Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1991 Jun 1;88(11):5051–5055. doi: 10.1073/pnas.88.11.5051

Allosteric transitions of the acetylcholine receptor probed at the amino acid level with a photolabile cholinergic ligand.

J L Galzi 1, F Revah 1, F Bouet 1, A Ménez 1, M Goeldner 1, C Hirth 1, J P Changeux 1
PMCID: PMC51805  PMID: 2052586

Abstract

Structural changes occurring upon desensitization of the Torpedo marmorata acetylcholine receptor were monitored with tritiated p-(N,N-dimethyl)aminobenzenediazonium fluoroborate, a reversible competitive antagonist in the dark, which may serve as a photoaffinity probe of the area of the receptor molecule with which cholinergic ligands interact. Addition of meproadifen, an allosteric effector that stabilizes the high-affinity desensitized state of the receptor upon binding to a site topographically distinct from the cholinergic ligand-binding domains, caused a major increase in labeling of the alpha subunit, a smaller increase in the delta subunit, and decreased labeling in the gamma subunit, thus revealing changes in the alpha and non-alpha subunits' contribution to cholinergic ligand binding. Also, in agreement with the tighter binding of cholinergic ligands to the desensitized receptor, differential labeling of three peptide loops of the alpha subunit was detected: while Tyr-190, Cys-192, and Cys-193 were labeled in a roughly identical manner in both resting and desensitized conformations, the labeling of Tyr-93 and Trp-149 increased up to 6-fold in the desensitized state. Tyr-93 and Trp-149 belong to separate regions containing strictly conserved "canonical" amino acids, common to all nicotinic, gamma-aminobutyrate, and glycine receptor subunits. These regions are thus likely to play a critical role in the regulation of ligand-gated ion channels.

Full text

PDF
5051

Images in this article

5055Image
on p.5055

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abramson S. N., Li Y., Culver P., Taylor P. An analog of lophotoxin reacts covalently with Tyr190 in the alpha-subunit of the nicotinic acetylcholine receptor. J Biol Chem. 1989 Jul 25;264(21):12666–12672. [PubMed] [Google Scholar]
  2. Betz H. Homology and analogy in transmembrane channel design: lessons from synaptic membrane proteins. Biochemistry. 1990 Apr 17;29(15):3591–3599. doi: 10.1021/bi00467a001. [DOI] [PubMed] [Google Scholar]
  3. Blount P., Merlie J. P. Molecular basis of the two nonequivalent ligand binding sites of the muscle nicotinic acetylcholine receptor. Neuron. 1989 Sep;3(3):349–357. doi: 10.1016/0896-6273(89)90259-6. [DOI] [PubMed] [Google Scholar]
  4. Boulter J., O'Shea-Greenfield A., Duvoisin R. M., Connolly J. G., Wada E., Jensen A., Gardner P. D., Ballivet M., Deneris E. S., McKinnon D. Alpha 3, alpha 5, and beta 4: three members of the rat neuronal nicotinic acetylcholine receptor-related gene family form a gene cluster. J Biol Chem. 1990 Mar 15;265(8):4472–4482. [PubMed] [Google Scholar]
  5. Boyd N. D., Cohen J. B. Kinetics of binding of [3H]acetylcholine and [3H]carbamoylcholine to Torpedo postsynaptic membranes: slow conformational transitions of the cholinergic receptor. Biochemistry. 1980 Nov 11;19(23):5344–5353. doi: 10.1021/bi00564a031. [DOI] [PubMed] [Google Scholar]
  6. Changeux J. P. The acetylcholine receptor: an "allosteric" membrane protein. Harvey Lect. 1979 1980;75:85–254. [PubMed] [Google Scholar]
  7. Chatrenet B., Trémeau O., Bontems F., Goeldner M. P., Hirth C. G., Ménez A. Topography of toxin-acetylcholine receptor complexes by using photoactivatable toxin derivatives. Proc Natl Acad Sci U S A. 1990 May;87(9):3378–3382. doi: 10.1073/pnas.87.9.3378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chothia C., Lesk A. M., Tramontano A., Levitt M., Smith-Gill S. J., Air G., Sheriff S., Padlan E. A., Davies D., Tulip W. R. Conformations of immunoglobulin hypervariable regions. Nature. 1989 Dec 21;342(6252):877–883. doi: 10.1038/342877a0. [DOI] [PubMed] [Google Scholar]
  9. Cooper E., Couturier S., Ballivet M. Pentameric structure and subunit stoichiometry of a neuronal nicotinic acetylcholine receptor. Nature. 1991 Mar 21;350(6315):235–238. doi: 10.1038/350235a0. [DOI] [PubMed] [Google Scholar]
  10. Damle V. N., Karlin A. Affinity labeling of one of two alpha-neurotoxin binding sites in acetylcholine receptor from Torpedo californica. Biochemistry. 1978 May 30;17(11):2039–2045. doi: 10.1021/bi00604a002. [DOI] [PubMed] [Google Scholar]
  11. Dennis M., Giraudat J., Kotzyba-Hibert F., Goeldner M., Hirth C., Chang J. Y., Lazure C., Chrétien M., Changeux J. P. Amino acids of the Torpedo marmorata acetylcholine receptor alpha subunit labeled by a photoaffinity ligand for the acetylcholine binding site. Biochemistry. 1988 Apr 5;27(7):2346–2357. doi: 10.1021/bi00407a016. [DOI] [PubMed] [Google Scholar]
  12. Galzi J. L., Revah F., Bessis A., Changeux J. P. Functional architecture of the nicotinic acetylcholine receptor: from electric organ to brain. Annu Rev Pharmacol Toxicol. 1991;31:37–72. doi: 10.1146/annurev.pa.31.040191.000345. [DOI] [PubMed] [Google Scholar]
  13. Galzi J. L., Revah F., Black D., Goeldner M., Hirth C., Changeux J. P. Identification of a novel amino acid alpha-tyrosine 93 within the cholinergic ligands-binding sites of the acetylcholine receptor by photoaffinity labeling. Additional evidence for a three-loop model of the cholinergic ligands-binding sites. J Biol Chem. 1990 Jun 25;265(18):10430–10437. [PubMed] [Google Scholar]
  14. Giraudat J., Dennis M., Heidmann T., Chang J. Y., Changeux J. P. Structure of the high-affinity binding site for noncompetitive blockers of the acetylcholine receptor: serine-262 of the delta subunit is labeled by [3H]chlorpromazine. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2719–2723. doi: 10.1073/pnas.83.8.2719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Heidmann T., Changeux J. P. Characterization of the transient agonist-triggered state of the acetylcholine receptor rapidly labeled by the noncompetitive blocker [3H]chlorpromazine: additional evidence for the open channel conformation. Biochemistry. 1986 Oct 7;25(20):6109–6113. doi: 10.1021/bi00368a041. [DOI] [PubMed] [Google Scholar]
  16. Heidmann T., Changeux J. P. Fast kinetic studies on the allosteric interactions between acetylcholine receptor and local anesthetic binding sites. Eur J Biochem. 1979 Feb 15;94(1):281–296. doi: 10.1111/j.1432-1033.1979.tb12894.x. [DOI] [PubMed] [Google Scholar]
  17. Heidmann T., Changeux J. P. Time-resolved photolabeling by the noncompetitive blocker chlorpromazine of the acetylcholine receptor in its transiently open and closed ion channel conformations. Proc Natl Acad Sci U S A. 1984 Mar;81(6):1897–1901. doi: 10.1073/pnas.81.6.1897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Heidmann T., Oswald R. E., Changeux J. P. Multiple sites of action for noncompetitive blockers on acetylcholine receptor rich membrane fragments from torpedo marmorata. Biochemistry. 1983 Jun 21;22(13):3112–3127. doi: 10.1021/bi00282a014. [DOI] [PubMed] [Google Scholar]
  19. Kao P. N., Dwork A. J., Kaldany R. R., Silver M. L., Wideman J., Stein S., Karlin A. Identification of the alpha subunit half-cystine specifically labeled by an affinity reagent for the acetylcholine receptor binding site. J Biol Chem. 1984 Oct 10;259(19):11662–11665. [PubMed] [Google Scholar]
  20. Krodel E. K., Beckman R. A., Cohen J. B. Identification of a local anesthetic binding site in nicotinic post-synaptic membranes isolated from Torpedo marmorata electric tissue. Mol Pharmacol. 1979 Mar;15(2):294–312. [PubMed] [Google Scholar]
  21. Kuhse J., Schmieden V., Betz H. A single amino acid exchange alters the pharmacology of neonatal rat glycine receptor subunit. Neuron. 1990 Dec;5(6):867–873. doi: 10.1016/0896-6273(90)90346-h. [DOI] [PubMed] [Google Scholar]
  22. Langenbuch-Cachat J., Bon C., Mulle C., Goeldner M., Hirth C., Changeux J. P. Photoaffinity labeling of the acetylcholine binding sites on the nicotinic receptor by an aryldiazonium derivative. Biochemistry. 1988 Apr 5;27(7):2337–2345. doi: 10.1021/bi00407a015. [DOI] [PubMed] [Google Scholar]
  23. Langosch D., Thomas L., Betz H. Conserved quaternary structure of ligand-gated ion channels: the postsynaptic glycine receptor is a pentamer. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7394–7398. doi: 10.1073/pnas.85.19.7394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nakanishi N., Shneider N. A., Axel R. A family of glutamate receptor genes: evidence for the formation of heteromultimeric receptors with distinct channel properties. Neuron. 1990 Nov;5(5):569–581. doi: 10.1016/0896-6273(90)90212-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Neubig R. R., Cohen J. B. Equilibrium binding of [3H]tubocurarine and [3H]acetylcholine by Torpedo postsynaptic membranes: stoichiometry and ligand interactions. Biochemistry. 1979 Nov 27;18(24):5464–5475. doi: 10.1021/bi00591a032. [DOI] [PubMed] [Google Scholar]
  26. Oswald R. E., Changeux J. P. Crosslinking of alpha-bungarotoxin to the acetylcholine receptor from Torpedo marmorata by ultraviolet light irradiation. FEBS Lett. 1982 Mar 22;139(2):225–229. doi: 10.1016/0014-5793(82)80857-0. [DOI] [PubMed] [Google Scholar]
  27. Papke R. L., Boulter J., Patrick J., Heinemann S. Single-channel currents of rat neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes. Neuron. 1989 Nov;3(5):589–596. doi: 10.1016/0896-6273(89)90269-9. [DOI] [PubMed] [Google Scholar]
  28. Pedersen S. E., Cohen J. B. d-Tubocurarine binding sites are located at alpha-gamma and alpha-delta subunit interfaces of the nicotinic acetylcholine receptor. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2785–2789. doi: 10.1073/pnas.87.7.2785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Shivers B. D., Killisch I., Sprengel R., Sontheimer H., Köhler M., Schofield P. R., Seeburg P. H. Two novel GABAA receptor subunits exist in distinct neuronal subpopulations. Neuron. 1989 Sep;3(3):327–337. doi: 10.1016/0896-6273(89)90257-2. [DOI] [PubMed] [Google Scholar]
  30. Stroud R. M., McCarthy M. P., Shuster M. Nicotinic acetylcholine receptor superfamily of ligand-gated ion channels. Biochemistry. 1990 Dec 18;29(50):11009–11023. doi: 10.1021/bi00502a001. [DOI] [PubMed] [Google Scholar]
  31. Unwin N., Toyoshima C., Kubalek E. Arrangement of the acetylcholine receptor subunits in the resting and desensitized states, determined by cryoelectron microscopy of crystallized Torpedo postsynaptic membranes. J Cell Biol. 1988 Sep;107(3):1123–1138. doi: 10.1083/jcb.107.3.1123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Verdoorn T. A., Draguhn A., Ymer S., Seeburg P. H., Sakmann B. Functional properties of recombinant rat GABAA receptors depend upon subunit composition. Neuron. 1990 Jun;4(6):919–928. doi: 10.1016/0896-6273(90)90145-6. [DOI] [PubMed] [Google Scholar]
  33. Wilson P. T., Lentz T. L., Hawrot E. Determination of the primary amino acid sequence specifying the alpha-bungarotoxin binding site on the alpha subunit of the acetylcholine receptor from Torpedo californica. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8790–8794. doi: 10.1073/pnas.82.24.8790. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES