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
. 1979 Mar;76(3):1064–1068. doi: 10.1073/pnas.76.3.1064

NBD-5-acylcholine: fluorescent analog of acetylcholine and agonist at the neuromuscular junction.

R Jürss, H Prinz, A Maelicke
PMCID: PMC383189  PMID: 312497

Abstract

We have studied the properties of N-7-(4-nitrobenzo-2-oxa-1,3-diazole)-omega-aminohexanoic acid beta-(N-trimethylammonium)ethyl ester, a fluorescent analog of acetylcholine at the cellular level by using pharmacological and electrophysiological techniques and at the molecular level by measuring the kinetics of interaction with solubilized acetylcholine receptor and with acetylcholine esterase (EC 3.1.1.7). The fluorescent drug is a powerful agonist of acetylcholine at the neuromuscular junction and also strongly desensitizes muscle fibers. Interaction with acetylcholine receptor is accompanied by large changes in the drug's fluorescence. From the kinetics of interaction studied by means of a stopped-flow fluorimeter with laser light source, we obtained a second-order forward rate constant in excess of 1 X 10(8) M-1 sec-1 and an initial dissociation rate constant (k1) of 0.5 sec-1 for receptor from Electrophorous electricus. Interaction of this analog with acetylcholine esterase from E. electricus is accompanied by a transient decrease in fluorescence followed by an increase leading to a stable plateau value at a level near the original one. The initial decrease in fluorescence followed second-order kinetics with k2 of the order of 10(9) M-1 sec-1. The slower consecutive reaction which could be blocked by phosphorylation of the esteratic site, was of first order with k1 = 0.05 sec-1.

Full text

PDF
1064

Selected References

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

  1. Adams P. R., Sakmann B. A comparison of current-voltage relations for full and partial agonists. J Physiol. 1978 Oct;283:621–644. doi: 10.1113/jphysiol.1978.sp012523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barrantes F. J. Intrinsic fluorescence of the membrane-bound acetylcholine receptor: its quenching by suberyldicholine. Biochem Biophys Res Commun. 1976 Sep 20;72(2):479–488. doi: 10.1016/s0006-291x(76)80067-8. [DOI] [PubMed] [Google Scholar]
  3. Barrantes F. J., Sakmann B., Bonner R., Eibl H., Jovin T. M. 1-Pyrene-butyrylcholine: a fluorescent probe for the cholinergic system. Proc Natl Acad Sci U S A. 1975 Aug;72(8):3097–3101. doi: 10.1073/pnas.72.8.3097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blake A., Peacocke A. R. The interaction of aminocridines with nucleic acids. Biopolymers. 1968;6(9):1225–1253. doi: 10.1002/bip.1968.360060902. [DOI] [PubMed] [Google Scholar]
  5. Bonner R., Barrantes F. J., Jovin T. M. Kinetics of agonist-induced intrinsic fluorescence changes in membrane-bound acetylcholine receptor. Nature. 1976 Sep 30;263(5576):429–431. doi: 10.1038/263429a0. [DOI] [PubMed] [Google Scholar]
  6. Cohen J. B., Changeux J. P. Interaction of a fluorescent ligand with membrane-bound cholinergic receptor from Torpedo marmorata. Biochemistry. 1973 Nov 20;12(24):4855–4864. doi: 10.1021/bi00748a008. [DOI] [PubMed] [Google Scholar]
  7. Eldefrawi M. E., Eldefrawi A. T., Wilson D. B. Tryptophan and cystein residues of the acetylcholine receptors of Torpedo species. Relationship to binding of cholinergic ligands. Biochemistry. 1975 Sep 23;14(19):4304–4310. doi: 10.1021/bi00690a026. [DOI] [PubMed] [Google Scholar]
  8. Froede H. C., Wilson I. B. On the subunit structure of acetylcholinesterase. Isr J Med Sci. 1970 Mar-Apr;6(2):179–184. [PubMed] [Google Scholar]
  9. Ghosh P. B., Whitehouse M. W. 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole: a new fluorigenic reagent for amino acids and other amines. Biochem J. 1968 Jun;108(1):155–156. doi: 10.1042/bj1080155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Grünhagen H. H., Changeux J. P. Studies on the electrogenic action of acetylcholine with Torpedo marmorata electric organ. IV. Quinacrine: a fluorescent probe for the conformational transitions of the cholinergic receptor protein in its membrane-bound state. J Mol Biol. 1976 Sep 25;106(3):497–516. doi: 10.1016/0022-2836(76)90249-7. [DOI] [PubMed] [Google Scholar]
  11. Klett R. P., Fulpius B. W., Cooper D., Smith M., Reich E., Possani L. D. The acetylcholine receptor. I. Purification and characterization of a macromolecule isolated from Electrophorus electricus. J Biol Chem. 1973 Oct 10;248(19):6841–6853. [PubMed] [Google Scholar]
  12. LePecq J. B., Paoletti C. A fluorescent complex between ethidium bromide and nucleic acids. Physical-chemical characterization. J Mol Biol. 1967 Jul 14;27(1):87–106. doi: 10.1016/0022-2836(67)90353-1. [DOI] [PubMed] [Google Scholar]
  13. Maelicke A., Fulpius B. W., Klett R. P., Reich E. Acetylcholine receptor. Responses to drug binding. J Biol Chem. 1977 Jul 25;252(14):4811–4830. [PubMed] [Google Scholar]
  14. Martinez-Carrion M., Raftery M. A. Use of a fluorescent probe for the study of ligand binding by the isolated cholinergic receptor of Torpedo californica. Biochem Biophys Res Commun. 1973 Dec 19;55(4):1156–1164. doi: 10.1016/s0006-291x(73)80016-6. [DOI] [PubMed] [Google Scholar]
  15. Neher E., Sakmann B. Single-channel currents recorded from membrane of denervated frog muscle fibres. Nature. 1976 Apr 29;260(5554):799–802. doi: 10.1038/260799a0. [DOI] [PubMed] [Google Scholar]
  16. Neher E., Sakmann B., Steinbach J. H. The extracellular patch clamp: a method for resolving currents through individual open channels in biological membranes. Pflugers Arch. 1978 Jul 18;375(2):219–228. doi: 10.1007/BF00584247. [DOI] [PubMed] [Google Scholar]
  17. Neher E., Steinbach J. H. Local anaesthetics transiently block currents through single acetylcholine-receptor channels. J Physiol. 1978 Apr;277:153–176. doi: 10.1113/jphysiol.1978.sp012267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Neumann E., Chang H. W. Dynamic properties of isolated acetylcholine receptor protein: kinetics of the binding of acetylcholine and Ca ions. Proc Natl Acad Sci U S A. 1976 Nov;73(11):3994–3998. doi: 10.1073/pnas.73.11.3994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Rosenberry T. L., Neumann E. Interaction of ligands with acetylcholinesterase. Use of temperature-jump relaxation kinetics in the binding of specific fluorescent ligands. Biochemistry. 1977 Aug 23;16(17):3870–3878. doi: 10.1021/bi00636a024. [DOI] [PubMed] [Google Scholar]
  20. Sator V., Raftery M. A., Martinez-Carrion M. Propidium as a probe of acetylcholine receptor binding sites. Arch Biochem Biophys. 1977 Nov;184(1):95–102. doi: 10.1016/0003-9861(77)90330-7. [DOI] [PubMed] [Google Scholar]
  21. TAKEUCHI A., TAKEUCHI N. Active phase of frog's end-plate potential. J Neurophysiol. 1959 Jul;22(4):395–411. doi: 10.1152/jn.1959.22.4.395. [DOI] [PubMed] [Google Scholar]
  22. Waksman G., Fournié-Zaluski M. C., Roques B. Synthesis of fluorescent acyl-cholines with agonistic properties: pharmacological activity on Electrophorus electroplaque and interaction in vitro with Torpedo receptor-rich membrane fragments. FEBS Lett. 1976 Sep 1;67(3):335–342. doi: 10.1016/0014-5793(76)80560-1. [DOI] [PubMed] [Google Scholar]
  23. Weber G., Borris D. P., De Robertis E., Barrantes F. J., La Torre J. L., Llorente de Carlin M. C. The use of a cholinergic fluorescent probe for the study of the receptor proteolipid. Mol Pharmacol. 1971 Sep;7(5):530–537. [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