AGONIST-INDUCED AFFINITY ALTERATIONS OF A CENTRAL NERVOUS SYSTEM NICOTINIC ACETYLCHOLINE RECEPTOR
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Conference Article| February 01 1991 Neuronal nicotinic receptors: functional correlates of ligand binding sites S. Wonnacott S. Wonnacott 1Department of Biochemistry, University of Bath, Bath BA2 7AY, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1991) 19 (1): 121–124. https://doi.org/10.1042/bst0190121 Article history Received: September 27 1990 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Cite Icon Cite Get Permissions Citation S. Wonnacott; Neuronal nicotinic receptors: functional correlates of ligand binding sites. Biochem Soc Trans 1 February 1991; 19 (1): 121–124. doi: https://doi.org/10.1042/bst0190121 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: nAChR, nicotinic acetylcholine receptor © 1991 Biochemical Society1991 Article PDF first page preview Close Modal You do not currently have access to this content.
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Species-specific agonist binding regions of nicotinic acetylcholine receptors (nAChR) were examined. Imidacloprid and physostigmine (Phy) selectively activated insect nAChR composed of Drosophila second alpha-like subunit (SAD) and chick β2, in contrast to rat α7 nAChR. The Phy-activated currents were α-bungarotoxin (α-BGT) sensitive, suggesting activation at the agonist binding loop C. Several SAD-α7 chimeras were constructed, by switching agonist binding regions, and expressed in oocytes. Though none of the chimeras was activated by a range of nicotinic agonists, [125I]α-BGT binding revealed homomeric assembly of all chimeric cDNAs. Phy differentially displaced [125I]α-BGT from the nAChR chimeras, suggesting that the β subunit is not involved in Phy binding, and that Phy targets the insect agonist binding loop C.
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α-Conotoxins, as nicotinic acetylcholine receptor (nAChR) antagonists, are powerful tools for dissecting biologic processes and guiding drug development. The α3β2 and α3β4 nAChR subtypes are expressed in the central and peripheral nervous systems and play a critical role in various pathophysiological conditions ranging from nicotine addiction to the development and progression of lung cancer. Here we used the α4/7-conotoxin RegIIA, a disulfide-bonded peptide from the venom of Conus regius, and its analog [N11A,N12A]RegIIA to probe the specific pharmacological properties of rat and human nAChR subtypes. nAChR subtypes were heterologously expressed in Xenopus oocytes and two-electrode voltage clamp recordings used to investigate the effects of the peptides on nAChR activity. RegIIA potently inhibited currents evoked by acetylcholine (ACh) at rat α3β2 (IC50 = 10.7 nM), whereas a 70-fold lower potency was observed at human α3β2 nAChR (IC50 = 704.1 nM). Conversely, there were no species-specific differences in sensitivity to RegIIA at the α3β4 nAChR. Receptor mutagenesis and molecular dynamics studies revealed that this difference can be attributed primarily to a single amino acid change: Glu198 on the rat α3 subunit corresponding to a proline on the human subunit. These findings reveal a novel species- and subunit-specific receptor-antagonist interaction.
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Recent evidence has indicated that the nicotinic acetylcholine receptor and the nicotinic α-bungarotoxin (α-BGT) site may be distinct in neuronal tissues. With regard to function, the former receptor appears to be involved in mediating synaptic events; however, the role of the nicotinic α-BGT site in nervous tissue is currently not known. Since the binding of α-BGT exhibits such high affinity and selectivity for a specific receptor, this may implicate an involvement of the toxin binding site in some aspect of neuronal activity with the receptor possibly mediating functions other than nicotinic cholinergic transmission. A further hypothesis to explain the nature of the toxin binding site may be that the natural ligand for the α-BGT site is one other than acetylcholine, with acetylcholine acting as a modulator of the site. Current studies in our laboratory are exploring these possibilities by determining whether specific peptides and/or polypeptides can interact at the nicotinic α-BGT site in nervous tissue. Studies using both in vivo and in vitro approaches suggest that thy-mopoietin may serve a role as a modulator of the nicotinic α-BGT site in neuronal tissues.
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Abstract: The TE671 human medulloblastoma cell line expresses a variety of characteristics of human neurons. Among these characteristics is the expression of membrane‐bound high‐affinity binding sites for α‐bungarotoxin, which is a potent antagonist of functional nicotinic acetylcholine receptors on these cells. These toxin binding sites represent a class of nicotinic receptor isotypes present in mammalian brain. Treatment of TE671 cells during proliferative growth phase with nicotine or carbamylcholine, but not with muscarine or d ‐tubocurarine, induced up to a fivefold increase in the density of radiolabeled toxin binding sites in crude membrane fractions. This effect was blocked by co‐incubation with the nicotinic antagonists d ‐tubocurarine and decamethonium, but not by mecamylamine or by muscarinic antagonists. Following a 10–13 h lag phase upon removal of agonist, recovery of the up‐regulated sites to control values occurred within an additional 10–20 h. These studies indicate that the expression of functional nicotinic acetylcholine receptors on TE671 cells is subject to regulation by nicotinic agonists. Studies of the murine CNS have consistently indicated nicotine‐induced up‐regulation of nicotinic acetylcholine receptors, thereby supporting the identification of the toxin binding site on these cells as the functional nicotinic receptor. Although a mechanism for this effect is not apparent, nicotine‐induced receptor blockade does not appear to be involved.
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