is caused by their sequestration/internalization.

    PubMed

    Tsuga, H; Kameyama, K; Haga, T

    1998-10-01

    Desensitization of human muscarinic acetylcholine receptor m2 subtypes (hm2 receptors) stably expressed in chinese hamster ovary cells was measured as decreases in the carbamylcholine-stimulated [35S]GTPgammaS binding activity in membrane preparations after pre-treatment of cells with carbamylcholine. The extent of carbamylcholine-stimulated [35S]GTPgammaS binding activity was found to decrease to 64% following pretreatment of cells with 10 microM carbamylcholine for 30 min, and under the same conditions 51-59% of hm2 receptors were sequestered/internalized as assessed by decreases in the [3H]N-methylscopolamine binding activity on the cell surface. A similar reduction in the carbamylcholine-stimulated [35S]GTPgammaS binding activity was observed by pretreatment of cells with 5 nM propylbenzylylcholine mustard, which irreversibly bound to and inactivated 58% of the hm2 receptors. When the cells were pretreated with 10 microM carbamylcholine in the presence of 0.32 M sucrose, which is known to inhibit clathrin-mediated endocytosis, no sequestration/internalization of hm2 receptors was observed, and the extent of carbamylcholine-stimulated [35S]GTPgammaS binding activity did not change. These results indicate that desensitization of hm2 receptors may be caused by reduction of receptor number on the cell surface through sequestration/internalization rather than by loss of the function of receptors.

  15. Some properties of human neuronal α7 nicotinic acetylcholine receptors fused to the green fluorescent protein

    PubMed Central

    Palma, Eleonora; Mileo, Anna M.; Martínez-Torres, Ataúlfo; Eusebi, Fabrizio; Miledi, Ricardo

    2002-01-01

    The functional properties and cellular localization of the human neuronal α7 nicotinic acetylcholine (AcCho) receptor (α7 AcChoR) and its L248T mutated (mut) form were investigated by expressing them alone or as gene fusions with the enhanced version of the green fluorescent protein (GFP). Xenopus oocytes injected with wild-type (wt), mutα7, or the chimeric subunit cDNAs expressed receptors that gated membrane currents when exposed to AcCho. As already known, AcCho currents generated by wtα7 receptors decay much faster than those elicited by the mutα7 receptors. Unexpectedly, the fusion of GFP to the wt and mutated α7 receptors led to opposite results: the AcCho-current decay of the wt receptors became slower, whereas that of the mutated receptors was accelerated. Furthermore, repetitive applications of AcCho led to a considerable “run-down” of the AcCho currents generated by mutα7-GFP receptors, whereas those of the wtα7-GFP receptors remained stable or increased in amplitude. The AcCho-current run-down of mutα7-GFP oocytes was accompanied by a marked decrease of α-bungarotoxin binding activity. Fluorescence, caused by the chimeric receptors expressed, was seen over the whole oocyte surface but was more intense and abundant in the animal hemisphere, whereas it was much weaker in the vegetal hemisphere. We conclude that fusion of GFP to wtα7 and mutα7 receptors provides powerful tools to study the distribution and function of α7 receptors. We also conclude that fused genes do not necessarily recapitulate all of the properties of the original receptors. This fact must be borne close in mind whenever reporter genes are attached to proteins. PMID:11891308

  16. Nicotine trapping causes the persistent desensitization of alpha4beta2 nicotinic receptors expressed in oocytes.

    PubMed

    Jia, Li; Flotildes, Karen; Li, Maureen; Cohen, Bruce N

    2003-02-01

    To determine whether prolonged nicotine exposure persistently inactivates rat alpha4beta2 nicotinic receptors expressed in Xenopus oocytes, we measured the voltage-clamped alpha4beta2 response to acetylcholine (ACh) before and 24 h after, 1-h or 12-h incubations in 10 microm nicotine. A 12-h incubation in 10 microm nicotine depressed the alpha4beta2 ACh response for 24 h without affecting total or surface alpha4beta2 expression. To determine whether oocyte-mediated nicotine release caused this depression, we co-incubated an alpha4beta2-expressing oocyte with an un-injected one (pre-incubated in 10 microm nicotine for 12 h) for 24 h and measured the change in the alpha4beta2 ACh response. The response decreased by the same factor after the co-incubation as it did after a 12-h incubation in 10 microm nicotine and a 24-h incubation in nicotine-free media. Thus, oocyte-mediated nicotine release caused the persistent desensitization we observed after a 12-h incubation in 10 microm nicotine. Consistent with this result, measurements of [3H]nicotine release show that oocytes release enough nicotine into the wash media to desensitize alpha4beta2 receptors and that prolonged incubation in 300 microm ACh (which cannot readily cross the membrane or accumulate in acidic vesicles) did not persistently depress the alpha4beta2 response.

  17. Rare human nicotinic acetylcholine receptor α4 subunit (CHRNA4) variants affect expression and function of high-affinity nicotinic acetylcholine receptors.

    PubMed

    McClure-Begley, T D; Papke, R L; Stone, K L; Stokes, C; Levy, A D; Gelernter, J; Xie, P; Lindstrom, J; Picciotto, M R

    2014-03-01

    Nicotine, the primary psychoactive component in tobacco smoke, produces its behavioral effects through interactions with neuronal nicotinic acetylcholine receptors (nAChRs). α4β2 nAChRs are the most abundant in mammalian brain, and converging evidence shows that this subtype mediates the rewarding and reinforcing effects of nicotine. A number of rare variants in the CHRNA4 gene that encode the α4 nAChR subunit have been identified in human subjects and appear to be underrepresented in a cohort of smokers. We compared three of these variants (α4R336C, α4P451L, and α4R487Q) to the common variant to determine their effects on α4β2 nAChR pharmacology. We examined [(3)H]epibatidine binding, interacting proteins, and phosphorylation of the α4 nAChR subunit with liquid chromatography and tandem mass spectrometry (LC-MS/MS) in HEK 293 cells and voltage-clamp electrophysiology in Xenopus laevis oocytes. We observed significant effects of the α4 variants on nAChR expression, subcellular distribution, and sensitivity to nicotine-induced receptor upregulation. Proteomic analysis of immunopurified α4β2 nAChRs incorporating the rare variants identified considerable differences in the intracellular interactomes due to these single amino acid substitutions. Electrophysiological characterization in X. laevis oocytes revealed alterations in the functional parameters of activation by nAChR agonists conferred by these α4 rare variants, as well as shifts in receptor function after incubation with nicotine. Taken together, these experiments suggest that genetic variation at CHRNA4 alters the assembly and expression of human α4β2 nAChRs, resulting in receptors that are more sensitive to nicotine exposure than those assembled with the common α4 variant. The changes in nAChR pharmacology could contribute to differences in responses to smoked nicotine in individuals harboring these rare variants.

  18. Role of Muscarinic Acetylcholine Receptor-2 in the Cerebellar Cortex in Cardiovascular Modulation in Anaesthetized Rats.

    PubMed

    Zhang, Changzheng; Sun, Tingzhe; Zhou, Peiling; Zhu, Qingfeng; Zhang, Liefeng

    2016-04-01

    Our previous investigations have demonstrated that microinjection of acetylcholine (ACh) or muscarinic ACh receptor activation in the cerebellar cortex induces a systemic blood pressure depressor response. This study aimed to determine the role of muscarinic ACh receptor-2 (M2 receptor) in the cerebellar cortex in cardiovascular function regulation in rats. A nonselective muscarinic receptor agonist (oxotremorine M, OXO; 30 mM), a selective M2 receptor agonist (arecaidine but-2-ynyl ester tosylate, ABET; 3, 10, and 30 mM), 30 mM OXO mixed with a selective M2 receptor antagonist (methoctramine hydrate, MCT; 0.3, 1, and 3 mM), and normal saline (0.9 % NaCl) were separately microinjected (0.5 µl/5 s) into the cerebellar cortex (lobule VI) of anaesthetized rats. We measured the mean arterial pressure (MAP), maximum change in MAP, and reactive time (RT; the duration required for the blood pressure to return to basal levels), heart rate (HR) and the maximum change in HR during the RT in response to drug activation. The results demonstrated that ABET dose-dependently decreased MAP and HR, increased the maximum change in MAP and the maximum change in HR, and prolonged the RT. Furthermore, MCT dose-dependently blocked the OXO-mediated cardiovascular depressor response. This study provides the first evidence that M2 receptors in the cerebellar cortex are involved in cardiovascular regulation, the activation of which evokes significant depressor and bradycardic responses.

  19. Subtype-selective nicotinic acetylcholine receptor agonists enhance the responsiveness to citalopram and reboxetine in the mouse forced swim test.

    PubMed

    Andreasen, Jesper T; Nielsen, Elsebet Ø; Christensen, Jeppe K; Olsen, Gunnar M; Peters, Dan; Mirza, Naheed R; Redrobe, John P

    2011-10-01

    Nicotine increases serotonergic and noradrenergic neuronal activity and facilitates serotonin and noradrenaline release. Accordingly, nicotine enhances antidepressant-like actions of reuptake inhibitors selective for serotonin or noradrenaline in the mouse forced swim test and the mouse tail suspension test. Both high-affinity α4β2 and low-affinity α7 nicotinic acetylcholine receptor subtypes are implicated in nicotine-mediated release of serotonin and noradrenaline. The present study therefore investigated whether selective agonism of α4β2 or α7 nicotinic acetylcholine receptors would affect the mouse forced swim test activity of two antidepressants with distinct mechanisms of action, namely the selective serotonin reuptake inhibitor citalopram and the noradrenaline reuptake inhibitor reboxetine. Subthreshold and threshold doses of citalopram (3 and 10 mg/kg) or reboxetine (10 and 20 mg/kg) were tested alone and in combination with the novel α4β2-selective partial nicotinic acetylcholine receptor agonist, NS3956 (0.3 and 1.0 mg/kg) or the α7-selective nicotinic acetylcholine receptor agonist, PNU-282987 (10 and 30 mg/kg). Alone, NS3956 and PNU-282987 were devoid of activity in the mouse forced swim test, but both 1.0 mg/kg NS3956 and 30 mg/kg PNU-282987 enhanced the effect of citalopram and also reboxetine. The data suggest that the activity of citalopram and reboxetine in the mouse forced swim test can be enhanced by agonists at either α4β2 or α7 nicotinic acetylcholine receptors, suggesting that both nicotinic acetylcholine receptor subtypes may be involved in the nicotine-enhanced action of antidepressants.

  20. The nicotinic receptor in the rat pineal gland is an alpha3beta4 subtype.

    PubMed

    Hernandez, Susan C; Vicini, Stefano; Xiao, Yingxian; Dávila-García, Martha I; Yasuda, Robert P; Wolfe, Barry B; Kellar, Kenneth J

    2004-10-01

    The rat pineal gland contains a high density of neuronal nicotinic acetylcholine receptors (nAChRs). We characterized the pharmacology of the binding sites and function of these receptors, measured the nAChR subunit mRNA, and used subunit-specific antibodies to establish the receptor subtype as defined by subunit composition. In ligand binding studies, [3H]epibatidine ([3H]EB) binds with an affinity of approximately 100 pM to nAChRs in the pineal gland, and the density of these sites is approximately 5 times that in rat cerebral cortex. The affinities of nicotinic drugs for binding sites in the pineal gland are similar to those at alpha3beta4 nAChRs heterologously expressed in human embryonic kidney 293 cells. In functional studies, the potencies and efficacies of nicotinic drugs to activate or block whole-cell currents in dissociated pinealocytes match closely their potencies and efficacies to activate or block 86Rb+ efflux in the cells expressing heterologous alpha3beta4 nAChRs. Measurements of mRNA indicated the presence of transcripts for alpha3, beta2, and beta4 nAChR subunits but not those for alpha2, alpha4, alpha5, alpha6, alpha7, or beta3 subunits. Immunoprecipitation with subunit-specific antibodies showed that virtually all [3H]EB-labeled nAChRs contained alpha3 and beta4 subunits associated in one complex. The beta2 subunit was not associated with this complex. Taken together, these results indicate that virtually all of the nAChRs in the rat pineal gland are the alpha3beta4 nAChR subtype and that the pineal gland can therefore serve as an excellent and convenient model in which to study the pharmacology and function of these receptors in a native tissue.

  1. CNS acetylcholine receptor activity in European medicinal plants traditionally used to improve failing memory.

    PubMed

    Wake, G; Court, J; Pickering, A; Lewis, R; Wilkins, R; Perry, E

    2000-02-01

    Certain Lamiaceous and Asteraceous plants have long histories of use as restoratives of lost or declining cognitive functions in western European systems of traditional medicine. Investigations were carried out to evaluate human CNS cholinergic receptor binding activity in extracts of those European medicinal plants reputed to enhance or restore mental functions including memory. Ethanolic extracts were prepared from accessions of these plants and a number of other species related by genus. Amongst the plant extracts screened for contents able to displace [3H]-(N)-nicotine and [3H]-(N)-scopolamine from nicotinic receptors and muscarinic receptors, respectively in homogenates of human cerebral cortical cell membranes, the most potent extracts, prepared from one accession of Melissa officinalis, three Salvia species and Artemisia absinthium had IC50 concentrations of < 1 mg/ml. The displacement curves of some extracts were comparable with that of carbamylcholine chloride, a potent acetylcholine analogue. Choline, a weak nicotinic ligand (IC50 = 3 x 10(-4) M) was found in extracts of all plants studied at concentrations of 10(-6)-10(-5) M. These concentrations could not account for not more than 5% of the displacement activity observed. Some extracts displayed differential displacement at nicotinic and muscarinic acetylcholine receptors, with M. officinalis 0033 having the highest [3H]-(N)-nicotine displacement value and Salvia elegans with the highest [3H]-(N)-scopolamine displacement value. There was also considerable variation in cholinoreceptor interactions between different accessions of a single plant species. Although most plant extracts screened showed some nicotinic and muscarinic activity, only some showed dose-dependent receptor activity typical of materials with genuine cholinergic activity.

  2. Identification of a hexapeptide that mimics a conformation-dependent binding site of acetylcholine receptor by use of a phage-epitope library.

    PubMed Central

    Balass, M; Heldman, Y; Cabilly, S; Givol, D; Katchalski-Katzir, E; Fuchs, S

    1993-01-01

    Monoclonal antibody (mAb) 5.5 is directed against the ligand-binding site of the nicotinic acetylcholine receptor. The epitope for this antibody is conformation-dependent, and the antibody does not react with synthetic peptides derived from the receptor sequence. We have identified a ligand peptide that mimics this conformation-dependent epitope from a phage-epitope library composed of filamentous phage displaying random hexapeptides. Among 38 positive phage clones, individually selected from the library, 34 positive clones carried the sequence Asp-Leu-Val-Trp-Leu-Leu (DLVWLL), 1 positive clone had the sequence Asp-Ile-Val-Trp-Leu-Leu (DIVWLL), and 3 positive clones expressed the sequence Leu-Ile-Glu-Trp-Leu-Leu (LIEWLL), none of which are significantly homologous with the nicotinic acetylcholine receptor alpha subunit sequence. All of these phages bind specifically to mAb 5.5. The synthetic peptide DLVWLL inhibits binding of mAb 5.5 to the related peptide-presenting phage and to the nicotinic acetylcholine receptor in a concentration-dependent manner; the IC50 value is of the order of 10(-4) M. Bioactivity of the peptide "mimotope" DLVWLL was demonstrated in vivo in hatched chickens by inhibition of the mAb 5.5 effect by the peptide. The neuromuscular block and myasthenia gravis-like symptoms that are induced in chicken by passive transfer of mAb 5.5 were specifically abolished by DLVWLL. This study shows the potential of a random peptide phage-epitope library for selecting a mimotope for an antibody that recognizes a folded form of the protein, where peptides from the linear amino acid sequence of the protein are not applicable. Images Fig. 5 PMID:7504273

  3. Activation of Muscarinic M1 Acetylcholine Receptors Induces Long-Term Potentiation in the Hippocampus

    PubMed Central

    Dennis, Siobhan H.; Pasqui, Francesca; Colvin, Ellen M.; Sanger, Helen; Mogg, Adrian J.; Felder, Christian C.; Broad, Lisa M.; Fitzjohn, Steve M.; Isaac, John T.R.; Mellor, Jack R.

    2016-01-01

    Muscarinic M1 acetylcholine receptors (M1Rs) are highly expressed in the hippocampus, and their inhibition or ablation disrupts the encoding of spatial memory. It has been hypothesized that the principal mechanism by which M1Rs influence spatial memory is by the regulation of hippocampal synaptic plasticity. Here, we use a combination of recently developed, well characterized, selective M1R agonists and M1R knock-out mice to define the roles of M1Rs in the regulation of hippocampal neuronal and synaptic function. We confirm that M1R activation increases input resistance and depolarizes hippocampal CA1 pyramidal neurons and show that this profoundly increases excitatory postsynaptic potential-spike coupling. Consistent with a critical role for M1Rs in synaptic plasticity, we now show that M1R activation produces a robust potentiation of glutamatergic synaptic transmission onto CA1 pyramidal neurons that has all the hallmarks of long-term potentiation (LTP): The potentiation requires NMDA receptor activity and bi-directionally occludes with synaptically induced LTP. Thus, we describe synergistic mechanisms by which acetylcholine acting through M1Rs excites CA1 pyramidal neurons and induces LTP, to profoundly increase activation of CA1 pyramidal neurons. These features are predicted to make a major contribution to the pro-cognitive effects of cholinergic transmission in rodents and humans. PMID:26472558

  4. Activation of Muscarinic M1 Acetylcholine Receptors Induces Long-Term Potentiation in the Hippocampus.

    PubMed

    Dennis, Siobhan H; Pasqui, Francesca; Colvin, Ellen M; Sanger, Helen; Mogg, Adrian J; Felder, Christian C; Broad, Lisa M; Fitzjohn, Steve M; Isaac, John T R; Mellor, Jack R

    2016-01-01

    Muscarinic M1 acetylcholine receptors (M1Rs) are highly expressed in the hippocampus, and their inhibition or ablation disrupts the encoding of spatial memory. It has been hypothesized that the principal mechanism by which M1Rs influence spatial memory is by the regulation of hippocampal synaptic plasticity. Here, we use a combination of recently developed, well characterized, selective M1R agonists and M1R knock-out mice to define the roles of M1Rs in the regulation of hippocampal neuronal and synaptic function. We confirm that M1R activation increases input resistance and depolarizes hippocampal CA1 pyramidal neurons and show that this profoundly increases excitatory postsynaptic potential-spike coupling. Consistent with a critical role for M1Rs in synaptic plasticity, we now show that M1R activation produces a robust potentiation of glutamatergic synaptic transmission onto CA1 pyramidal neurons that has all the hallmarks of long-term potentiation (LTP): The potentiation requires NMDA receptor activity and bi-directionally occludes with synaptically induced LTP. Thus, we describe synergistic mechanisms by which acetylcholine acting through M1Rs excites CA1 pyramidal neurons and induces LTP, to profoundly increase activation of CA1 pyramidal neurons. These features are predicted to make a major contribution to the pro-cognitive effects of cholinergic transmission in rodents and humans.

  5. Whole-cell patch-clamp recording of nicotinic acetylcholine receptors in adult Brugia malayi muscle

    PubMed Central

    Robertson, A. P.; Buxton, S. K.; Martin, R. J.

    2013-01-01

    Lymphatic filariasis is a debilitating disease caused by clade III parasites like Brugia malayi and Wuchereria bancrofti. Current recommended treatment regimen for this disease relies on albendazole, ivermectin and diethylcarbamazine, none of which targets the nicotinic acetylcholine receptors in these parasitic nematodes. Our aim therefore has been to develop adult B. malayi for electrophysiological recordings to aid in characterizing the ion channels in this parasite as anthelmintic target sites. In that regard, we recently demonstrated the amenability of adult B. malayi to patch-clamp recordings and presented results on the single-channel properties of nAChR in this nematode. We have built on this by recording whole-cell nAChR currents from adult B. malayi muscle. Acetylcholine, levamisole, pyrantel, bephenium and tribendimidine activated the receptors on B. malayi muscle, producing robust currents ranging from > 200 pA to ~1.5 nA. Levamisole completely inhibited motility of the adult B. malayi within 10 min and after 60 min, motility had recovered back to control values. PMID:23562945

  6. Nicotine enhances alcohol intake and dopaminergic responses through β2* and β4* nicotinic acetylcholine receptors

    PubMed Central

    Tolu, Stefania; Marti, Fabio; Morel, Carole; Perrier, Carole; Torquet, Nicolas; Pons, Stephanie; de Beaurepaire, Renaud; Faure, Philippe

    2017-01-01

    Alcohol and nicotine are the most widely co-abused drugs. Both modify the activity of dopaminergic (DA) neurons of the Ventral Tegmental Area (VTA) and lead to an increase in DA release in the Nucleus Accumbens, thereby affecting the reward system. Evidences support the hypothesis that distinct nicotinic acetylcholine receptors (nAChRs), the molecular target of acetylcholine (ACh) and exogenous nicotine, are also in addition implicated in the response to alcohol. The precise molecular and neuronal substrates of this interaction are however not well understood. Here we used in vivo electrophysiology in the VTA to characterise acute and chronic interactions between nicotine and alcohol. Simultaneous injections of the two drugs enhanced their responses on VTA DA neuron firing and chronic exposure to nicotine increased alcohol-induced DA responses and alcohol intake. Then, we assessed the role of β4 * nAChRs, but not β2 * nAChRs, in mediating acute responses to alcohol using nAChR subtypes knockout mice (β2−/− and β4−/− mice). Finally, we showed that nicotine-induced modifications of alcohol responses were absent in β2−/− and β4−/− mice, suggesting that nicotine triggers β2* and β4 * nAChR-dependent neuroadaptations that subsequently modify the responses to alcohol and thus indicating these receptors as key mediators in the complex interactions between these two drugs. PMID:28332590

  7. The physiology of the nicotinic acetylcholine receptor and its importance in the administration of anesthesia.

    PubMed

    Rossman, Amanda C

    2011-10-01

    The nicotinic acetylcholine receptor (nAChR) can be found widely throughout the body. Although the activation of this receptor leads to multiple functions dependent on its location within the body and subunit composition, all nAChRs aid in the communication between the extracellular and intracellular compartments. The nAChR is composed of 3 domains: the extracellular, transmembrane, and intracellular. The receptor functions in response to ligands that act as an agonist or antagonist that binds to the extracellular domain causing activation or inactivation of the receptor. The activation of the nAChR causes a twisting motion of the receptor, which opens a gate allowing for the passage of sodium, potassium, and calcium cations through the cell membrane. The muscle-type nAChR and neuronal-type nAChR have important roles during the administration of anesthesia. The muscle-type nAChR, located in the neuromuscular junction, is the target of neuromuscular blockers and local anesthetics to prevent muscle contraction. General anesthetics affect the neuronal-type nAChR by inhibiting functions of the central nervous system, including memory formation. The importance of the nAChR cannot be underestimated, for it is through the manipulation of this receptor that many anesthetic goals are achieved.

  8. Analysis of acetylcholine receptor phosphorylation sites using antibodies to synthetic peptides and monoclonal antibodies.

    PubMed Central

    Safran, A; Neumann, D; Fuchs, S

    1986-01-01

    Three peptides corresponding to residues 354-367, 364-374, 373-387 of the acetylcholine receptor (AChR) delta subunit were synthesized. These peptides represent the proposed phosphorylation sites of the cAMP-dependent protein kinase, the tyrosine-specific protein kinase and the calcium/phospholipid-dependent protein kinase respectively. Using these peptides as substrates for phosphorylation by the catalytic subunit of cAMP-dependent protein kinase it was shown that only peptides 354-367 was phosphorylated whereas the other two were not. These results verify the location of the cAMP-dependent protein kinase phosphorylation site within the AChR delta subunit. Antibodies elicited against these peptides reacted with the delta subunit. The antipeptide antibodies and two monoclonal antibodies (7F2, 5.46) specific for the delta subunit were tested for their binding to non-phosphorylated receptor and to receptor phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. Antibodies to peptide 354-367 were found to react preferentially with non-phosphorylated receptor whereas the two other anti-peptide antibodies bound equally to phosphorylated and non-phosphorylated receptors. Monoclonal antibody 7F2 reacted preferentially with the phosphorylated form of the receptor whereas monoclonal antibody 5.46 did not distinguish between the two forms. Images Fig. 2. Fig. 4. Fig. 5. PMID:3816758

  9. Minimum number of lipids are required to support the functional properties of the nicotinic acetylcholine receptor

    SciTech Connect

    Jones, O.T.; Eubanks, J.H.; Earnest, J.P.; McNamee, M.G.

    1988-05-17

    The detergent sodium cholate was used to both solubilize and partially delipidate the nicotinic acetylcholine receptor from Torpedo californica. Using both native membranes and reconstituted membranes, it is shown that the detergent to lipid molar ratio is the most important parameter in determining the effect of the detergent on the functional properties of the receptor. Receptor-lipid complexes were quantitatively separated from detergent and excess lipids by centrifugation through detergent-free sucrose gradients. The lipid to protein molar ratio of the complexes could be precisely controlled by adjusting the cholate and lipid concentrations of the starting membranes. Analyses of both ion influx activity and ligand binding revealed that a minimum of 45 lipids per receptor was required for stabilization of the receptor in a fully functional state. Progressive irreversible inactivation occurred as the lipid to protein mole ratio was decreased below 45, and complete inactivation occurred below a ratio of 20. The results are consistent with a functional requirement for a single shell of lipids around the perimeter of the receptor.

  10. Steroids induce acetylcholine receptors on cultured human muscle: implications for myasthenia gravis.

    PubMed Central

    Kaplan, I; Blakely, B T; Pavlath, G K; Travis, M; Blau, H M

    1990-01-01

    Antibodies to the acetylcholine receptor (AChR), which are diagnostic of the human autoimmune disease myasthenia gravis, block AChR function and increase the rate of AChR degradation leading to impaired neuromuscular transmission. Steroids are frequently used to alleviate symptoms of muscle fatigue and weakness in patients with myasthenia gravis because of their well-documented immunosuppressive effects. We show here that the steroid dexamethasone significantly increases total surface AChRs on cultured human muscle exposed to myasthenia gravis sera. Our results suggest that the clinical improvement observed in myasthenic patients treated with steroids is due not only to an effect on the immune system but also to a direct effect on muscle. We propose that the identification and development of pharmacologic agents that augment receptors and other proteins that are reduced by human genetic or autoimmune disease will have broad therapeutic applications. Images PMID:2236023

  11. Steroids induce acetylcholine receptors on cultured human muscle: Implications for myasthenia gravis

    SciTech Connect

    Kaplan, I.; Blakely, B.T.; Pavlath, G.K.; Travis, M.; Blau, H.M. )

    1990-10-01

    Antibodies to the acetylcholine receptor (AChR), which are diagnostic of the human autoimmune disease myasthenia gravis, block AChR function and increase the rate of AChR degradation leading to impaired neuromuscular transmission. Steroids are frequently used to alleviate symptoms of muscle fatigue and weakness in patients with myasthenia gravis because of their well-documented immunosuppressive effects. The authors show here that the steroid dexamethasone significantly increases total surface AChRs on cultured human muscle exposed to myasthenia gravis sera. The results suggest that the clinical improvement observed in myasthenic patients treated with steroids is due not only to an effect on the immune system but also a direct effect on muscle. They propose that the identification and development of pharmacologic agents that augment receptors and other proteins that are reduced by human genetic or autoimmune disease will have broad therapeutic applications.

  12. A model of the closed form of the nicotinic acetylcholine receptor m2 channel pore.

    PubMed

    Kim, Sanguk; Chamberlain, Aaron K; Bowie, James U

    2004-08-01

    The nicotinic acetylcholine receptor is a neurotransmitter-gated ion channel in the postsynaptic membrane. It is composed of five homologous subunits, each of which contributes one transmembrane helix--the M2 helix--to create the channel pore. The M2 helix from the delta subunit is capable of forming a channel by itself. Although a model of the receptor was recently proposed based on a low-resolution, cryo-electron microscopy density map, we found that the model does not explain much of the other available experimental data. Here we propose a new model of the M2 channel derived solely from helix packing and symmetry constraints. This model agrees well with experimental results from solid-state NMR, chemical reactivity, and mutagenesis experiments. The model depicts the channel pore, the channel gate, and the residues responsible for cation specificity.

  13. Bispyridinium Compounds Inhibit Both Muscle and Neuronal Nicotinic Acetylcholine Receptors in Human Cell Lines

    PubMed Central

    Ring, Avi; Strom, Bjorn Oddvar; Turner, Simon R.; Timperley, Christopher M.; Bird, Michael; Green, A. Christopher; Chad, John E.; Worek, Franz; Tattersall, John E. H.

    2015-01-01

    Standard treatment of poisoning by organophosphorus anticholinesterases uses atropine to reduce the muscarinic effects of acetylcholine accumulation and oximes to reactivate acetylcholinesterase (the effectiveness of which depends on the specific anticholinesterase), but does not directly address the nicotinic effects of poisoning. Bispyridinium molecules which act as noncompetitive antagonists at nicotinic acetylcholine receptors have been identified as promising compounds and one has been shown to improve survival following organophosphorus poisoning in guinea-pigs. Here, we have investigated the structural requirements for antagonism and compared inhibitory potency of these compounds at muscle and neuronal nicotinic receptors and acetylcholinesterase. A series of compounds was synthesised, in which the length of the polymethylene linker between the two pyridinium moieties was increased sequentially from one to ten carbon atoms. Their effects on nicotinic receptor-mediated calcium responses were tested in muscle-derived (CN21) and neuronal (SH-SY5Y) cells. Their ability to inhibit acetylcholinesterase activity was tested using human erythrocyte ghosts. In both cell lines, the nicotinic response was inhibited in a dose-dependent manner and the inhibitory potency of the compounds increased with greater linker length between the two pyridinium moieties, as did their inhibitory potency for human acetylcholinesterase activity in vitro. These results demonstrate that bispyridinium compounds inhibit both neuronal and muscle nicotinic receptors and that their potency depends on the length of the hydrocarbon chain linking the two pyridinium moieties. Knowledge of structure-activity relationships will aid the optimisation of molecular structures for therapeutic use against the nicotinic effects of organophosphorus poisoning. PMID:26274808

  14. The α6 nicotinic acetylcholine receptor subunit influences ethanol-induced sedation.

    PubMed

    Kamens, Helen M; Hoft, Nicole R; Cox, Ryan J; Miyamoto, Jill H; Ehringer, Marissa A

    2012-08-01

    Alcohol and nicotine are often co-used and data from human and animals studies have demonstrated that common genes underlie responses to these two drugs. Recently, the genes that code for the subunits of the nicotinic acetylcholine receptors have been implicated as a common genetic mediator for alcohol and nicotine responses. The mammalian genes that code for the α6 and β3 subunits of the nicotinic acetylcholine receptor (Chrna6 and Chrnb3, respectively) are located adjacent to each other on human and mouse chromosome 8. These subunits have gained attention as potential regulators of drug behaviors because of their expression in the striatum where they have been shown to modulate dopamine release. Human genetic studies have shown that variation in these genes is associated with alcohol phenotypes. In the current experiments, mice lacking the Chrna6 or Chrnb3 gene were tested for three ethanol behaviors: choice ethanol consumption, ataxia, and sedation. Wildtype (WT), heterozygous (HET), and knockout (KO) mice of each strain went through a standard 2-bottle choice drinking paradigm, the balance beam, and the Loss of Righting Reflex (LORR) paradigm. No genotypic effects on any of the 3 behavioral tasks were observed in Chrnb3 animals. While the Chrna6 gene did not significantly influence ethanol consumption (g/kg) or ataxia, mice lacking the α6 subunit took significantly longer to recover their righting reflex than WT animals. These data provide evidence that receptors containing this subunit modulate the sedative effects of ethanol. Further work examining other models of ethanol consumption and behavioral responses to ethanol is needed to fully characterize the role of these receptor subunits in modulating ethanol responses.

  15. Activation of muscarinic and nicotinic acetylcholine receptors in the nucleus accumbens core is necessary for the acquisition of drug reinforcement.

    PubMed

    Crespo, Jose A; Sturm, Katja; Saria, Alois; Zernig, Gerald

    2006-05-31

    Neurotransmitter release in the nucleus accumbens core (NACore) during the acquisition of remifentanil or cocaine reinforcement was determined in an operant runway procedure by simultaneous tandem mass spectrometric analysis of dopamine, acetylcholine, and remifentanil or cocaine itself. Run times for remifentanil or cocaine continually decreased over the five consecutive runs of the experiment. Intra-NACore dopamine, acetylcholine, and drug peaked with each intravenous remifentanil or cocaine self-administration and decreased to pre-run baseline with half-lives of approximately 10 min. As expected, remifentanil or cocaine peaks did not vary between the five runs. Surprisingly, however, drug-contingent dopamine peaks also did not change over the five runs, whereas acetylcholine peaks did. Thus, the acquisition of drug reinforcement was paralleled by a continuous increase in acetylcholine overflow in the NACore, whereas the overflow of dopamine, the expected prime neurotransmitter candidate for conditioning in drug reinforcement, did not increase. Local intra-accumbens administration by reverse microdialysis of either atropine or mecamylamine completely and reversibly blocked the acquisition of remifentanil reinforcement. Our findings suggest that activation of muscarinic and nicotinic acetylcholine receptors in the NACore by acetylcholine volume transmission is necessary during the acquisition phase of drug reinforcement conditioning.

  16. Multiple binding sites in the nicotinic acetylcholine receptors: An opportunity for polypharmacolgy.

    PubMed

    Iturriaga-Vásquez, Patricio; Alzate-Morales, Jans; Bermudez, Isabel; Varas, Rodrigo; Reyes-Parada, Miguel

    2015-11-01

    For decades, the development of selective compounds has been the main goal for chemists and biologists involved in drug discovery. However, diverse lines of evidence indicate that polypharmacological agents, i.e. those that act simultaneously at various protein targets, might show better profiles than selective ligands, regarding both efficacy and side effects. On the other hand, the availability of the crystal structure of different receptors allows a detailed analysis of the main interactions between drugs and receptors in a specific binding site. Neuronal nicotinic acetylcholine receptors (nAChRs) constitute a large and diverse family of ligand-gated ion channels (LGICs) that, as a product of its modulation, regulate neurotransmitter release, which in turns produce a global neuromodulation of the central nervous system. nAChRs are pentameric protein complexes in such a way that expression of compatible subunits can lead to various receptor assemblies or subtypes. The agonist binding site, located at the extracellular region, exhibits different properties depending on the subunits that conform the receptor. In the last years, it has been recognized that nAChRs could also contain one or more allosteric sites which could bind non-classical nicotinic ligands including several therapeutically useful drugs. The presence of multiple binding sites in nAChRs offers an interesting possibility for the development of novel polypharmacological agents with a wide spectrum of actions.

  17. Identification of nicotinic acetylcholine receptor recycling and its role in maintaining receptor density at the neuromuscular junction in vivo.

    PubMed

    Bruneau, Emile; Sutter, David; Hume, Richard I; Akaaboune, Mohammed

    2005-10-26

    In the CNS, receptor recycling is critical for synaptic plasticity; however, the recycling of receptors has never been observed at peripheral synapses. Using a novel imaging technique, we show here that nicotinic acetylcholine receptors (AChRs) recycle into the postsynaptic membrane of the neuromuscular junction. By sequentially labeling AChRs with biotin-bungarotoxin and streptavidin-fluorophore conjugates, we were able to distinguish recycled, preexisting, and new receptor pools at synapses in living mice. Time-lapse imaging revealed that recycled AChRs were incorporated into the synapse within hours of initial labeling, and their numbers increased with time. At fully functional synapses, AChR recycling was robust and comparable in magnitude with the insertion of newly synthesized receptors, whereas chronic synaptic activity blockade nearly abolished receptor recycling. Finally, using the same sequential labeling method, we found that acetylcholinesterase, another synaptic component, does not recycle. These results identify an activity-dependent AChR-recycling mechanism that enables the regulation of receptor density, which could lead to rapid alterations in synaptic efficacy.

  18. Expression and functional properties of α7 acetylcholine nicotinic receptors are modified in the presence of other receptor subunits.

    PubMed

    Criado, Manuel; Valor, Luis M; Mulet, José; Gerber, Susana; Sala, Salvador; Sala, Francisco

    2012-11-01

    Although α7 nicotinic receptors are predominantly homopentamers, previous reports have indicated that α7 and β2 subunits are able to form heteromers. We have studied whether other nicotinic receptor subunits can also assemble with α7 subunits and the effect of this potential association. Coexpression of α7 with α2, α3, or β4 subunits reduced to about half, surface α-bungarotoxin binding sites and acetylcholine-gated currents. This is probably because of inhibition of membrane trafficking, as the total amount of α7 subunits was similar in all cases and a significant proportion of mature α7 receptors was present inside the cell. Only β4 subunits appeared to directly associate with α7 receptors at the membrane and these heteromeric receptors showed some kinetic and pharmacological differences when compared with homomeric α7 receptors. Finally, we emulated the situation of bovine chromaffin cells in Xenopus laevis oocytes by using the same proportion of α3, β4, α5, and α7 mRNAs, finding that α-bungarotoxin binding was similarly reduced in spite of increased currents, apparently mediated by α3β4(α5) receptors.

  19. Sperm Epidermal Growth Factor Receptor (EGFR) Mediates α7 Acetylcholine Receptor (AChR) Activation to Promote Fertilization

    PubMed Central

    Jaldety, Yael; Glick, Yair; Orr-Urtreger, Avi; Ickowicz, Debby; Gerber, Doron; Breitbart, Haim

    2012-01-01

    To attain fertilization the spermatozoon binds to the egg zona pellucida (ZP) via sperm receptor(s) and undergoes an acrosome reaction (AR). Several sperm receptors have been described in the literature; however, the identity of this receptor is not yet certain. In this study, we suggest that the α7 nicotinic acetylcholine receptor (α7nAChR) might be a sperm receptor activated by ZP to induce epidermal growth factor receptor (EGFR)-mediated AR. We found that isolated ZP or α7 agonists induced the AR in sperm from WT but not α7-null spermatozoa, and the induced AR was inhibited by α7 or EGFR antagonists. Moreover, α7-null sperm showed very little binding to the egg, and microfluidic affinity in vitro assay clearly showed that α7nAChR, as well as EGFR, interacted with ZP3. Induction of EGFR activation and the AR by an α7 agonist was inhibited by a Src family kinase (SFK) inhibitor. In conclusion we suggest that activation of α7 by ZP leads to SFK-dependent EGFR activation, Ca2+ influx, and the acrosome reaction. PMID:22577141

  20. Disruption and reformation of the acetylcholine receptor clusters of cultured rat myotubes occur in two distinct stages.

    PubMed

    Pumplin, D W; Bloch, R J

    1987-01-01

    We have examined the redistribution of acetylcholine receptor (AChR) intramembrane particles (IMPs) when AChR clusters of cultured rat myotubes are experimentally disrupted and allowed to reform. In control myotubes, the AChR IMPs are evenly distributed within the AChR domains of cluster membrane. Shortly after addition of azide to disrupt clusters, IMPs become unevenly scattered, with some microaggregation. After longer treatment, IMPs are depleted from AChR domains with no further change in IMP distribution. Contact domains of clusters are relatively poor in IMPs both before and after cluster dispersal. Upon visualization with fluorescent alpha-bungarotoxin, some AChR in azide-treated samples appear as small, bright spots. These spots do not correspond to microaggregates seen in freeze-fracture replicas, and probably represent receptors that have been internalized. The internalization rate is insufficient to account completely for the loss of IMPs from clusters, however. During reformation of AChR clusters upon removal of azide, IMP concentration in receptor domains increases. At early stages of reformation, IMPs appear in small groups containing compact microaggregates. At later times, AChR domains enlarge and IMPs within them assume the evenly spaced distribution characteristic of control clusters. These observations suggest that the disruption of clusters is accompanied by mobilization of AChR from a fixed array, allowing AChR IMPs to diffuse away from the clusters, to form microaggregates, and to become internalized. Cluster reformation appears to be the reverse of this process. Our results are thus consistent with a two-step model for AChR clustering, in which the concentration of IMPs into a small membrane region precedes their rearrangement into evenly spaced sites.

  1. Azemiopsin from Azemiops feae Viper Venom, a Novel Polypeptide Ligand of Nicotinic Acetylcholine Receptor*

    PubMed Central

    Utkin, Yuri N.; Weise, Christoph; Kasheverov, Igor E.; Andreeva, Tatyana V.; Kryukova, Elena V.; Zhmak, Maxim N.; Starkov, Vladislav G.; Hoang, Ngoc Anh; Bertrand, Daniel; Ramerstorfer, Joachim; Sieghart, Werner; Thompson, Andrew J.; Lummis, Sarah C. R.; Tsetlin, Victor I.

    2012-01-01

    Azemiopsin, a novel polypeptide, was isolated from the Azemiops feae viper venom by combination of gel filtration and reverse-phase HPLC. Its amino acid sequence (DNWWPKPPHQGPRPPRPRPKP) was determined by means of Edman degradation and mass spectrometry. It consists of 21 residues and, unlike similar venom isolates, does not contain cysteine residues. According to circular dichroism measurements, this peptide adopts a β-structure. Peptide synthesis was used to verify the determined sequence and to prepare peptide in sufficient amounts to study its biological activity. Azemiopsin efficiently competed with α-bungarotoxin for binding to Torpedo nicotinic acetylcholine receptor (nAChR) (IC50 0.18 ± 0.03 μm) and with lower efficiency to human α7 nAChR (IC50 22 ± 2 μm). It dose-dependently blocked acetylcholine-induced currents in Xenopus oocytes heterologously expressing human muscle-type nAChR and was more potent against the adult form (α1β1ϵδ) than the fetal form (α1β1γδ), EC50 being 0.44 ± 0.1 μm and 1.56 ± 0.37 μm, respectively. The peptide had no effect on GABAA (α1β3γ2 or α2β3γ2) receptors at a concentration up to 100 μm or on 5-HT3 receptors at a concentration up to 10 μm. Ala scanning showed that amino acid residues at positions 3–6, 8–11, and 13–14 are essential for binding to Torpedo nAChR. In biological activity azemiopsin resembles waglerin, a disulfide-containing peptide from the Tropidechis wagleri venom, shares with it a homologous C-terminal hexapeptide, but is the first natural toxin that blocks nAChRs and does not possess disulfide bridges. PMID:22613724

  2. Electrophysiological perspectives on the therapeutic use of nicotinic acetylcholine receptor partial agonists.

    PubMed

    Papke, Roger L; Trocmé-Thibierge, Caryn; Guendisch, Daniela; Al Rubaiy, Shehd Abdullah Abbas; Bloom, Stephen A

    2011-05-01

    Partial agonist therapies rely variously on two hypotheses: the partial agonists have their effects through chronic low-level receptor activation or the partial agonists work by decreasing the effects of endogenous or exogenous full agonists. The relative significance of these activities probably depends on whether acute or chronic effects are considered. We studied nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus laevis oocytes to test a model for the acute interactions between acetylcholine (ACh) and weak partial agonists. Data were best-fit to a basic competition model that included an additional factor for noncompetitive inhibition. Partial agonist effects were compared with the nAChR antagonist bupropion in prolonged bath application experiments that were designed to mimic prolonged drug exposure typical of therapeutic drug delivery. A primary effect of prolonged application of nicotine was to decrease the response of all nAChR subtypes to acute applications of ACh. In addition, nicotine, cytisine, and varenicline produced detectable steady-state activation of α4β2* [(α4)(2)(β2)(3), (α4)(3)(β2)(2), and (α4)(2)(β2)(2)α5)] receptor subtypes that was not seen with other test compounds. Partial agonists produced no detectable steady-state activation of α7 nAChR, but seemed to show small potentiation of ACh-evoked responses; however, "run-up" of α7 ACh responses was also sometimes observed under control conditions. Potential off-target effects of the partial agonists therefore included the modulation of α7 responses by α4β2 partial agonists and decreases in α4β2* responses by α7-selective agonists. These data indicate the dual effects expected for α4β2* partial agonists and provide models and insights for utility of partial agonists in therapeutic development.

  3. Electrophysiology-Based Assays to Detect Subtype-Selective Modulation of Human Nicotinic Acetylcholine Receptors

    PubMed Central

    Kirsch, Glenn E.; Fedorov, Nikolai B.; Kuryshev, Yuri A.; Liu, Zhiqi; Orr, Michael S.

    2016-01-01

    Abstract The Family Smoking Prevention and Tobacco Control Act of 2009 (Public Law 111-31) gave the US Food and Drug Administration (FDA) the responsibility for regulating tobacco products. Nicotine is the primary addictive component of tobacco and its effects can be modulated by additional ingredients in manufactured products. Nicotine acts by mimicking the neurotransmitter acetylcholine on neuronal nicotinic acetylcholine receptors (nAChRs), which function as ion channels in cholinergic modulation of neurotransmission. Subtypes within the family of neuronal nAChRs are defined by their α- and β-subunit composition. The subtype-selective profiles of tobacco constituents are largely unknown, but could be essential for understanding the physiological effects of tobacco products. In this report, we report the development and validation of electrophysiology-based high-throughput screens (e-HTS) for human nicotinic subtypes, α3β4, α3β4α5, α4β2, and α7 stably expressed in Chinese Hamster Ovary cells. Assessment of agonist sensitivity and acute desensitization gave results comparable to those obtained by conventional manual patch clamp electrophysiology assays. The potency of reference antagonists for inhibition of the receptor channels and selectivity of positive allosteric modulators also were very similar between e-HTS and conventional manual patch voltage clamp data. Further validation was obtained in pilot screening of a library of FDA-approved drugs that identified α7 subtype-selective positive allosteric modulation by novel compounds. These assays provide new tools for profiling of nicotinic receptor selectivity. PMID:27505073

  4. [Estrogen receptor alpha in obesity and diabetes].

    PubMed

    Cahua-Pablo, José Ángel; Flores-Alfaro, Eugenia; Cruz, Miguel

    2016-01-01

    Estradiol (E2) is an important hormone in reproductive physiology, cardiovascular, skeletal and in the central nervous system (CNS). In human and rodents, E2 and its receptors are involved in the control of energy and glucose metabolism in health and metabolic diseases. The estrogen receptor (ER) belongs to the superfamily of nuclear receptors (NR), which are transcription factors that regulate gene expression. Three ER, ER-alpha, ER-beta and the G protein-coupled ER (GPER; also called GPR30) in tissues are involved in glucose and lipid homeostasis. Also, it may have important implications for risk factors associated with metabolic syndrome (MS), insulin resistance (IR), obesity and type 2 diabetes (T2D).

  5. Desensitization and internalization of the m2 muscarinic acetylcholine receptor are directed by independent mechanisms.

    PubMed

    Pals-Rylaarsdam, R; Xu, Y; Witt-Enderby, P; Benovic, J L; Hosey, M M

    1995-12-01

    The phenomenon of acute desensitization of G-protein-coupled receptors has been associated with several events, including receptor phosphorylation, loss of high affinity agonist binding, receptor:G-protein uncoupling, and receptor internalization. However, the biochemical events underlying these processes are not fully understood, and their contributions to the loss of signaling remain correlative. In addition, the nature of the kinases and the receptor domains which are involved in modulation of activity have only begun to be investigated. In order to directly measure the role of G-protein-coupled receptor kinases (GRKs) in the desensitization of the m2 muscarinic acetylcholine receptor (m2 mAChR), a dominant-negative allele of GRK2 was used to inhibit receptor phosphorylation by endogenous GRK activity in a human embryonic kidney cell line. The dominant-negative GRK2K220R reduced agonist-dependent phosphorylation of the m2 mAChR by approximately 50% and prevented acute desensitization of the receptor as measured by the ability of the m2 mAChR to attenuate adenylyl cyclase activity. In contrast, the agonist-induced internalization of the m2 mAChR was unaffected by the GRK2K220R construct. Further evidence linking receptor phosphorylation to acute receptor desensitization was obtained when two deletions of the third intracellular loop were made which created m2 mAChRs that did not become phosphorylated in an agonist-dependent manner and did not desensitize. However, the mutant mAChRs retained the ability to internalize. These data provide the first direct evidence that GRK-mediated receptor phosphorylation is necessary for m2 mAChR desensitization; the likely sites of in vivo phosphorylation are in the central portion of the third intracellular loop (amino acids 282-323). These results also indicate that internalization of the m2 receptor is not a key event in desensitization and is mediated by mechanisms distinct from GRK phosphorylation of the receptor.

  6. Muscarinic Acetylcholine Receptor M3 Modulates Odorant Receptor Activity via Inhibition of β-Arrestin-2 Recruitment

    PubMed Central

    Jiang, Yue; Li, Yun Rose; Tian, Huikai; Ma, Minghong; Matsunami, Hiroaki

    2015-01-01

    The olfactory system in rodents serves a critical function in social, reproductive, and survival behaviors. Processing of chemosensory signals in the brain is dynamically regulated in part by an animal's physiological state. We previously reported that type 3 muscarinic acetylcholine receptors (M3-Rs) physically interact with odorant receptors (ORs) to promote odor-induced responses in a heterologous expression system. However, it is not known how M3-Rs affect the ability of olfactory sensory neurons (OSNs) to respond to odors. Here, we show that an M3-R antagonist attenuates odor-induced responses in OSNs from wild-type, but not M3-R-null mice. Using a novel molecular assay, we demonstrate that the activation of M3-Rs inhibits the recruitment of β-arrestin-2 to ORs, resulting in a potentiation of odor-induced response in OSNs. These results suggest a role for acetylcholine in modulating olfactory processing at the initial stages of signal transduction in the olfactory system. PMID:25800153

  7. Spontaneous opening of the acetylcholine receptor channel in developing muscle cells from normal and dystrophic mice

    SciTech Connect

    Franco-Obregon, A.; Lansman, J.B.

    1995-12-31

    Single-channel activity was recorded from cell-attached patches on skeletal muscle cells isolated from wild-type mice and from mice carrying the dy or mdx mutations. Spontaneous openings of the nicotinic acetylcholine receptor channel (nAChR) were detected in virtually all recordings from either 4v/dy or dyl + myotubes. but only infrequently from wild-type or mdx myotubes. Spontaneous openings were also present in most recordings from undifferentiated myoblasts from all of the mouse strains studied. The biophysical properties of the spontaneous activity were similar to those of the embryonic form of the nAChR in the presence of acetylcholine (ACh). Examination of the single-channel currents evoked by low concentrations of ACh showed a reduced sensitivity to the agonist in the dystrophic dy and mdx myotubes. but not in wild- type myotubes. The results suggest that alterations in nAChR function are associated with the pathogenesis of muscular dystrophy in the dy mouse.

  8. Mode of action of triflumezopyrim: A novel mesoionic insecticide which inhibits the nicotinic acetylcholine receptor.

    PubMed

    Cordova, Daniel; Benner, Eric A; Schroeder, Mark E; Holyoke, Caleb W; Zhang, Wenming; Pahutski, Thomas F; Leighty, Robert M; Vincent, Daniel R; Hamm, Jason C

    2016-07-01

    Triflumezopyrim, a newly commercialized molecule from DuPont Crop Protection, belongs to the novel class of mesoionic insecticides. This study characterizes the biochemical and physiological action of this novel insecticide. Using membranes from the aphid, Myzus persicae, triflumezopyrim was found to displace (3)H-imidacloprid with a Ki value of 43 nM with competitive binding results indicating that triflumezopyrim binds to the orthosteric site of the nicotinic acetylcholine receptor (nAChR). In voltage clamp studies using dissociated Periplaneta americana neurons, triflumezopyrim inhibits nAChR currents with an IC50 of 0.6 nM. Activation of nAChR currents was minimal and required concentrations ≥100 μM. Xenopus oocytes expressing chimeric nAChRs (Drosophila α2/chick β2) showed similar inhibitory effects from triflumezopyrim. In P. americana neurons, co-application experiments with acetylcholine reveal the inhibitory action of triflumezopyrim to be rapid and prolonged in nature. Such physiological action is distinct from other insecticides in IRAC Group 4 in which the toxicological mode of action is attributed to nAChR agonism. Mesoionic insecticides act via inhibition of the orthosteric binding site of the nAChR despite previous beliefs that such action would translate to poor insect control. Triflumezopyrim is the first commercialized insecticide from this class and provides outstanding control of hoppers, including the brown planthopper, Nilaparvata lugens, which is already displaying strong resistance to neonicotinoids such as imidacloprid.

  9. Activation and desensitization of peripheral muscle and neuronal nicotinic acetylcholine receptors by selected, naturally-occurring pyridine alkaloids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Teratogenic alkaloids can cause developmental defects due to inhibition of fetal movement that results from desensitization of fetal muscletype nicotinic acetylcholine receptors (nAChRs). We investigated the ability of two known teratogens, the piperidinyl-pyridine anabasine and its 1,2-dehydropiper...

  10. The role of the a7 subunit of the nicotinic acetylcholine receptor in the acute toxicosis of methyllycaconitine in mice.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The adverse physiological effects of methyllycaconitine (MLA) have been attributed to its competitive antagonism of nicotinic acetylcholine receptors (nAChRs). Recent research demonstrated a correlation between the LD50 of MLA and the amount of a7 nAChR in various mouse strains, suggesting that mice...

  11. Neuronal Nicotinic Acetylcholine Receptor Structure and Function and Response to Nicotine.

    PubMed

    Dani, John A

    2015-01-01

    Nicotinic acetylcholine receptors (nAChRs) belong to the "Cys-loop" superfamily of ligand-gated ion channels that includes GABAA, glycine, and serotonin (5-HT3) receptors. There are 16 homologous mammalian nAChR subunits encoded by a multigene family. These subunits combine to form many different nAChR subtypes with various expression patterns, diverse functional properties, and differing pharmacological characteristics. Because cholinergic innervation is pervasive and nAChR expression is extremely broad, practically every area of the brain is impinged upon by nicotinic mechanisms. This review briefly examines the structural and functional properties of the receptor/channel complex itself. The review also summarizes activation and desensitization of nAChRs by the low nicotine concentrations obtained from tobacco. Knowledge of the three-dimensional structure and the structural characteristics of channel gating has reached an advanced stage. Likewise, the basic functional properties of the channel also are reasonably well understood. It is these receptor/channel properties that underlie the participation of nAChRs in nearly every anatomical region of the mammalian brain.

  12. Amino acids outside of the loops that define the agonist binding site are important for ligand binding to insect nicotinic acetylcholine receptors.

    PubMed

    Liu, Zewen; Han, Zhaojun; Liu, Shuhua; Zhang, Yixi; Song, Feng; Yao, Xiangmei; Gu, Jianhua

    2008-07-01

    Nicotinic acetylcholine (ACh) receptors (nAChRs) are the targets of several kinds of insecticides. Based on the mutagenesis studies of Torpedo californica nAChRs and solved structure of a molluscan, glial-derived soluble ACh-binding protein, a model of the agonist site was constructed with contributing amino acids from three distinct loops (A, B, and C) of the alpha subunits and another three loops (D, E, and F) of the non-alpha subunits. According to this model, most insect nAChR subunits can form the functional heteromeric or homomeric receptors. Actually, insect subunits themselves did not form any functional receptor at various combinations as yet, and only part of them can form the functional receptors with vertebrate non-alpha subunits. These findings suggested that the agonist binding for insect nAChRs was not only contributed by those key amino acids in six loops, but also some unidentified amino acids from other regions. In our previous studies on nAChRs for Nilaparvata lugens, a target-site mutation (Y151S) was found within two alpha subunits (Nlalpha1 and Nlalpha3). In Drosophila S2 cells and Xenopus oocytes, Nlalpha1 can form functional receptors with rat beta2 subunit. However, the same thing was not observed in Nlalpha3. In the present paper, by exchanging the corresponding regions between Nlalpha1 and Nlalpha3 to generate different chimeras, amino acid residues or residue clusters in the regions outside the six loops were found to play essential roles in agonist binding, especially for the amino acid clusters between loop B and C. This result indicated that the residues in the six loops could be necessary, but not enough for the activity of agonist binding.

  13. The molecular basis of oligomeric organization of the human M3 muscarinic acetylcholine receptor.

    PubMed

    Liste, María José Varela; Caltabiano, Gianluigi; Ward, Richard J; Alvarez-Curto, Elisa; Marsango, Sara; Milligan, Graeme

    2015-06-01

    G protein-coupled receptors, including the M3 muscarinic acetylcholine receptor, can form homo-oligomers. However, the basis of these interactions and the overall organizational structure of such oligomers are poorly understood. Combinations of site-directed mutagenesis and homogenous time-resolved fluorescence resonance energy transfer studies that assessed interactions between receptor protomers at the surface of transfected cells indicated important contributions of regions of transmembrane domains I, IV, V, VI, and VII as well as intracellular helix VIII to the overall organization. Molecular modeling studies based on both these results and an X-ray structure of the inactive state of the M3 receptor bound by the antagonist/inverse agonist tiotropium were then employed. The results could be accommodated fully by models in which a proportion of the cell surface M3 receptor population is a tetramer with rhombic, but not linear, orientation. This is consistent with previous studies based on spectrally resolved, multiphoton fluorescence resonance energy transfer. Modeling studies furthermore suggest an important role for molecules of cholesterol at the dimer + dimer interface of the tetramer, which is consistent with the presence of cholesterol at key locations in many G protein-coupled receptor crystal structures. Mutants that displayed disrupted quaternary organization were often poorly expressed and showed immature N-glycosylation. Sustained treatment of cells expressing such mutants with the muscarinic receptor inverse agonist atropine increased cellular levels and restored both cell surface delivery and quaternary organization to many of the mutants. These observations suggest that organization as a tetramer may occur before plasma membrane delivery and may be a key step in cellular quality control assessment.

  14. Distribution of muscarinic acetylcholine receptors and presynaptic nerve terminals in amphibian heart

    PubMed Central

    1980-01-01

    At many synapses, neurotransmitter receptor molecules in the postsynaptic membrane are selectively concentrated at a site directly opposite the presynaptic nerve terminal. In this paper, I examine acetylcholine (ACh) receptor distribution in cardiac muscle in relatin to the distribution of presynaptic axonal varicosities. The density of varicosities, stained with zinc iodide and osmium, ranges from 0.7/100 micrometer 2 in ventricle to 1.9/100 micrometer 2 in sinus venosus. It is estimated that < 3% of the muscle surface is apposed to presynaptic varicosities. ACh receptors, however, are randomly distributed on the muscle surface and not concentrated in patche. ACh receptor distribution was determined by iontophoretic application of ACh and mapping of ACh sensitivity and by [3H]QNB (quinuclidinyl benzilate) binding and autoradiography [3H]QNB binds with > 90% specificity to a single, saturable, high-affinity (Kd = 11.1 pM at 21 degrees C) class of binding sites. QNB binding sites are thought to correspond to ACh receptors, because muscarinic agonists compete for [3H]QNB binding and produce a hyperpolarization in the sinus venosus with the same order of potency. The concentrations of QNB binding sites in the sinus and atria are about twice those found in ventricle. The receptor density corresponds to the density of innervation measured by zinc iodide and osmium staining. Autoradiographic experiments show that [3H]QNB binding sites are distributed randomly over the entire surface of the muscle. This distribution of ACh receptors in cardiac muscle has important implications for the function of the cardiac neuroeffector junction. PMID:6968315

  15. Brain α4β2 nicotinic acetylcholine receptors are involved in the secretion of noradrenaline and adrenaline from adrenal medulla in rats.

    PubMed

    Shimizu, Takahiro; Tanaka, Kenjiro; Hasegawa, Takashi; Yokotani, Kunihiko

    2011-03-11

    Recently, we reported that intracerebroventricularly (i.c.v.) administered (±)-epibatidine (a non-selective agonist of nicotinic acetylcholine receptors) elevates plasma noradrenaline and adrenaline through brain nicotinic acetylcholine receptor-mediated mechanisms in rats. In the present study, we characterized the receptors involved in these responses using selective agonists and antagonists of nicotinic acetylcholine receptor subtypes in anesthetized rats. (±)-Epibatidine (5 and 10nmol/animal, i.c.v.) and (-)-nicotine (250 and 500nmol/animal, i.c.v.) both elevated plasma noradrenaline and adrenaline (adrenaline>noradrenaline) but the former was more efficient than the latter. The (±)-epibatidine (5nmol/animal, i.c.v.)-induced elevation of plasma catecholamines was reduced by dihydro-β-erythroidine (a selective antagonist of α4β2 nicotinic acetylcholine receptors) (100 and 300nmol/animal, i.c.v.), while methyllycaconitine (a selective antagonist of α7 nicotinic acetylcholine receptors) (100 and 300nmol/animal, i.c.v.) had no effect on the (±)-epibatidine-induced responses. RJR-2403 (a selective agonist of α4β2 nicotinic acetylcholine receptors) (2.5 and 5μmol/animal, i.c.v.) elevated plasma noradrenaline and adrenaline (adrenaline>noradrenaline), while PNU-282987 (a selective agonist of α7 nicotinic acetylcholine receptors) (2.5 and 5μmol/animal, i.c.v.) had no effect. Furthermore, the RJR-2403 (5μmol/animal, i.c.v.)-induced responses were abolished by acute bilateral adrenalectomy. Immunohistochemical procedures demonstrated the expression of α4 and β2 nicotinic acetylcholine receptor subunits on the spinally projecting hypothalamic paraventricular neurons. Taken together, brain α4β2 nicotinic acetylcholine receptors seem to be involved in the secretion of noradrenaline and adrenaline from adrenal medulla in rats.

  16. Single nucleotide polymorphisms and genotypes of transient receptor potential ion channel and acetylcholine receptor genes from isolated B lymphocytes in myalgic encephalomyelitis/chronic fatigue syndrome patients.

    PubMed

    Marshall-Gradisnik, Sonya; Johnston, Samantha; Chacko, Anu; Nguyen, Thao; Smith, Peter; Staines, Donald

    2016-12-01

    Objective The pathomechanism of chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) is unknown; however, a small subgroup of patients has shown muscarinic antibody positivity and reduced symptom presentation following anti-CD20 intervention. Given the important roles of calcium (Ca(2+)) and acetylcholine (ACh) signalling in B cell activation and potential antibody development, we aimed to identify relevant single nucleotide polymorphisms (SNPs) and genotypes in isolated B cells from CFS/ME patients. Methods A total of 11 CFS/ME patients (aged 31.82 ± 5.50 years) and 11 non-fatigued controls (aged 33.91 ± 5.06 years) were included. Flow cytometric protocols were used to determine B cell purity, followed by SNP and genotype analysis for 21 mammalian TRP ion channel genes and nine mammalian ACh receptor genes. SNP association and genotyping analysis were performed using ANOVA and PLINK analysis software. Results Seventy-eight SNPs were identified in nicotinic and muscarinic acetylcholine receptor genes in the CFS/ME group, of which 35 were in mAChM3. The remaining SNPs were identified in nAChR delta (n = 12), nAChR alpha 9 (n = 5), TRPV2 (n = 7), TRPM3 (n = 4), TRPM4 (n = 1) mAChRM3 2 (n = 2), and mAChRM5 (n = 3) genes. Nine genotypes were identified from SNPs in TRPM3 (n = 1), TRPC6 (n = 1), mAChRM3 (n = 2), nAChR alpha 4 (n = 1), and nAChR beta 1 (n = 4) genes, and were located in introns and 3' untranslated regions. Odds ratios for these specific genotypes ranged between 7.11 and 26.67 for CFS/ME compared with the non-fatigued control group. Conclusion This preliminary investigation identified a number of SNPs and genotypes in genes encoding TRP ion channels and AChRs from B cells in patients with CFS/ME. These may be involved in B cell functional changes, and suggest a role for Ca(2+) dysregulation in AChR and TRP ion channel signalling in the pathomechanism of CFS/ME.

  17. M1 muscarinic acetylcholine receptor agonism alters sleep without affecting memory consolidation.

    PubMed

    Nissen, Christoph; Power, Ann E; Nofzinger, Eric A; Feige, Bernd; Voderholzer, Ulrich; Kloepfer, Corinna; Waldheim, Bernhard; Radosa, Marc-Philipp; Berger, Mathias; Riemann, Dieter

    2006-11-01

    Preclinical studies have implicated cholinergic neurotransmission, specifically M1 muscarinic acetylcholine receptor (mAChR) activation, in sleep-associated memory consolidation. In the present study, we investigated the effects of administering the direct M1 mAChR agonist RS-86 on pre-post sleep memory consolidation. Twenty healthy human participants were tested in a declarative word-list task and a procedural mirror-tracing task. RS-86 significantly reduced rapid eye movement (REM) sleep latency and slow wave sleep (SWS) duration in comparison with placebo. Presleep acquisition and postsleep recall rates were within the expected ranges. However, recall rates in both tasks were almost identical for the RS-86 and placebo conditions. These results indicate that selective M1 mAChR activation in healthy humans has no clinically relevant effect on pre-post sleep consolidation of declarative or procedural memories at a dose that reduces REM sleep latency and SWS duration.

  18. Correlation between acetylcholine receptor antibody levels and thymic pathology in myasthenia gravis: a review.

    PubMed

    Huang, G Z; Lo, Y L

    2013-06-01

    Myasthenia gravis is the most common chronic autoimmune neuromuscular disease. Anti-acetylcholine receptor (AChR) antibodies are found in at least 80% of patients with generalized myasthenia and have been implicated in disease pathogenesis. Thymic abnormalities are frequently found in seropositive patients, and the thymus is thought to be involved in generation of autoimmunity. This article reviews existing literature on the role of AChR antibodies in the pathogenesis of myasthenia gravis, and the correlation between AChR antibody titers and thymic pathology. Most studies found that highest titers are seen in thymic hyperplasia, followed by intermediate titers in thymoma, and lowest titers in atrophic or normal thymus. One publication found no difference between titers in thymoma and normal thymus.

  19. Dynamic heterogeneity and non-Gaussian statistics for acetylcholine receptors on live cell membrane

    NASA Astrophysics Data System (ADS)

    He, W.; Song, H.; Su, Y.; Geng, L.; Ackerson, B. J.; Peng, H. B.; Tong, P.

    2016-05-01

    The Brownian motion of molecules at thermal equilibrium usually has a finite correlation time and will eventually be randomized after a long delay time, so that their displacement follows the Gaussian statistics. This is true even when the molecules have experienced a complex environment with a finite correlation time. Here, we report that the lateral motion of the acetylcholine receptors on live muscle cell membranes does not follow the Gaussian statistics for normal Brownian diffusion. From a careful analysis of a large volume of the protein trajectories obtained over a wide range of sampling rates and long durations, we find that the normalized histogram of the protein displacements shows an exponential tail, which is robust and universal for cells under different conditions. The experiment indicates that the observed non-Gaussian statistics and dynamic heterogeneity are inherently linked to the slow-active remodelling of the underlying cortical actin network.

  20. Design and synthesis of isoxazole containing bioisosteres of epibatidine as potent nicotinic acetylcholine receptor agonists.

    PubMed

    Singh, S; Avor, K S; Pouw, B; Seale, T W; Basmadjian, G P

    1999-10-01

    An efficient synthesis of isoxazole containing isosteres of epibatidine is described. The synthesis proceeded from N-tert-butoxycarbonyl (Boc)-exo-2-(methoxycarbonyl)-7-azabicyclo[2.2.1]heptane (9). Compound 9 was reacted with the dilithium salt of an appropriately substituted oxime in tetrahydrofuran (THF). Cyclodehydration of the resultant beta-keto oxime and deprotection of the N-Boc group in 5 N aqueous HCl afforded the isoxazole containing isosteres of epibatidine (6-8). The binding affinities of these compounds were determined at the nicotinic acetylcholine receptor for the displacement of [3H]cystisine. The unsubstituted isoxazole containing isostere (6) showed the lower binding potency compared to the 3'-methylisoxazole isostere (7). Substitution with a phenyl group at the 3'-position of the isoxazole significantly reduced the binding potency. The in vivo toxicological studies of these analogs were also performed. The LD50 of the analogs ranged in the order: Me > H > Ph.

  1. Cooperation between the products of different nuclei in hybrid myotubes produces localized acetylcholine receptor clusters.

    PubMed Central

    Gordon, H; Ralston, E; Hall, Z W

    1992-01-01

    Cultured myotubes form clusters of acetylcholine receptors (AChRs) spontaneously and at sites of nerve-muscle contact. To investigate the cellular mechanisms by which spontaneous clusters are formed, we have made hybrid myotubes between a mouse muscle cell variant, S27, that does not cluster AChRs, and one that does not make AChRs. We have also made hybrid myotubes using S27 and quail muscle cells. In both cases, clusters of AChRs were found near the non-S27 nuclei; in the case of the interspecific hybrids, mouse AChRs were associated with extracellular matrix components contributed by the quail nuclei. Our results suggest that AChRs made by one nucleus can be clustered by localized extracellular matrix produced by a different nucleus and provide an example of nuclear cooperation between the products of different nuclei within multinucleated muscle fibers. Images PMID:1631161

  2. [Molecular dynamics simulations of migration of ions and molecules through the acetylcholine receptor channel].

    PubMed

    Shaĭtan, K V; Li, A; Tershkina, K B; Kirpichnikov, M P

    2007-01-01

    A dynamic model of the channel of an acetylcholine receptor in a closed state has been proposed. The channel is formed by five a-helices of subunit M2 and stabilized by the cyclic hydrocarbon (CH2)105. The migration of charged and unchanged van der Waals particles with a diameter of 7.72 A equivalent to the diameter of a hydrated sodium ion has been studied. The migration occurred by the action of external force applied to the complex along the channel axis. In the closed state, the inhibition of ions is due to two components: electrostatic interaction and steric constraints. The van der Waals channel gate is formed by residues 13'-A-Val255, B-Val261, C-Val269, D-Val255, and E-Ile264, and the negatively changed residues occurring in the upper part of the channel have a great effect on ion selectivity.

  3. A method for soluble overexpression of the 7 nicotinic acetylcholine receptor extracellular domain

    NASA Astrophysics Data System (ADS)

    Fischer, Markus; Corringer, Pierre-Jean; Schott, Karin; Bacher, Adelbert; Changeux, Jean-Pierre

    2001-03-01

    We describe the construction of a soluble protein carrying the N-terminal extracellular domain (ECD) of the 7 subunit of the nicotinic acetylcholine receptor. The approach was to fuse the 7 ECD at the C and N termini of several monomeric and pentameric soluble carrier proteins and to investigate the soluble expression of the product in Escherichia coli. An initial screening of six carrier proteins resulted in the selection of a fusion protein comprising, from the N to the C terminus, the maltose binding protein, a 17-aa linker containing an enterokinase binding site, and the α7 ECD. This protein is soluble upon expression in bacteria and is purified by affinity chromatography. It binds the competitive nicotinic antagonist α-bungarotoxin with 2.5 μM affinity and displays a CD spectrum corresponding to a folded protein. The method might be suitable to produce large quantities of protein for crystallization and immunochemical experiments.

  4. Role of Nicotinic Acetylcholine Receptor on Efferent Inhibition in Cochlear Hair Cell

    PubMed Central

    2012-01-01

    The α9α10 nicotinic acetylcholine receptors (nAChRs) mediates efferent inhibition of hair cell function within the auditory sensory organ. Gating of the nAChRs leads to activation of calcium-dependent potassium channels to hyperpolarize the hair cell. In efferent system, main calcium providers to SK channel are nAChR and synaptic cistern, which contribution to efferent inhibition is different between avian and mammalian species. Calcium permeation is more effective in nAChRs of mammalian cochlea than avian cochlea, and mammalian calcium permeability of nAChRs is about 3 times more than avian hair cell. Thus, mammalian nAChRs is a main component of efferent inhibition in cochlear hair cell system. PMID:24653883

  5. The essential oil of Eucalyptus tereticornis, and its constituents alpha- and beta-pinene, potentiate acetylcholine-induced contractions in isolated rat trachea.

    PubMed

    Lima, Francisco J B; Brito, Teresinha S; Freire, Walter B S; Costa, Roberta C; Linhares, Maria I; Sousa, Francisca C F; Lahlou, Saad; Leal-Cardoso, José H; Santos, Armênio A; Magalhães, Pedro J C

    2010-09-01

    The effects of the essential oil of Eucalyptus tereticornis (EOET), especially the effects of its constituents alpha- and beta-pinene, were studied on rat trachea in vitro. In tracheal rings, EOET, alpha- or beta-pinene potentiated the contractions induced by acetylcholine (ACh). Contractions induced by K(+) (60mM) were also potentiated by alpha- and beta-pinene, but were reduced by EOET. Our findings show that EOET has myorelaxant effects on rat airways, but potentiates ACh-induced contractions. Monoterpenes alpha- and beta-pinene are involved in its potentiating actions, but are not responsible for its myorelaxant effects. A putative inhibition of the acetylcholinesterase enzyme is involved.

  6. Stoichiometry and pharmacology of two human alpha4beta2 nicotinic receptor types.

    PubMed

    Moroni, Mirko; Bermudez, Isabel

    2006-01-01

    The alpha4beta2 nicotinic acetylcholine receptor (nAChR) is the most abundant nAChR subtype in the brain, where it forms the high-affinity binding site for nicotine. The alpha4beta2 nAChR belongs to a gene family of ligand-gated ion channels that also includes muscle nAChRs, GABAA receptors, and glycine receptors and that assembles into pentameric structures. alpha4 and beta2 nAChR subunits expressed heterologously in Xenopus laevis oocytes assemble into a mixture of high- and low-affinity functional receptors, giving rise to biphasic ACh concentration-response curves (Zwart and Vijverberg, 1998; Buisson and Bertrand, 2001; Houlihan et al., 2001). High- and low-affinity alpha4beta2 nAChRs differ significantly in their functional and pharmacological properties (Zwart and Vijverberg, 1998; Buisson and Bertrand, 2001; Houlihan et al., 2001; Nelson et al., 2003) and result from the assembly of alpha4 and beta2 subunits into two distinct stoichiometric arrangements: (alpha4)2(beta2)3(high-affinity subtype) and (alpha4)3(beta2)2 (low-affinity subtype) (Nelson et al., 2003). In this study we have examined the functional and pharmacological properties of high- and low-affinity alpha4beta2 receptors using two-electrode voltage clamp procedures on Xenopus oocytes transfected with high (1:10) or low (10:1) ratios of alpha4/beta2 cDNAs, which yield high (1:10)- or low (10:1)- affinity receptors with monophasic ACh concentration- response curves. Furthermore, to determine the stoichiometry of high- and low-affinity receptors expressed heterologously by Xenopus oocytes, we have determined the stoichiometry of high- and low-affinity alpha4beta2 receptors by mutating a highly conserved hydrophobic residue in the middle (position 9') of the pore-lining domain, which increases agonist potency in a manner that allows predictions on subunit composition (Cooper et al., 1991; Revah et al., 1991; Labarca et al., 1995; Boorman et al., 2000).

  7. Computational determination of the binding mode of α-conotoxin to nicotinic acetylcholine receptor

    NASA Astrophysics Data System (ADS)

    Tabassum, Nargis; Yu, Rilei; Jiang, Tao

    2016-12-01

    Conotoxins belong to the large families of disulfide-rich peptide toxins from cone snail venom, and can act on a broad spectrum of ion channels and receptors. They are classified into different subtypes based on their targets. The α-conotoxins selectively inhibit the current of the nicotinic acetylcholine receptors. Because of their unique selectivity towards distinct nAChR subtypes, α-conotoxins become valuable tools in nAChR study. In addition to the X-ray structures of α-conotoxins in complex with acetylcholine-binding protein, a homolog of the nAChR ligand-binding domain, the high-resolution crystal structures of the extracellular domain of the α1 and α9 subunits are also obtained. Such structures not only revealed the details of the configuration of nAChR, but also provided higher sequence identity templates for modeling the binding modes of α-conotoxins to nAChR. This mini-review summarizes recent modeling studies for the determination of the binding modes of α-conotoxins to nAChR. As there are not crystal structures of the nAChR in complex with conotoxins, computational modeling in combination of mutagenesis data is expected to reveal the molecular recognition mechanisms that govern the interactions between α-conotoxins and nAChR at molecular level. An accurate determination of the binding modes of α-conotoxins on AChRs allows rational design of α-conotoxin analogues with improved potency or selectivity to nAChRs.

  8. Modes of action, resistance and toxicity of insecticides targeting nicotinic acetylcholine receptors.

    PubMed

    Ihara, Makoto; Buckingham, Steven D; Matsuda, Kazuhiko; Sattelle, David B

    2017-02-06

    Nicotinic acetylcholine receptors (nAChRs) are members of the cys-loop superfamily of ligand-gated ion channels (cys-loop LGICs) and mediate fast cholinergic synaptic transmission in the nervous system of insects. The completion of many insect genome projects has greatly enhanced our understanding of the individual subunits that make up nAChR gene families from an insect genetic model organism (Drosophila melanogaster), crop pests, disease vectors and beneficial (pollinator) species. In addition to considerable insect nAChR subunit diversity, individual subunits can be subject to alternative splicing and RNA editing and these post-transcriptional modifications can add significantly to the diversity of nAChR receptor subtypes. The actions of insecticides targeting nAChRs, notably cartap, neonicotinoids, sulfoximines, flupyradifurone, spinosyns and triflumezopyrim are reviewed. Structural studies obtained using an acetylcholine binding protein (AChBP) co-crystallised with neonicotinoids have yielded important new insights into the requirements for neonicotinoid insecticide - nAChR interactions. The persistent application of insecticides to crop pests leads to the onset of resistance and several examples of resistance to insecticides targeting nAChRs have been documented. Understanding the molecular basis of resistance can inform our understanding of the mechanism of insecticide action. It also provides an important driver for the development of new chemistry, diagnostic tests for resistance and the adoption of application strategies designed to attenuate such problems. Finally, we consider toxicity issues relating to nAChR-active insecticides, with particular reference to beneficial insect species (pollinators) as well as mammalian and avian toxicity. This review is part of the special issue "Insecticide Mode of Action: From Insect to Mammalian Toxicity.".

  9. GLUTAMATERGIC SYNAPSE FORMATION IS PROMOTED BY α7-CONTAINING NICOTINIC ACETYLCHOLINE RECEPTORS

    PubMed Central

    Lozada, Adrian F.; Wang, Xulong; Gounko, Natalia V.; Massey, Kerri A.; Duan, Jingjing; Liu, Zhaoping; Berg, Darwin K.

    2012-01-01

    Glutamate is the primary excitatory transmitter in adult brain, acting through synapses on dendritic spines and shafts. Early in development, however, when glutamatergic synapses are only beginning to form, nicotinic cholinergic excitation is already widespread; it is mediated by acetylcholine activating nicotinic acetylcholine receptors (nAChRs) that generate waves of activity across brain regions. A major class of nAChRs contributing at this time is a species containing α7 subunits (α7-nAChRs). These receptors are highly permeable to calcium, influence a variety of calcium-dependent events, and are diversely distributed throughout the developing CNS. Here we show that α7-nAChRs unexpectedly promote formation of glutamatergic synapses during development. The dependence on α7-nAChRs becomes clear when comparing wild-type mice with mice constitutively lacking the α7-nAChR gene. Ultrastructural analysis, immunostaining, and patch-clamp recording all reveal synaptic deficits when α7-nAChR input is absent. Similarly, nicotinic activation of α7-nAChRs in wild-type organotypic culture, as well as cell culture, increases the number of glutamatergic synapses. RNA interference demonstrates that the α7-nAChRs must be expressed in the neuron being innervated for normal innervation to occur. Moreover the deficits persist throughout the developmental period of major de novo synapse formation and are still fully apparent in the adult. GABAergic synapses, in contrast, are undiminished in number under such conditions. As a result, mice lacking α7-nAChRs have an altered balance in the excitatory/inhibitory input they receive. This ratio represents a fundamental feature of neural networks and shows for the first time that endogenous nicotinic cholinergic signaling plays a key role in network construction. PMID:22649244

  10. Block of nicotinic acetylcholine receptors by philanthotoxins is strongly dependent on their subunit composition

    PubMed Central

    Kachel, Hamid S.; Patel, Rohit N.; Franzyk, Henrik; Mellor, Ian R.

    2016-01-01

    Philanthotoxin-433 (PhTX-433) is an active component of the venom from the Egyptian digger wasp, Philanthus triangulum. PhTX-433 inhibits several excitatory ligand-gated ion channels, and to improve selectivity two synthetic analogues, PhTX-343 and PhTX-12, were developed. Previous work showed a 22-fold selectivity of PhTX-12 over PhTX-343 for embryonic muscle-type nicotinic acetylcholine receptors (nAChRs) in TE671 cells. We investigated their inhibition of different neuronal nAChR subunit combinations as well as of embryonic muscle receptors expressed in Xenopus oocytes. Whole-cell currents in response to application of acetylcholine alone or co-applied with PhTX analogue were studied by using two-electrode voltage-clamp. α3β4 nAChRs were most sensitive to PhTX-343 (IC50 = 12 nM at −80 mV) with α4β4, α4β2, α3β2, α7 and α1β1γδ being 5, 26, 114, 422 and 992 times less sensitive. In contrast α1β1γδ was most sensitive to PhTX-12 along with α3β4 (IC50 values of 100 nM) with α4β4, α4β2, α3β2 and α7 being 3, 3, 26 and 49 times less sensitive. PhTX-343 inhibition was strongly voltage-dependent for all subunit combinations except α7, whereas this was not the case for PhTX-12 for which weak voltage dependence was observed. We conclude that PhTX-343 mainly acts as an open-channel blocker of nAChRs with strong subtype selectivity. PMID:27901080

  11. Upregulation of surface alpha4beta2 nicotinic receptors is initiated by receptor desensitization after chronic exposure to nicotine.

    PubMed

    Fenster, C P; Whitworth, T L; Sheffield, E B; Quick, M W; Lester, R A

    1999-06-15

    It is hypothesized that desensitization of neuronal nicotinic acetylcholine receptors (nAChRs) induced by chronic exposure to nicotine initiates upregulation of nAChR number. To test this hypothesis directly, oocytes expressing alpha4beta2 receptors were chronically incubated (24-48 hr) in nicotine, and the resulting changes in specific [3H]nicotine binding to surface receptors on intact oocytes were compared with functional receptor desensitization. Four lines of evidence strongly support the hypothesis. (1) The half-maximal nicotine concentration necessary to produce desensitization (9.7 nM) was the same as that needed to induce upregulation (9.9 nM). (2) The concentration of [3H]nicotine for half-maximal binding to surface nAChRs on intact oocytes was also similar (11.1 nM), as predicted from cyclical desensitization models. (3) Functional desensitization of alpha3beta4 receptors required 10-fold higher nicotine concentrations, and this was mirrored by a 10-fold shift in concentrations necessary for upregulation. (4) Mutant alpha4beta2 receptors that do not recover fully from desensitization, but not wild-type channels, were upregulated after acute (1 hr) applications of nicotine. Interestingly, the nicotine concentration required for half-maximal binding of alpha4beta2 receptors in total cell membrane homogenates was 20-fold lower than that measured for surface nAChRs in intact oocytes. These data suggest that cell homogenate binding assays may not accurately reflect the in vivo desensitization affinity of surface nAChRs and may account for some of the previously reported differences in the efficacy of nicotine for inducing nAChR desensitization and upregulation.

  12. Acetylcholine Receptor and Ion Conductance Modulator Sites at the Murine Neuromuscular Junction: Evidence from Specific Toxin Reactions

    PubMed Central

    Albuquerque, Edson X.; Barnard, Eric A.; Chiu, Tieh H.; Lapa, Antonio J.; Dolly, J. Oliver; Jansson, Sten-Erik; Daly, John; Witkop, Bernhard

    1973-01-01

    The perhydro derivative of histrionicotoxin reversibly blocks the excitatory ionic transduction system in the synaptic and sarcolemmal membranes of mammalian skeletal muscle cells. The efficacy of perhydrohistrionicotoxin as an antagonist at the post-synaptic membrane is increased by the transient presence of acetylcholine in the endplate of innervated muscles and at extrajunctional receptors in denervated muscles. α-Bungarotoxin and [3H]monoacetyl-α-bungarotoxin block the endplate acetylcholine receptors, each binding to the same extent. The effect of bungarotoxin is partially reversible. These electrophysiological results, together with the effects of perhydrohistrionicotoxin and/or d-tubocurarine on the binding of [3H]monoacetyl-α-bungarotoxin at endplates of murine diaphragm muscle and on the bungarotoxin-elicited irreversible blockade of neuromuscular transmission, suggest that at least two types of sites participate in the synaptic excitation by acetylcholine. One site, competitively blocked by bungarotoxin and by curare, is presumably the acetylcholine receptor. Binding of bungarotoxin at this site is responsible for an irreversible blockade of neuromuscular transmission. The second site, competitively blocked by bungarotoxin and perhydrohistrionicotoxin, is proposed to be part of the cholinergic ion conductance modulator. Binding of bungarotoxin to this site does not result in an irreversible blockade. Images PMID:4351811

  13. Long-term effects of methamphetamine exposure on cognitive function and muscarinic acetylcholine receptor levels in mice.

    PubMed

    Siegel, Jessica A; Craytor, Michael J; Raber, Jacob

    2010-10-01

    Exposure to methamphetamine during brain development impairs cognition in humans and rodents. In mice, these impairments are more severe in females than males. Genetic factors, such as apolipoprotein E genotype, may modulate the cognitive effects of methamphetamine. Methamphetamine-induced alterations in the brain acetylcholine system may contribute to the cognitive effects of methamphetamine and may also be modulated by apolipoprotein E isoform. We assessed the long-term effects of methamphetamine exposure during brain development on cognitive function and muscarinic acetylcholine receptors in mice, and whether apolipoprotein E isoform modulates these effects. Mice expressing human apolipoprotein E3 or E4 were exposed to methamphetamine (5 mg/kg) or saline once a day from postnatal days 11-20 and behaviorally tested in adulthood. Muscarinic acetylcholine receptor binding was measured in the hippocampus and cortex. Methamphetamine exposure impaired novel location recognition in female, but not male, mice. Methamphetamine-exposed male and female mice showed impaired novel object recognition and increased number of muscarinic acetylcholine receptors in the hippocampus. The cognitive and cholinergic effects of methamphetamine were similar in apolipoprotein E3 and E4 mice. Thus, the cholinergic system, but not apolipoprotein E isoform, might play an important role in the long-term methamphetamine-induced cognitive deficits in adulthood.

  14. Pharmacological and ionic characterizations of the muscarinic receptors modulating (/sup 3/H)acetylcholine release from rat cortical synaptosomes

    SciTech Connect

    Meyer, E.M.; Otero, D.H.

    1985-05-01

    The muscarinic receptors that modulate acetylcholine release from rat cortical synaptosomes were characterized with respect to sensitivity to drugs that act selectively at M1 or M2 receptor subtypes, as well as to changes in ionic strength and membrane potential. The modulatory receptors appear to be of the M2 type, since they are activated by carbachol, acetylcholine, methacholine, oxotremorine, and bethanechol, but not by pilocarpine, and are blocked by atropine, scopolamine, and gallamine (at high concentrations), but not by pirenzepine or dicyclomine. The ED50S for carbachol, acetylcholine, and oxotremorine are less than 10 microM, suggesting that the high affinity state of the receptor is functional. High ionic strength induced by raising the NaCl concentration has no effect on agonist (oxotremorine) potency, but increases the efficacy of this compound, which disagrees with receptor-binding studies. On the other hand, depolarization with either KCl or with veratridine (20 microM) reduces agonist potencies by approximately an order of magnitude, suggesting a potential mechanism for receptor regulation.

  15. Symposium overview: mechanism of action of nicotine on neuronal acetylcholine receptors, from molecule to behavior.

    PubMed

    Narahashi, T; Fenster, C P; Quick, M W; Lester, R A; Marszalec, W; Aistrup, G L; Sattelle, D B; Martin, B R; Levin, E D

    2000-10-01

    Nicotine has long been known to interact with nicotinic acetylcholine (ACh) receptors since Langley used it extensively to chart sympathetic ganglia a century ago. It has also been used as an effective insecticide. However, it was not until the 1990s that the significance of nicotine was increasingly recognized from the toxicological, pharmacological, and environmental points of view. This is partly because studies of neuronal nicotinic ACh receptors are rapidly emerging from orphan status, fueled by several lines of research. Since Alzheimer's disease is known to be associated with down-regulation of cholinergic activity in the brain, a variety of nicotine derivatives are being tested and developed for treatment of the disease. Public awareness of the adverse effects of nicotine has reached the highest level recently. Since insect resistance to insecticides is one of the most serious issues in the pest-control arena, it is an urgent requirement to develop new insecticides that act on target sites not shared by the existing insecticides. The neuronal nicotinic ACh receptor is one of them, and new nicotinoids are being developed. Thus, the time is ripe to discuss the mechanism of action of nicotine from a variety of angles, including the molecular, physiological, and behavioral points of view. This Symposium covered a wide area of nicotine studies: genetic, genomic, and functional aspects of nicotinic ACh receptors were studied, as related to anthelmintics and insecticides; interactions between ethanol and nicotine out the ACh receptor were analyzed, in an attempt to explain the well-known heavy drinker-heavy smoker correlation; the mechanisms that underlie the desensitization of ACh receptors were studied as related to nicotine action; selective pharmacological profiles of nicotine, and descriptions of some derivatives were described; and chronic nicotine infusion effects on memory were examined using animal models.

  16. Modulation of nicotinic acetylcholine and N-methyl-d-aspartate receptors by some Hymenopteran venoms.

    PubMed

    Zalat, Samy; Elbana, Shereen; Rizzoli, Silvio; Schmidt, Justin O; Mellor, Ian R

    2005-09-01

    The effect of 19 venoms from solitary wasps, solitary bees, social wasps and ants were investigated for their effects on nicotinic acetylcholine receptors (nAChR) and ionotropic glutamate receptors (IGRs) of both the N-methyl-d-aspartate (NMDAR) and non-NMDAR type. Whole-cell patch clamp of human muscle TE671 cells was used to study nAChR, and of rat cortical and cerebellar granule cells for IGRs. Solitary wasp venoms caused significant voltage-dependent antagonism of nAChR responses to 10 microM ACh and NMDAR responses to 100 microM NMDA (+10 microM glycine) when co-applied at 1 microg/ml with the agonists. At positive holding potentials (V(H)) potentiation of these receptors was observed with some venoms. Solitary bee venoms only affected nAChR by causing either voltage-independent antagonism or potentiation of their responses to 10 microM ACh. Of four social wasp venoms, one acted on nAChR by potentiating responses to 10 ACh, while another generated an ACh-like response when applied alone. They had no effect on IGRs. Of the two ant venoms, one caused voltage-independent inhibition of nAChR. Neither affected IGRs. The data indicate the presence of nAChR agonists and antagonists and NMDAR antagonists in Hymenopteran venoms and warrant further investigation to separate and identify these venom components.

  17. Inhibitory effect of strychnine on acetylcholine receptor activation in bovine adrenal medullary chromaffin cells.

    PubMed Central

    Kuijpers, G A; Vergara, L A; Calvo, S; Yadid, G

    1994-01-01

    1. Strychnine, which is known as a potent and selective antagonist of the inhibitory glycine receptor in the central nervous system, inhibits the nicotinic stimulation of catecholamine release from bovine cultured adrenal chromaffin cells in a concentration-dependent (1-100 microM) manner. At 10 microM nicotine, the IC50 value for strychnine is approximately 30 microM. Strychnine also inhibits the nicotine-induced membrane depolarization and increase in intracellular Ca2+ concentration. 2. The inhibitory action of strychnine is reversible and is selective for nicotinic stimulation, with no effect observed on secretion elicited by a high external K+ concentration, histamine or angiotensin II. 3. Strychnine competes with nicotine in its effect, but not modify the apparent positive cooperatively of the nicotine binding sites. In the absence of nicotine, strychnine has no effect on catecholamine release. Glycine does not affect catecholamine release nor the inhibitory action of strychnine on this release. 4. These results suggest that strychnine interacts with the agonist binding site of the nicotinic acetylcholine receptor in chromaffin cells, thus exerting a pharmacological effect independently of the glycine receptor. PMID:7834198

  18. Unraveling a molecular determinant for clathrin-independent internalization of the M2 muscarinic acetylcholine receptor

    PubMed Central

    Wan, Min; Zhang, Wenhua; Tian, Yangli; Xu, Chanjuan; Xu, Tao; Liu, Jianfeng; Zhang, Rongying

    2015-01-01

    Endocytosis and postendocytic sorting of G-protein-coupled receptors (GPCRs) is important for the regulation of both their cell surface density and signaling profile. Unlike the mechanisms of clathrin-dependent endocytosis (CDE), the mechanisms underlying the control of GPCR signaling by clathrin-independent endocytosis (CIE) remain largely unknown. Among the muscarinic acetylcholine receptors (mAChRs), the M4 mAChR undergoes CDE and recycling, whereas the M2 mAChR is internalized through CIE and targeted to lysosomes. Here we investigated the endocytosis and postendocytic trafficking of M2 mAChR based on a comparative analysis of the third cytoplasmic domain in M2 and M4 mAChRs. For the first time, we identified that the sequence 374KKKPPPS380 servers as a sorting signal for the clathrin-independent internalization of M2 mAChR. Switching 374KKKPPPS380 to the i3 loop of the M4 mAChR shifted the receptor into lysosomes through the CIE pathway; and therefore away from CDE and recycling. We also found another previously unidentified sequence that guides CDE of the M2 mAChR, 361VARKIVKMTKQPA373, which is normally masked in the presence of the downstream sequence 374KKKPPPS380. Taken together, our data indicate that endocytosis and postendocytic sorting of GPCRs that undergo CIE could be sequence-dependent. PMID:26094760

  19. Binding affinities of anti-acetylcholine receptor autoantibodies in myasthenia gravis

    SciTech Connect

    Bray, J.J.; Drachman, D.B.

    1982-01-01

    Antibodies directed against acetylcholine (ACh) receptors are present in the sera of nearly 90% of patients with myasthenia gravis (MG), and are involved in the pathogenesis of this autoimmune disease. However, the antibody titers measured by the standard radioimmunoassay correspond poorly with the clinical severity of the disease. To determine whether this disparity could be accounted for by differences in the binding affinities of anti-ACh receptor antibodies in different patients, we have measured the binding affinities of these autoantibodies in 15 sera from MG patients. The affinity constants (K/sub o/), as determined by Scatchard analysis, were all in the range of 10/sup 10/ M/sup -1/, comparable to the highest values reported in immunized animals. The affinity constants were truly representative of the population of autoantibodies detected by the radioimmunoassay, as shown by the remarkable linearity of the Scatchard plots (r/sup 2/>0.90) and the close correlation between the antibody titers determined by extrapolation of the Scatchard plots and by saturation analysis (r = 0.99; p < 0.001). There was only a 6-fold variation in affinity constants measured in this series of patients despite widely differing antibody titers and severity of the disease. Factors other than the titer and affinity of anti-ACh receptor antibodies may correlate better with the clinical manifestations of MG.

  20. Molecular Modeling of the M3 Acetylcholine Muscarinic Receptor and Its Binding Site

    PubMed Central

    Martinez-Archundia, Marlet; Cordomi, Arnau; Garriga, Pere; Perez, Juan J.

    2012-01-01

    The present study reports the results of a combined computational and site mutagenesis study designed to provide new insights into the orthosteric binding site of the human M3 muscarinic acetylcholine receptor. For this purpose a three-dimensional structure of the receptor at atomic resolution was built by homology modeling, using the crystallographic structure of bovine rhodopsin as a template. Then, the antagonist N-methylscopolamine was docked in the model and subsequently embedded in a lipid bilayer for its refinement using molecular dynamics simulations. Two different lipid bilayer compositions were studied: one component palmitoyl-oleyl phosphatidylcholine (POPC) and two-component palmitoyl-oleyl phosphatidylcholine/palmitoyl-oleyl phosphatidylserine (POPC-POPS). Analysis of the results suggested that residues F222 and T235 may contribute to the ligand-receptor recognition. Accordingly, alanine mutants at positions 222 and 235 were constructed, expressed, and their binding properties determined. The results confirmed the role of these residues in modulating the binding affinity of the ligand. PMID:22500107

  1. Unraveling a molecular determinant for clathrin-independent internalization of the M2 muscarinic acetylcholine receptor.

    PubMed

    Wan, Min; Zhang, Wenhua; Tian, Yangli; Xu, Chanjuan; Xu, Tao; Liu, Jianfeng; Zhang, Rongying

    2015-06-22

    Endocytosis and postendocytic sorting of G-protein-coupled receptors (GPCRs) is important for the regulation of both their cell surface density and signaling profile. Unlike the mechanisms of clathrin-dependent endocytosis (CDE), the mechanisms underlying the control of GPCR signaling by clathrin-independent endocytosis (CIE) remain largely unknown. Among the muscarinic acetylcholine receptors (mAChRs), the M4 mAChR undergoes CDE and recycling, whereas the M2 mAChR is internalized through CIE and targeted to lysosomes. Here we investigated the endocytosis and postendocytic trafficking of M2 mAChR based on a comparative analysis of the third cytoplasmic domain in M2 and M4 mAChRs. For the first time, we identified that the sequence (374)KKKPPPS(380) servers as a sorting signal for the clathrin-independent internalization of M2 mAChR. Switching (374)KKKPPPS(380) to the i3 loop of the M4 mAChR shifted the receptor into lysosomes through the CIE pathway; and therefore away from CDE and recycling. We also found another previously unidentified sequence that guides CDE of the M2 mAChR, (361)VARKIVKMTKQPA(373), which is normally masked in the presence of the downstream sequence (374)KKKPPPS(380). Taken together, our data indicate that endocytosis and postendocytic sorting of GPCRs that undergo CIE could be sequence-dependent.

  2. Effects of isoflurane on the actions of neuromuscular blockers on the muscle nicotine acetylcholine receptors.

    PubMed

    Li, Chuanxiang; Yao, Shanglong; Nie, Hui; Lü, Bin

    2004-01-01

    In this study, we tested the hypothesis that volatile anesthetic enhancement of muscle relaxation is the result of combined drug effects on the nicotinic acetylcholine receptors. The poly A m RNA from muscle by isolation were microinjected into Xenopus oocytes for receptor expression. Concentration-effect curves for the inhibition of Ach-induced currents were established for vecuronium, rocuranium, and isoflurane. Subsequently, inhibitory effects of NDMRs were studied in the presence of the isoflurane at a concentration equivalent to half the concentration producing a 50% inhibition alone. All tested drugs produced rapid and readily reversible concentration-dependent inhibition. The 50% inhibitory concentration values were 889 micromol/L (95% CI: 711-1214 micromol). 33.4 micromol (95% CI: 27.1-41.7 nmol) and 9.2 nmol (95% CI: 7.9-12.3 nmol) for isoflurane. rocuranium and vecuronium, respectively. Coapplication of isoflurane significantly enhanced the inhibitory effects of rocuranium and vecuronium, and it was especially so at low concentration of NMDRs. Isoflurane increases the potency of NDMRs, possibly by enhancing antagonist affinity at the receptor site.

  3. Fixation of allosteric states of the nicotinic acetylcholine receptor by chemical cross-linking

    PubMed Central

    Watty, Anke; Methfessel, Christoph; Hucho, Ferdinand

    1997-01-01

    Receptor activity can be described in terms of ligand-induced transitions between functional states. The nicotinic acetylcholine receptor (nAChR), a prototypic ligand-gated ion channel, is an “unconventional allosteric protein” which exists in at least three interconvertible conformations, referred to as resting (low agonist affinity, closed channel), activated (open channel), and desensitized (high agonist affinity, closed channel). Here we show that 3,3′-dimethyl suberimidate (DMS) is an agonistic bifunctional cross-linking reagent, which irreversibly “freezes” the nAChR in a high agonist affinity/closed-channel state. The monofunctional homologue methyl acetoimidate, which is also a weak cholinergic agonist, has no such irreversible effect. Glutardialdehyde, a cross-linker that is not a cholinergic effector, fixes the receptor in a low-affinity state in the absence of carbamoylcholine, but, like DMS, in a high-affinity state in its presence. Covalent cross-linking thus allows us to arrest the nAChR in defined conformational states. PMID:9223339

  4. Stoichiometry for α-bungarotoxin block of α7 acetylcholine receptors

    PubMed Central

    daCosta, Corrie J. B.; Free, Chris R.; Sine, Steven M.

    2015-01-01

    α-Bungarotoxin (α-Btx) binds to the five agonist binding sites on the homopentameric α7-acetylcholine receptor, yet the number of bound α-Btx molecules required to prevent agonist-induced channel opening remains unknown. To determine the stoichiometry for α-Btx blockade, we generate receptors comprised of wild-type and α-Btx-resistant subunits, tag one of the subunit types with conductance mutations to report subunit stoichiometry, and following incubation with α-Btx, monitor opening of individual receptor channels with defined subunit stoichiometry. We find that a single α-Btx-sensitive subunit confers nearly maximal suppression of channel opening, despite four binding sites remaining unoccupied by α-Btx and accessible to the agonist. Given structural evidence that α-Btx locks the agonist binding site in an inactive conformation, we conclude that the dominant mechanism of antagonism is non-competitive, originating from conformational arrest of the binding sites, and that the five α7 subunits are interdependent and maintain conformational symmetry in the open channel state. PMID:26282895

  5. Mice Lacking M1 and M3 Muscarinic Acetylcholine Receptors Have Impaired Odor Discrimination and Learning

    PubMed Central

    Chan, Wilson; Singh, Sanmeet; Keshav, Taj; Dewan, Ramita; Eberly, Christian; Maurer, Robert; Nunez-Parra, Alexia; Araneda, Ricardo C.

    2017-01-01

    The cholinergic system has extensive projections to the olfactory bulb (OB) where it produces a state-dependent regulation of sensory gating. Previous work has shown a prominent role of muscarinic acetylcholine (ACh) receptors (mAChRs) in regulating the excitability of OB neurons, in particular the M1 receptor. Here, we examined the contribution of M1 and M3 mAChR subtypes to olfactory processing using mice with a genetic deletion of these receptors, the M1−/− and the M1/M3−/− knockout (KO) mice. Genetic ablation of the M1 and M3 mAChRs resulted in a significant deficit in odor discrimination of closely related molecules, including stereoisomers. However, the discrimination of dissimilar molecules, social odors (e.g., urine) and novel object recognition was not affected. In addition the KO mice showed impaired learning in an associative odor-learning task, learning to discriminate odors at a slower rate, indicating that both short and long-term memory is disrupted by mAChR dysfunction. Interestingly, the KO mice exhibited decreased olfactory neurogenesis at younger ages, a deficit that was not maintained in older animals. In older animals, the olfactory deficit could be restored by increasing the number of new born neurons integrated into the OB after exposing them to an olfactory enriched environment, suggesting that muscarinic modulation and adult neurogenesis could be two different mechanism used by the olfactory system to improve olfactory processing. PMID:28210219

  6. Nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induce cyclooxygenase-2 activity in human gastric cancer cells: Involvement of nicotinic acetylcholine receptor (nAChR) and {beta}-adrenergic receptor signaling pathways

    SciTech Connect

    Shin, Vivian Yvonne; Jin, H.C.; Ng, Enders K.O.; Yu Jun; Leung, W.K.; Cho, C.H.; Sung, J.J.Y.

    2008-12-01

    Induction of cyclooxygenase-2 (COX-2) associates with cigarette smoke exposure in many malignancies. Nicotine and its derivative, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are the two important components in cigarette smoke that contributes to cancer development. However, the molecular mechanism(s) by which nicotine or NNK promotes gastric carcinogenesis remains largely unknown. We found that nicotine and NNK significantly enhanced cell proliferation in AGS cells that expressed both alpha7 nicotinic acetylcholine receptor ({alpha}7 nAChR) and {beta}-adrenergic receptors. Treatment of cells with {alpha}-bungarotoxin ({alpha}-BTX, {alpha}7nAChR antagonist) or propranolol ({beta}-adrenergic receptor antagonist) blocked NNK-induced COX-2/PGE{sub 2} and cell proliferation, while nicotine-mediated cell growth and COX-2/PGE{sub 2} induction can only be suppressed by propranolol, but not {alpha}-BTX. Moreover, in contrast to the dependence of growth promoting effect of nicotine on Erk activation, inhibitor of p38 mitogen-activated protein kinase (MAPK) repressed NNK-induced COX-2 upregulation and resulted in suppression of cell growth. In addition, nicotine and NNK mediated COX-2 induction via different receptors to modulate several G1/S transition regulatory proteins and promote gastric cancer cell growth. Selective COX-2 inhibitor (SC-236) caused G1 arrest and abrogated nicotine/NNK-induced cell proliferation. Aberrant expression of cyclin D1 and other G1 regulatory proteins are reversed by blockade of COX-2. These results pointed to the importance of adrenergic and nicotinic receptors in gastric tumor growth through MAPK/COX-2 activation, which may perhaps provide a chemoprevention strategy for cigarette smoke-related gastric carcinogenesis.

  7. 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.

    PubMed

    Taillebois, Emiliane; Thany, Steeve H

    2016-09-01

    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.

  8. Subunit Interfaces Contribute Differently to Activation and Allosteric Modulation of Neuronal Nicotinic Acetylcholine Receptors

    PubMed Central

    Short, Caitlin A.; Cao, Angela T.; Wingfield, Molly A.; Doers, Matthew E.; Jobe, Emily M.; Wang, Nan; Levandoski, Mark M.

    2015-01-01

    Neuronal nicotinic acetylcholine receptors (nAChRs) are widely distributed in the nervous system and are implicated in many normal and pathological processes. The structural determinants of allostery in nAChRs are not well understood. One class of nAChR allosteric modulators, including the small molecule morantel (Mor), acts from a site that is structurally homologous to the canonical agonist site but exists in the β(+)/α(–) subunit interface. We hypothesized that all nAChR subunits move with respect to each other during channel activation and allosteric modulation. We therefore studied five pairs of residues predicted to span the interfaces of α3β2 receptors, one at the agonist interface and four at the modulator interface. Substituting cysteines in these positions, we used disulfide trapping to perturb receptor function. The pair α3Y168-β2D190, involving the C loop region of the β2 subunit, mediates modulation and agonist activation, because evoked currents were reduced up to 50% following oxidation (H2O2) treatment. The pair α3S125-β2Q39, below the canonical site, is also involved in channel activation, in accord with previous studies of the muscle-type receptor; however, the pair is differentially sensitive to ACh activation and Mor modulation (currents decreased 60% and 80%, respectively). The pairs α3Q37-β2A127 and α3E173-β2R46, both in the non-canonical interface, showed increased currents following oxidation, suggesting that subunit movements are not symmetrical. Together, our results from disulfide trapping and further mutation analysis indicate that subunit interface movement is important for allosteric modulation of nAChRs, but that the two types of interfaces contribute unequally to receptor activation. PMID:25486620

  9. Tracking the Molecular Evolution of Calcium Permeability in a Nicotinic Acetylcholine Receptor

    PubMed Central

    Lipovsek, Marcela; Fierro, Angélica; Pérez, Edwin G.; Boffi, Juan C.; Millar, Neil S.; Fuchs, Paul A.; Katz, Eleonora; Elgoyhen, Ana Belén

    2014-01-01

    Nicotinic acetylcholine receptors are a family of ligand-gated nonselective cationic channels that participate in fundamental physiological processes at both the central and the peripheral nervous system. The extent of calcium entry through ligand-gated ion channels defines their distinct functions. The α9α10 nicotinic cholinergic receptor, expressed in cochlear hair cells, is a peculiar member of the family as it shows differences in the extent of calcium permeability across species. In particular, mammalian α9α10 receptors are among the ligand-gated ion channels which exhibit the highest calcium selectivity. This acquired differential property provides the unique opportunity of studying how protein function was shaped along evolutionary history, by tracking its evolutionary record and experimentally defining the amino acid changes involved. We have applied a molecular evolution approach of ancestral sequence reconstruction, together with molecular dynamics simulations and an evolutionary-based mutagenesis strategy, in order to trace the molecular events that yielded a high calcium permeable nicotinic α9α10 mammalian receptor. Only three specific amino acid substitutions in the α9 subunit were directly involved. These are located at the extracellular vestibule and at the exit of the channel pore and not at the transmembrane region 2 of the protein as previously thought. Moreover, we show that these three critical substitutions only increase calcium permeability in the context of the mammalian but not the avian receptor, stressing the relevance of overall protein structure on defining functional properties. These results highlight the importance of tracking evolutionarily acquired changes in protein sequence underlying fundamental functional properties of ligand-gated ion channels. PMID:25193338

  10. Tracking the molecular evolution of calcium permeability in a nicotinic acetylcholine receptor.

    PubMed

    Lipovsek, Marcela; Fierro, Angélica; Pérez, Edwin G; Boffi, Juan C; Millar, Neil S; Fuchs, Paul A; Katz, Eleonora; Elgoyhen, Ana Belén

    2014-12-01

    Nicotinic acetylcholine receptors are a family of ligand-gated nonselective cationic channels that participate in fundamental physiological processes at both the central and the peripheral nervous system. The extent of calcium entry through ligand-gated ion channels defines their distinct functions. The α9α10 nicotinic cholinergic receptor, expressed in cochlear hair cells, is a peculiar member of the family as it shows differences in the extent of calcium permeability across species. In particular, mammalian α9α10 receptors are among the ligand-gated ion channels which exhibit the highest calcium selectivity. This acquired differential property provides the unique opportunity of studying how protein function was shaped along evolutionary history, by tracking its evolutionary record and experimentally defining the amino acid changes involved. We have applied a molecular evolution approach of ancestral sequence reconstruction, together with molecular dynamics simulations and an evolutionary-based mutagenesis strategy, in order to trace the molecular events that yielded a high calcium permeable nicotinic α9α10 mammalian receptor. Only three specific amino acid substitutions in the α9 subunit were directly involved. These are located at the extracellular vestibule and at the exit of the channel pore and not at the transmembrane region 2 of the protein as previously thought. Moreover, we show that these three critical substitutions only increase calcium permeability in the context of the mammalian but not the avian receptor, stressing the relevance of overall protein structure on defining functional properties. These results highlight the importance of tracking evolutionarily acquired changes in protein sequence underlying fundamental functional properties of ligand-gated ion channels.

  11. Activation of nematode G protein GOA-1 by the human muscarinic acetylcholine receptor M2 subtype. Functional coupling of G-protein-coupled receptor and G protein originated from evolutionarily distant animals.

    PubMed

    Minaba, Masaomi; Ichiyama, Susumu; Kojima, Katsura; Ozaki, Mamiko; Kato, Yusuke

    2006-12-01

    Signal transduction mediated by heterotrimeric G proteins regulates a wide variety of physiological functions. We are interested in the manipulation of G-protein-mediating signal transduction using G-protein-coupled receptors, which are derived from evolutionarily distant organisms and recognize unique ligands. As a model, we tested the functionally coupling GOA-1, G alpha(i/o) ortholog in the nematode Caenorhabditis elegans, with the human muscarinic acetylcholine receptor M2 subtype (M2), which is one of the mammalian G alpha(i/o)-coupled receptors. GOA-1 and M2 were prepared as a fusion protein using a baculovirus expression system. The affinity of the fusion protein for GDP was decreased by addition of a muscarinic agonist, carbamylcholine and the guanosine 5'-[3-O-thio]triphosphate ([35S]GTPgammaS) binding was increased with an increase in the carbamylcholine concentrations in a dose-dependent manner. These effects evoked by carbamylcholine were completely abolished by a full antagonist, atropine. In addition, the affinity for carbamylcholine decreased under the presence of GTP as reported for M2-G alpha(i/o) coupling. These results indicate that the M2 activates GOA-1 as well as G alpha(i/o).

  12. The effective opening of nicotinic acetylcholine receptors with single agonist binding sites

    PubMed Central

    Williams, Dustin K.; Stokes, Clare; Horenstein, Nicole A.

    2011-01-01

    We have identified a means by which agonist-evoked responses of nicotinic receptors can be conditionally eliminated. Modification of α7L119C mutants by the sulfhydryl reagent 2-aminoethyl methanethiosulfonate (MTSEA) reduces responses to acetylcholine (ACh) by more than 97%, whereas corresponding mutations in muscle-type receptors produce effects that depend on the specific subunits mutated and ACh concentration. We coexpressed α7L119C subunits with pseudo wild-type α7C116S subunits, as well as ACh-insensitive α7Y188F subunits with wild-type α7 subunits in Xenopus laevis oocytes using varying ratios of cRNA. When mutant α7 cRNA was coinjected at a 5:1 ratio with wild-type cRNA, net charge responses to 300 µM ACh were retained by α7L119C-containing mutants after MTSEA modification and by the ACh-insensitive Y188F-containing mutants, even though the expected number of ACh-sensitive wild-type binding sites would on average be fewer than two per receptor. Responses of muscle-type receptors with one MTSEA-sensitive subunit were reduced at low ACh concentrations, but much less of an effect was observed when ACh concentrations were high (1 mM), indicating that saturation of a single binding site with agonist can evoke strong activation of nicotinic ACh receptors. Single-channel patch clamp analysis revealed that the burst durations of fetal wild-type and α1β1γδL121C receptors were equivalent until the α1β1γδL121C mutants were exposed to MTSEA, after which the majority (81%) of bursts were brief (≤2 ms). The longest duration events of the receptors modified at only one binding site were similar to the long bursts of native receptors traditionally associated with the activation of receptors with two sites containing bound agonists. PMID:21444659

  13. Conformational properties of the acetylcholine receptor as revealed by studies with constrained depolarizing ligands

    PubMed Central

    Wassermann, N. H.; Bartels, E.; Erlanger, B. F.

    1979-01-01

    Conformational aspects of the acetylcholine receptor (AcChoR) of Electrophorus electricus have been examined by studies of its interaction with structurally related, constrained aromatic bis quaternary compounds. Among the compounds synthesized was 3,3′-bis[α-(trimethylammonium)-methyl]azobenzene dibromide (3,3′-bisQ). This compound is photochromic and can exist in a cis or trans isomeric form, both of which have now been isolated in pure form. Trans-3,3′-bisQ is the most potent activator known, producing a 60-mV depolarization at 0.2 μM and 50% activity at 0.06 μM. The cis isomer is less than 1% as active. Its high activity and constrained structure suggest that trans-3,3′-bisQ can be considered to be a “template” of the combining site of AcChoR, when the latter is in the activated state. The following conclusions can then be drawn concerning the AcChoR binding site. (i) Depolarization can occur by interaction with reagents that are essentially inflexible. (ii) The binding site has a planar hydrophobic region that interacts with methylene groups of acetylcholine and with hydrophobic areas in general. (iii) In the same plane as the hydrophobic area is a site that interacts with electron-donating functional groups including the carbonyl oxygen of acetylcholine and the azo nitrogens of trans-3,3′-bisQ. (iv) About 1.5 Å out of the plane of the hydrophobic and the electron acceptor site is an anionic site; when the AcChoR is in the activated state, this site is separated from the electron acceptor site by 5.2 Å and from another anionic site by 11 Å. (v) The anionic sites are located within a cleft of limited size, sufficient to accommodate quaternary methyl groups. (vi) Although depolarization can occur with reagents that possess only hydrophobic and cationic groups if their geometric arrangement is proper, the highest activity resides in compounds capable of all of the interactions cited above. Images PMID:284340

  14. An Accessory Agonist Binding Site Promotes Activation of α4β2* Nicotinic Acetylcholine Receptors*

    PubMed Central

    Wang, Jingyi; Kuryatov, Alexander; Sriram, Aarati; Jin, Zhuang; Kamenecka, Theodore M.; Kenny, Paul J.; Lindstrom, Jon

    2015-01-01

    Neuronal nicotinic acetylcholine receptors containing α4, β2, and sometimes other subunits (α4β2* nAChRs) regulate addictive and other behavioral effects of nicotine. These nAChRs exist in several stoichiometries, typically with two high affinity acetylcholine (ACh) binding sites at the interface of α4 and β2 subunits and a fifth accessory subunit. A third low affinity ACh binding site is formed when this accessory subunit is α4 but not if it is β2. Agonists selective for the accessory ACh site, such as 3-[3-(3-pyridyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS9283), cannot alone activate a nAChR but can facilitate more efficient activation in combination with agonists at the canonical α4β2 sites. We therefore suggest categorizing agonists according to their site selectivity. NS9283 binds to the accessory ACh binding site; thus it is termed an accessory site-selective agonist. We expressed (α4β2)2 concatamers in Xenopus oocytes with free accessory subunits to obtain defined nAChR stoichiometries and α4/accessory subunit interfaces. We show that α2, α3, α4, and α6 accessory subunits can form binding sites for ACh and NS9283 at interfaces with α4 subunits, but β2 and β4 accessory subunits cannot. To permit selective blockage of the accessory site, α4 threonine 126 located on the minus side of α4 that contributes to the accessory site, but not the α4β2 sites, was mutated to cysteine. Alkylation of this cysteine with a thioreactive reagent blocked activity of ACh and NS9283 at the accessory site. Accessory agonist binding sites are promising drug targets. PMID:25869137

  15. Acetylcholine receptors from human muscle as pharmacological targets for ALS therapy

    PubMed Central

    Palma, Eleonora; Reyes-Ruiz, Jorge Mauricio; Lopergolo, Diego; Roseti, Cristina; Bertollini, Cristina; Ruffolo, Gabriele; Cifelli, Pierangelo; Onesti, Emanuela; Limatola, Cristina; Miledi, Ricardo; Inghilleri, Maurizio

    2016-01-01

    Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting motor neurons that leads to progressive paralysis of skeletal muscle. Studies of ALS have revealed defects in expression of acetylcholine receptors (AChRs) in skeletal muscle that occur even in the absence of motor neuron anomalies. The endocannabinoid palmitoylethanolamide (PEA) modified the clinical conditions in one ALS patient, improving muscle force and respiratory efficacy. By microtransplanting muscle membranes from selected ALS patients into Xenopus oocytes, we show that PEA reduces the desensitization of acetylcholine-evoked currents after repetitive neurotransmitter application (i.e., rundown). The same effect was observed using muscle samples from denervated (non-ALS) control patients. The expression of human recombinant α1β1γδ (γ-AChRs) and α1β1εδ AChRs (ε-AChRs) in Xenopus oocytes revealed that PEA selectively affected the rundown of ACh currents in ε-AChRs. A clear up-regulation of the α1 subunit in muscle from ALS patients compared with that from non-ALS patients was found by quantitative PCR, but no differential expression was found for other subunits. Clinically, ALS patients treated with PEA showed a lower decrease in their forced vital capacity (FVC) over time as compared with untreated ALS patients, suggesting that PEA can enhance pulmonary function in ALS. In the present work, data were collected from a cohort of 76 ALS patients and 17 denervated patients. Our results strengthen the evidence for the role of skeletal muscle in ALS pathogenesis and pave the way for the development of new drugs to hamper the clinical effects of the disease. PMID:26929355

  16. Functional characterisation of a nicotinic acetylcholine receptor α subunit from the brown dog tick, Rhipicephalus sanguineus☆

    PubMed Central

    Lees, Kristin; Jones, Andrew K.; Matsuda, Kazuhiko; Akamatsu, Miki; Sattelle, David B.; Woods, Debra J.; Bowman, Alan S.

    2014-01-01

    Ticks and tick-borne diseases have a major impact on human and animal health worldwide. Current control strategies rely heavily on the use of chemical acaricides, most of which target the CNS and with increasing resistance, new drugs are urgently needed. Nicotinic acetylcholine receptors (nAChRs) are targets of highly successful insecticides. We isolated a full-length nAChR α subunit from a normalised cDNA library from the synganglion (brain) of the brown dog tick, Rhipicephalus sanguineus. Phylogenetic analysis has shown this R. sanguineus nAChR to be most similar to the insect α1 nAChR group and has been named Rsanα1. Rsanα1 is distributed in multiple tick tissues and is present across all life-stages. When expressed in Xenopus laevis oocytes Rsanα1 failed to function as a homomer, with and without the addition of either Caenorhabditis elegans resistance-to-cholinesterase (RIC)-3 or X. laevis RIC-3. When co-expressed with chicken β2 nAChR, Rsanα1 evoked concentration-dependent, inward currents in response to acetylcholine (ACh) and showed sensitivity to nicotine (100 μM) and choline (100 μM). Rsanα1/β2 was insensitive to both imidacloprid (100 μM) and spinosad (100 μM). The unreliable expression of Rsanα1 in vitro suggests that additional subunits or chaperone proteins may be required for more robust expression. This study enhances our understanding of nAChRs in arachnids and may provide a basis for further studies on the interaction of compounds with the tick nAChR as part of a discovery process for novel acaricides. PMID:24291321

  17. Molecular-Dynamics Simulations of ELIC a Prokaryotic Homologue of the Nicotinic Acetylcholine Receptor

    SciTech Connect

    Cheng, Xiaolin; Ivanov, Ivaylo N; Wang, Hailong; McCammon, Jonathan

    2009-01-01

    The ligand-gated ion channel from Erwinia chrysanthemi (ELIC) is a prokaryotic homolog of the eukaryotic nicotinic acetylcholine receptor (nAChR) that responds to the binding of neurotransmitter acetylcholine and mediates fast signal transmission. ELIC is similar to the nAChR in its primary sequence and overall subunit organization, but despite their structural similarity, it is not clear whether these two ligand-gated ion channels operate in a similar manner. Further, it is not known to what extent mechanistic insights gleaned from the ELIC structure translate to eukaryotic counterparts such as the nAChR. Here we use molecular-dynamics simulations to probe the conformational dynamics and hydration of the transmembrane pore of ELIC. The results are compared with those from our previous simulation of the human ?7 nAChR. Overall, ELIC displays increased stability compared to the nAChR, whereas the two proteins exhibit remarkable similarity in their global motion and flexibility patterns. The majority of the increased stability of ELIC does not stem from the deficiency of the models used in the simulations, and but rather seems to have a structural basis. Slightly altered dynamical correlation features are also observed among several loops within the membrane region. In sharp contrast to the nAChR, ELIC is completely dehydrated from the pore center to the extracellular end throughout the simulation. Finally, the simulation of an ELIC mutant substantiates the important role of F246 on the stability, hydration and possibly function of the ELIC channel.

  18. Acetylcholine receptors from human muscle as pharmacological targets for ALS therapy.

    PubMed

    Palma, Eleonora; Reyes-Ruiz, Jorge Mauricio; Lopergolo, Diego; Roseti, Cristina; Bertollini, Cristina; Ruffolo, Gabriele; Cifelli, Pierangelo; Onesti, Emanuela; Limatola, Cristina; Miledi, Ricardo; Inghilleri, Maurizio

    2016-03-15

    Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting motor neurons that leads to progressive paralysis of skeletal muscle. Studies of ALS have revealed defects in expression of acetylcholine receptors (AChRs) in skeletal muscle that occur even in the absence of motor neuron anomalies. The endocannabinoid palmitoylethanolamide (PEA) modified the clinical conditions in one ALS patient, improving muscle force and respiratory efficacy. By microtransplanting muscle membranes from selected ALS patients into Xenopus oocytes, we show that PEA reduces the desensitization of acetylcholine-evoked currents after repetitive neurotransmitter application (i.e., rundown). The same effect was observed using muscle samples from denervated (non-ALS) control patients. The expression of human recombinant α1β1γδ (γ-AChRs) and α1β1εδ AChRs (ε-AChRs) in Xenopus oocytes revealed that PEA selectively affected the rundown of ACh currents in ε-AChRs. A clear up-regulation of the α1 subunit in muscle from ALS patients compared with that from non-ALS patients was found by quantitative PCR, but no differential expression was found for other subunits. Clinically, ALS patients treated with PEA showed a lower decrease in their forced vital capacity (FVC) over time as compared with untreated ALS patients, suggesting that PEA can enhance pulmonary function in ALS. In the present work, data were collected from a cohort of 76 ALS patients and 17 denervated patients. Our results strengthen the evidence for the role of skeletal muscle in ALS pathogenesis and pave the way for the development of new drugs to hamper the clinical effects of the disease.

  19. Activation and modulation of human α4β2 nicotinic acetylcholine receptors by the neonicotinoids clothianidin and imidacloprid.

    PubMed

    Li, Ping; Ann, Jason; Akk, Gustav

    2011-08-01

    Neonicotinoids are synthetic, nicotine-derived insecticides used for agricultural and household pest control. Though highly effective at activating insect nicotinic receptors, many neonicotinoids are also capable of directly activating and/or modulating the activation of vertebrate nicotinic receptors. In this study, we have investigated the actions of the neonicotinoids clothianidin (CTD) and imidacloprid (IMI) on human neuronal α4β2 nicotinic acetylcholine receptors. The data demonstrate that the compounds are weak agonists of the human receptors with relative peak currents of 1-4% of the response to 1 mM acetylcholine (ACh). Coapplication of IMI strongly inhibited currents elicited by ACh. From Schild plot analysis, we estimate that the affinity of IMI for the human α4β2 receptor is 18 μM. The application of low concentrations of CTD potentiated responses to low concentrations of ACh, suggesting that receptors occupied by one ACh and one CTD molecule have a higher gating efficacy than receptors with one ACh bound. Interestingly, subunit stoichiometry affected inhibition by CTD, with (α4)(2) (β2)(3) receptors significantly more strongly inhibited than the (α4)(3) (β2)(2) receptors.

  20. Nicotinic binding in rat brain: autoradiographic comparison of (/sup 3/H)acetylcholine, (/sup 3/H)nicotine, and (/sup 125/I)-alpha-bungarotoxin

    SciTech Connect

    Clarke, P.B.; Schwartz, R.D.; Paul, S.M.; Pert, C.B.; Pert, A.

    1985-05-01

    Three radioligands have been commonly used to label putative nicotinic cholinoceptors in the mammalian central nervous system: the agonists (/sup 3/H)nicotine and (/sup 3/H)acetylcholine ((/sup 3/H)ACh--in the presence of atropine to block muscarinic receptors), and the snake venom extract, (/sup 125/I)-alpha-bungarotoxin((/sup 125/I)BTX), which acts as a nicotinic antagonist at the neuromuscular junction. Binding studies employing brain homogenates indicate that the regional distributions of both (/sup 3/H)nicotine and (/sup 3/H)ACh differ from that of (/sup 125/I)BTX. The possible relationship between brain sites bound by (/sup 3/H)nicotine and (/sup 3/H)ACh has not been examined directly. The authors have used the technique of autoradiography to produce detailed maps of (/sup 3/H)nicotine, (/sup 3/H)ACh, and (/sup 125/I)BTX labeling; near-adjacent tissue sections were compared at many levels of the rat brain. The maps of high affinity agonist labeling are strikingly concordant, with highest densities in the interpeduncular nucleus, most thalamic nuclei, superior colliculus, medial habenula, presubiculum, cerebral cortex (layers I and III/IV), and the substantia nigra pars compacta/ventral tegmental area. The pattern of (/sup 125/I)BTX binding is strikingly different, the only notable overlap with agonist binding being the cerebral cortex (layer I) and superior colliculus. (/sup 125/I)BTX binding is also dense in the inferior colliculus, cerebral cortex (layer VI), hypothalamus, and hippocampus, but is virtually absent in thalamus. Various lines of evidence suggest that the high affinity agonist-binding sites in brain correspond to nicotinic cholinergic receptors similar to those found at autonomic ganglia; BTX-binding sites may also serve as receptors for nicotine and are possibly related to neuromuscular nicotinic cholinoceptors.

  1. What is the effect of nicotinic acetylcholine receptor stimulation on osteoarthritis in a rodent animal model?

    PubMed Central

    Bock, Kilian; Plaass, Christian; Coger, Vincent; Peck, Claas-Tido; Reimers, Kerstin; Stukenborg-Colsman, Christina; Claassen, Leif

    2016-01-01

    Objectives: Despite the rising number of patients with osteoarthritis, no sufficient chondroprotective and prophylactic therapy for osteoarthritis has been established yet. The purpose of this study was to verify whether stimulation of the nicotinic acetylcholine receptor via nicotine has a beneficial effect on cartilage degeneration in the development of osteoarthritis and is capable of reducing the expression of proinflammatory cytokines and cartilage degrading enzymes in synovial membranes after osteoarthritis induction. Methods: Experimental osteoarthritis was induced in Lewis rats using a standardized osteoarthritis model with monoiodoacetate. A total of 16 Lewis rats were randomized into four groups: control, sham + nicotine application, osteoarthritis, and osteoarthritis + nicotine application. Nicotine (0.625 mg/kg twice daily) was administered intraperitoneally for 42 days. We analyzed histological sections, radiological images and the expression of the proinflammatory cytokines, such as interleukin-1β, tumor necrosis factor-α and interleukin-6, and of matrix metalloproteases 3, 9 and 13 and tissue inhibitors of metalloprotease-1 in synovial membranes via quantitative polymerase chain reaction. Results: Histological and x-ray examination revealed cartilage degeneration in the osteoarthritis group compared to control or sham + nicotine groups (histological control vs osteoarthritis: p = 0.002 and x-ray control vs osteoarthritis: p = 0.004). Nicotine treatment reduced the cartilage degeneration without significant differences. Osteoarthritis induction led to a higher expression of proinflammatory cytokines and matrix metalloproteases as compared to control groups. This effect was attenuated after nicotine administration. The differences of proinflammatory cytokines and matrix metalloproteases did not reach statistical significance. Conclusion: With the present small-scale study, we could not prove a positive effect of nicotinic

  2. Drug-dependent behaviors and nicotinic acetylcholine receptor expressions in Caenorhabditis elegans following chronic nicotine exposure.

    PubMed

    Polli, Joseph R; Dobbins, Dorothy L; Kobet, Robert A; Farwell, Mary A; Zhang, Baohong; Lee, Myon-Hee; Pan, Xiaoping

    2015-03-01

    Nicotine, the major psychoactive compound in tobacco, targets nicotinic acetylcholine receptors (nAChRs) and results in drug dependence. The nematode Caenorhabditis elegans' (C. elegans) genome encodes conserved and extensive nicotinic receptor subunits, representing a useful system to investigate nicotine-induced nAChR expressions in the context of drug dependence. However, the in vivo expression pattern of nAChR genes under chronic nicotine exposure has not been fully investigated. To define the role of nAChR genes involved in nicotine-induced locomotion changes and the development of tolerance to these effects, we characterized the locomotion behavior combining the use of two systems: the Worm Tracker hardware and the WormLab software. Our results indicate that the combined system is an advantageous alternative to define drug-dependent locomotion behavior in C. elegans. Chronic (24-h dosing) nicotine exposure at 6.17 and 61.7μM induced nicotine-dependent behaviors, including drug stimulation, tolerance/adaption, and withdrawal responses. Specifically, the movement speed of naïve worms on nicotine-containing environments was significantly higher than on nicotine-free environments, suggesting locomotion stimulation by nicotine. In contrast, the 24-h 6.17μM nicotine-treated worms exhibited significantly higher speeds on nicotine-free plates than on nicotine-containing plates. Furthermore significantly increased locomotion behavior during nicotine cessation was observed in worms treated with a higher nicotine concentration of 61.7μM. The relatively low locomotion speed of nicotine-treated worms on nicotine-containing environments also indicates adaption/tolerance of worms to nicotine following chronic nicotine exposure. In addition, this study provides useful information regarding the comprehensive in vivo expression profile of the 28 "core" nAChRs following different dosages of chronic nicotine treatments. Eleven genes (lev-1, acr-6, acr-7, acr-11, lev-8, acr

  3. Agarwood induced laxative effects via acetylcholine receptors on loperamide-induced constipation in mice.

    PubMed

    Kakino, Mamoru; Izuta, Hiroshi; Ito, Tetsuro; Tsuruma, Kazuhiro; Araki, Yoko; Shimazawa, Masamitsu; Oyama, Masayoshi; Iinuma, Munekazu; Hara, Hideaki

    2010-01-01

    Agarwood (Aquilaria sinensis, Aquilaria crasna) is well known as an incense in the oriental region such as Thailand, Taiwan, and Cambodia, and is used as a digestive in traditional medicine. We investigated the laxative effects and mechanism of agarwood leaves extracted with ethanol (EEA-1, Aquilaria sinensis; EEA-2, Aquilaria crasna). EEA-1, EEA-2, the main constituents of EEAs (mangiferin, and genkwanin-5-O-primeveroside), and senna increased the frequency and weight of stools in loperamide-induced constipation model mice. EEA-1 and EEA-2 did not induce diarrhea as a side effect, but senna induced severe diarrhea. EEA-1 and senna increased gastro-intestinal (GI) transit in the model mice. EEA-1, but not senna, also increased the intestinal tension of isolated jejunum and ileum in guinea pigs, and the tension increase was blocked by atropine, a muscarinic receptor antagonist, but not by other inhibitors (granicetron, pyrilamine, or bradykinin-antagonist peptide). Furthermore, the increase in frequency and weight of stools induced by EEA-1 were blocked by pre-administration of atropine in the model mice. These findings indicate that EEAs exerted a laxative effect via acetylcholine receptors in the mouse constipation model.

  4. Preparation and characterization of muscarinic-acetylcholine-receptor-enriched membranes from pig atria.

    PubMed Central

    Peterson, G L; Schimerlik, M I

    1982-01-01

    A procedure was developed for the large scale preparation of membranes from pig atria which are enriched 10-13 fold in the muscarinic acetylcholine receptor. The procedure involved differential centrifugation and sucrose-gradient centrifugation in solutions containing 150 mM-NaClO4 and 5 mM-EDTA to minimize membrane aggregation. The final membrane preparation bound about 1.1 pmol of L-quinuclidinyl benzilate/mg of protein. Comparable results were obtained with either fresh or frozen tissue. About the same yield (120 pmol of L-quinuclidinyl benzilate sites/100 g of tissue) and specific activity of membranes were obtained from different regions of the atria. The final preparation was stable at -80 degrees C in buffered sucrose solutions. The membranes appeared mostly as sheets or fragments and partly as closed vesicles in the electron microscope and were heterogeneous in isopycnic Percoll gradients. Marker enzyme studies showed that the receptor was enriched in parallel with the plasma membrane markers guanylate cyclase (particulate form) and (Na+ + K+)-activated ATPase. Some contamination by mitochondrial outer and endoplasmic reticulum membranes was evident from the distribution of monoamine oxidase and glucose-6-phosphatase activity, but the preparation was largely free of sarcoplasmic reticulum, mitochondrial inner, and lysosomal membranes. PMID:7092826

  5. Heteromeric α7β2 Nicotinic Acetylcholine Receptors in the Brain

    PubMed Central

    Wu, Jie; Liu, Qiang; Tang, Pei; Mikkelsen, Jens D.; Shen, Jianxin; Whiteaker, Paul; Yakel, Jerrel L.

    2016-01-01

    The α7 nicotinic acetylcholine receptor (α7 nAChR) is highly expressed in the brain, where it maintains various neuronal functions including (but not limited to) learning and memory. In addition, the protein expression levels of α7 nAChRs are altered in various brain disorders. The classic rule governing α7 nAChR assembly in the mammalian brain was that it was assembled from five α7 subunits to form a homomeric receptor pentamer. However, emerging evidence demonstrates the presence of heteromeric α7 nAChRs in heterologously expressed systems and naturally in brain neurons, where α7 subunits are co-assembled with β2 subunits to form a novel type of α7β2 nAChR. Interestingly, the α7β2 nAChR exhibits distinctive function and pharmacology from traditional homomeric α7 nAChRs. We review recent advances in probing the distribution, function, pharmacology, pathophysiology, and stoichiometry of the heteromeric α7β2 nAChR, which have provided new insights into the understanding of a novel target of cholinergic signaling. PMID:27179601

  6. Gating of Long-Term Potentiation by Nicotinic Acetylcholine Receptors at the Cerebellum Input Stage

    PubMed Central

    Prestori, Francesca; Bonardi, Claudia; Mapelli, Lisa; Lombardo, Paola; Goselink, Rianne; De Stefano, Maria Egle; Gandolfi, Daniela; Mapelli, Jonathan; Bertrand, Daniel; Schonewille, Martijn; De Zeeuw, Chris; D’Angelo, Egidio

    2013-01-01

    The brain needs mechanisms able to correlate plastic changes with local circuit activity and internal functional states. At the cerebellum input stage, uncontrolled induction of long-term potentiation or depression (LTP or LTD) between mossy fibres and granule cells can saturate synaptic capacity and impair cerebellar functioning, which suggests that neuromodulators are required to gate plasticity processes. Cholinergic systems innervating the cerebellum are thought to enhance procedural learning and memory. Here we show that a specific subtype of acetylcholine receptors, the α7-nAChRs, are distributed both in cerebellar mossy fibre terminals and granule cell dendrites and contribute substantially to synaptic regulation. Selective α7-nAChR activation enhances the postsynaptic calcium increase, allowing weak mossy fibre bursts, which would otherwise cause LTD, to generate robust LTP. The local microperfusion of α7-nAChR agonists could also lead to in vivo switching of LTD to LTP following sensory stimulation of the whisker pad. In the cerebellar flocculus, α7-nAChR pharmacological activation impaired vestibulo-ocular-reflex adaptation, probably because LTP was saturated, preventing the fine adjustment of synaptic weights. These results show that gating mechanisms mediated by specific subtypes of nicotinic receptors are required to control the LTD/LTP balance at the mossy fibre-granule cell relay in order to regulate cerebellar plasticity and behavioural adaptation. PMID:23741401

  7. Conformational analysis of a toxic peptide from Trimeresurus wagleri which blocks the nicotinic acetylcholine receptor.

    PubMed Central

    Sellin, L C; Mattila, K; Annila, A; Schmidt, J J; McArdle, J J; Hyvönen, M; Rantala, T T; Kivistö, T

    1996-01-01

    The 22-residue toxic peptide (WTX1) from the venom of the Southeast Asian snake Trimeresurus wagleri has multiple sites of action, but its lethal effect has been attributed to blocking the postsynaptic acetylcholine receptor at the neuromuscular junction. The 3-dimensional structure of WTX1 was studied using 2-dimensional nuclear magnetic resonance spectroscopy, circular dichroism, and computer simulations. In aqueous solution, WTX1 was shown to have extended and flexible "tails" defined by a short, rigid disulfide-bonded loop. The flexible regions can undergo structural rearrangement when moved from an aqueous to a less polar environment and may contribute to its effectiveness at different receptor sites. By substituting Gly or Phe for His at position 10, significant effects on the disulfide bond formation and, thereby, the activity of the peptide were observed. These results suggest that even subtle differences in single residues can have profound effects on the dynamics of folding, disulfide bond formation, and activity of this toxic peptide. Images FIGURE 10 FIGURE 12 PMID:8770182

  8. Modulation of acetylcholine release from rat striatal slices by the GABA/benzodiazepine receptor complex

    SciTech Connect

    Supavilai, P.; Karobath, M.

    1985-02-04

    GABA, THIP and muscimol enhance spontaneous and inhibit electrically induced release of tritium labelled compounds from rat striatal slices which have been pre-labelled with /sup 3/H-choline. Baclofen is inactive in this model. Muscimol can inhibit electrically induced release of tritiated material by approximately 75% with half maximal effects at 2 ..mu..M. The response to muscimol can be blocked by the GABA antagonists bicuculline methobromide, picrotoxin, anisatin, R 5135 and CPTBO (cyclopentylbicyclophosphate). Drugs which act on the benzodiazepine receptor (BR) require the presence of muscimol to be effective and they modulate the effects of muscimol in a bidirectional manner. Thus BR agonists enhance and inverse BR agonists attenuate the inhibitory effects of muscimol on electrically induced release. Ro15-1788, a BR antagonist, does not modulate the inhibitory effects of muscimol but antagonizes the actions of clonazepam, a BR agonist, and of DMCM, an inverse BR agonist. These results demonstrate that a GABA/benzodiazepine receptor complex can modulate acetylcholine release from rat striatal slices in vitro. 24 references, 3 figures, 5 table.

  9. Antagonist pharmacology of desensitizing and non-desensitizing nicotinic acetylcholine receptors in cockroach neurons.

    PubMed

    Salgado, Vincent L

    2016-09-01

    Two α-bungarotoxin-sensitive nicotinic acetylcholine (ACh) receptor subtypes in neurons of the American cockroach have been identified as desensitizing (nAChD) and selectively inhibitable with 100nM imidacloprid, and non-desensitizing (nAChN) and selectively inhibitable with 100pM methyllycaconitine. In this paper, the single-electrode voltage-clamp technique was used to measure concentration-response relations for the action of ACh and five antagonists on pharmacologically separated nAChD and nAChN receptors of acutely dissociated neurons from thoracic ganglia of the American cockroach. A dual bath and U-tube perfusion system was used to achieve rapid application of ACh in the continued presence of antagonists, which was essential to accurately measure inhibition by rapidly-reversible antagonists. ACh activated both receptors with an EC50 of 7μM and the antagonist potencies were (nAChD/nAChN in nM): dihydro-β-erythroidine: 1.0/5.6, d-tubocurarine: 1000/34, condelphine: 0.39/0.65, phencyclidine: 74/980 and mecamylamine 47/1150. While each of these antagonists displayed some subtype selectivity, none are selective enough to be used as subtype-selective tools. These results bring to a total of 16 the number of nicotinic compounds that have been measured on nAChD and nAChN currents. Characterization of these receptors is important for understanding the role of nAChRs in the insect nervous system and the mechanism of action of insecticides.

  10. Physiological and biochemical studies of newly synthesized muscarinic acetylcholine receptors in embryonic chicken heart

    SciTech Connect

    Hunter, D.D.

    1986-01-01

    Exposure of either chicken embryos in ovo or cultured embryonic chicken cardiac cells in vitro to the muscarinic agonist carbachol results in a 70-90% decrease in the number of muscarinic acetylcholine receptors (mAChR) expressed in cardiac cells. Block of agonist-receptor interactions in ovo with the antagonist atropine or removal of the agonist in vitro results in a gradual increase in mAChR number, reaching the control level in 14 hr. Measurements of physiological sensitivity of atria or cultured cells show that, even after the complete recovery of receptor number, the sensitivity to agonist is reduced. The sensitivity of the mAChR-mediated inhibition of adenylate cyclase is also decreased at this time. Newly synthesized mAChR which appear following affinity alkylation in cultured cells are also poorly coupled to the stimulation of /sup 86/Rb/sup +/ efflux, indicating that decreased physiological sensitivity is not due to an unknown effect of long-term agonist exposure on general cellular function, but rather reflects an intrinsic property of newly synthesized mAChR. This increase in sensitivity is also not blocked by cycloheximide. The increase in sensitivity of the mAChR-mediated responses is due neither to a lack of expression of newly synthesized mAChR on the surface nor to reduced agonist affinity of the mAChR. The diminished sensitivity and subsequent maturation observed in cells containing newly synthesized receptors is due either to a small change in mAChR, or to a change in an as-yet-undefined component of the mAChR transduction system; this alteration represents a novel locus for modulation of cholinergic signals in the heart.

  11. Molecular Determinants for Competitive Inhibition of α4β2 Nicotinic Acetylcholine Receptors

    PubMed Central

    Carbone, Annalisa; García-Beltrán, Olimpo; Livingstone, Phil D.; Biggin, Philip C.; Cassels, Bruce K.; Wonnacott, Susan; Zapata-Torres, Gerald; Bermudez, Isabel

    2010-01-01

    The Erythrina alkaloids erysodine and dihydro-β-erythroidine (DHβE) are potent and selective competitive inhibitors of α4β2 nicotinic acetylcholine receptors (nAChRs), but little is known about the molecular determinants of the sensitivity of this receptor subtype to inhibition by this class of antagonists. We addressed this issue by examining the effects of DHβE and a range of aromatic Erythrina alkaloids on [3H]cytisine binding and receptor function in conjunction with homology models of the α4β2 nAChR, mutagenesis, and functional assays. The lactone group of DHβE and a hydroxyl group at position C-16 in aromatic Erythrina alkaloids were identified as major determinants of potency, which was decreased when the conserved residue Tyr126 in loop A of the α4 subunit was substituted by alanine. Sensitivity to inhibition was also decreased by substituting the conserved aromatic residues α4Trp182 (loop B), α4Tyr230 (loop C), and β2Trp82 (loop D) and the nonconserved β2Thr84; however, only α4Trp182 was predicted to contact bound antagonist, suggesting α4Tyr230, β2Trp82, and β2Thr84 contribute allosterically to the closed state elicited by bound antagonist. In addition, homology modeling predicted strong ionic interactions between the ammonium center of the Erythrina alkaloids and β2Asp196, leading to the uncapping of loop C. Consistent with this, β2D196A abolished sensitivity to inhibition by DHβE or erysodine but not by epierythratidine, which is not predicted to form ionic bonds with β2Asp196. This residue is not conserved in subunits that comprise nAChRs with low sensitivity to inhib