Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 1993 Feb;64(2):325–338. doi: 10.1016/S0006-3495(93)81373-0

Analysis of cyclic and acyclic nicotinic cholinergic agonists using radioligand binding, single channel recording, and nuclear magnetic resonance spectroscopy.

K A McGroddy 1, A A Carter 1, M M Tubbert 1, R E Oswald 1
PMCID: PMC1262337  PMID: 8457664

Abstract

The relationship between the structure and function of a series of nicotinic cholinergic agonists has been studied using radioligand binding, single channel recording, and nuclear magnetic resonance spectroscopy. The cyclic compound 1,1-dimethyl-4-acetylpiperazinium iodide and its trifluoromethyl analogue (F3-PIP) interact with nicotinic acetylcholine receptors (nAChRs) from both Torpedo electroplaque and BC3H-1 cells at lower concentrations than the acyclic derivatives, N,N,N,N'-tetramethyl-N'-acetylethylenediamine iodide and its fluorinated analogue (F3-TED). The magnitude of the difference in potencies depends on the type of measurement. In binding experiments, the differences between the two classes of compounds depends mainly on the conditions of the experiment. In measurements of the initial interaction with the nAChR, the PIP compounds have an affinity approximately one order of magnitude higher than that of the TED compounds. Longer incubations indicated that the PIP compounds were able to induce a time-dependent shift in receptor affinity consistent with desensitization, whereas the TED compounds were unable to induce such a shift. The activation of single channel currents by the cyclic compounds occurs at concentrations approximately two orders of magnitude lower than for the acyclic compounds, but the TED compounds exhibit a larger degree of channel blockade than the PIP compounds. Previous work (McGroddy, K.A., and R.E. Oswald. 1992. Biophys. J. 64:314-324) has shown that the TED compounds can exist in two energetically distinct conformational states related by an isomerization of the amide bond. 19F nuclear magnetic resonance experiments suggest that the higher energy population of the TED compounds may interact preferentially with the ACh binding sites on the nAChRs and that a significant fraction of the difference between the initial affinity of the PIP and TED compounds may be accounted for by the predominance in solution of a conformational state less able to interact with the ACh binding sites on nAChRs.

Full text

PDF
325

Images in this article

331FIGURE 3
on p.331
335FIGURE 6
on p.335

Selected References

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

  1. Altschuh D., Vix O., Rees B., Thierry J. C. A conformation of cyclosporin A in aqueous environment revealed by the X-ray structure of a cyclosporin-Fab complex. Science. 1992 Apr 3;256(5053):92–94. doi: 10.1126/science.1566062. [DOI] [PubMed] [Google Scholar]
  2. Beers W. H., Reich E. Structure and activity of acetylcholine. Nature. 1970 Dec 5;228(5275):917–922. doi: 10.1038/228917a0. [DOI] [PubMed] [Google Scholar]
  3. Behling R. W., Yamane T., Navon G., Jelinski L. W. Conformation of acetylcholine bound to the nicotinic acetylcholine receptor. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6721–6725. doi: 10.1073/pnas.85.18.6721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Colquhoun D., Hawkes A. G. On the stochastic properties of single ion channels. Proc R Soc Lond B Biol Sci. 1981 Mar 6;211(1183):205–235. doi: 10.1098/rspb.1981.0003. [DOI] [PubMed] [Google Scholar]
  6. Colquhoun D., Sakmann B. Fast events in single-channel currents activated by acetylcholine and its analogues at the frog muscle end-plate. J Physiol. 1985 Dec;369:501–557. doi: 10.1113/jphysiol.1985.sp015912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dunn S. M., Blanchard S. G., Raftery M. A. Kinetics of carbamylcholine binding to membrane-bound acetylcholine receptor monitored by fluorescence changes of a covalently bound probe. Biochemistry. 1980 Nov 25;19(24):5645–5652. doi: 10.1021/bi00565a029. [DOI] [PubMed] [Google Scholar]
  8. Ealick S. E., Babu Y. S., Bugg C. E., Erion M. D., Guida W. C., Montgomery J. A., Secrist J. A., 3rd Application of crystallographic and modeling methods in the design of purine nucleoside phosphorylase inhibitors. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11540–11544. doi: 10.1073/pnas.88.24.11540. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Freeman J. A., Schmidt J. T., Oswald R. E. Effect of alpha-bungarotoxin on retinotectal synaptic transmission in the goldfish and the toad. Neuroscience. 1980;5(5):929–942. doi: 10.1016/0306-4522(80)90161-x. [DOI] [PubMed] [Google Scholar]
  10. Galzi J. L., Bertrand D., Devillers-Thiéry A., Revah F., Bertrand S., Changeux J. P. Functional significance of aromatic amino acids from three peptide loops of the alpha 7 neuronal nicotinic receptor site investigated by site-directed mutagenesis. FEBS Lett. 1991 Dec 9;294(3):198–202. doi: 10.1016/0014-5793(91)80668-s. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Hamill O. P., Marty A., Neher E., Sakmann B., Sigworth F. J. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981 Aug;391(2):85–100. doi: 10.1007/BF00656997. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Horn R., Lange K. Estimating kinetic constants from single channel data. Biophys J. 1983 Aug;43(2):207–223. doi: 10.1016/S0006-3495(83)84341-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jackson M. B. Dependence of acetylcholine receptor channel kinetics on agonist concentration in cultured mouse muscle fibres. J Physiol. 1988 Mar;397:555–583. doi: 10.1113/jphysiol.1988.sp017019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jorgensen W. L. Rusting of the lock and key model for protein-ligand binding. Science. 1991 Nov 15;254(5034):954–955. doi: 10.1126/science.1719636. [DOI] [PubMed] [Google Scholar]
  17. Labarca P., Montal M. S., Lindstrom J. M., Montal M. The occurrence of long openings in the purified cholinergic receptor channel increases with acetylcholine concentration. J Neurosci. 1985 Dec;5(12):3409–3413. doi: 10.1523/JNEUROSCI.05-12-03409.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Magleby K. L., Weiss D. S. Estimating kinetic parameters for single channels with simulation. A general method that resolves the missed event problem and accounts for noise. Biophys J. 1990 Dec;58(6):1411–1426. doi: 10.1016/S0006-3495(90)82487-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. McGroddy K. A., Oswald R. E. Solution structure and dynamics of cyclic and acyclic cholinergic agonists. Biophys J. 1993 Feb;64(2):314–324. doi: 10.1016/S0006-3495(93)81372-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Ni F., Konishi Y., Scheraga H. A. Thrombin-bound conformation of the C-terminal fragments of hirudin determined by transferred nuclear Overhauser effects. Biochemistry. 1990 May 8;29(18):4479–4489. doi: 10.1021/bi00470a030. [DOI] [PubMed] [Google Scholar]
  22. Olson E. N., Glaser L., Merlie J. P., Sebanne R., Lindstrom J. Regulation of surface expression of acetylcholine receptors in response to serum and cell growth in the BC3H1 muscle cell line. J Biol Chem. 1983 Nov 25;258(22):13946–13953. [PubMed] [Google Scholar]
  23. Oswald R. E., Millhauser G. L., Carter A. A. Diffusion model in ion channel gating. Extension to agonist-activated ion channels. Biophys J. 1991 May;59(5):1136–1142. doi: 10.1016/S0006-3495(91)82328-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Papke R. L., Millhauser G., Lieberman Z., Oswald R. E. Relationships of agonist properties to the single channel kinetics of nicotinic acetylcholine receptors. Biophys J. 1988 Jan;53(1):1–10. doi: 10.1016/S0006-3495(88)83059-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Papke R. L., Oswald R. E. Mechanisms of noncompetitive inhibition of acetylcholine-induced single-channel currents. J Gen Physiol. 1989 May;93(5):785–811. doi: 10.1085/jgp.93.5.785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Saitoh T., Oswald R., Wennogle L. P., Changeux J. P. Conditions for the selective labelling of the 66 000 dalton chain of the acetylcholine receptor by the covalent non-competitive blocker 5-azido-[3H]trimethisoquin. FEBS Lett. 1980 Jul 11;116(1):30–36. doi: 10.1016/0014-5793(80)80522-9. [DOI] [PubMed] [Google Scholar]
  27. Schmidt J., Raftery M. A. A simple assay for the study of solubilized acetylcholine receptors. Anal Biochem. 1973 Apr;52(2):349–354. doi: 10.1016/0003-2697(73)90036-5. [DOI] [PubMed] [Google Scholar]
  28. Sheridan R. P., Nilakantan R., Dixon J. S., Venkataraghavan R. The ensemble approach to distance geometry: application to the nicotinic pharmacophore. J Med Chem. 1986 Jun;29(6):899–906. doi: 10.1021/jm00156a005. [DOI] [PubMed] [Google Scholar]
  29. Sigworth F. J., Sine S. M. Data transformations for improved display and fitting of single-channel dwell time histograms. Biophys J. 1987 Dec;52(6):1047–1054. doi: 10.1016/S0006-3495(87)83298-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sine S. M., Claudio T., Sigworth F. J. Activation of Torpedo acetylcholine receptors expressed in mouse fibroblasts. Single channel current kinetics reveal distinct agonist binding affinities. J Gen Physiol. 1990 Aug;96(2):395–437. doi: 10.1085/jgp.96.2.395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sine S. M., Steinbach J. H. Activation of acetylcholine receptors on clonal mammalian BC3H-1 cells by high concentrations of agonist. J Physiol. 1987 Apr;385:325–359. doi: 10.1113/jphysiol.1987.sp016496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sine S. M., Steinbach J. H. Activation of acetylcholine receptors on clonal mammalian BC3H-1 cells by low concentrations of agonist. J Physiol. 1986 Apr;373:129–162. doi: 10.1113/jphysiol.1986.sp016039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sine S., Taylor P. Functional consequences of agonist-mediated state transitions in the cholinergic receptor. Studies in cultured muscle cells. J Biol Chem. 1979 May 10;254(9):3315–3325. [PubMed] [Google Scholar]
  34. Spivak C. E., Yadav J. S., Shang W. C., Hermsmeier M., Gund T. M. Carbamyl analogues of potent nicotinic agonists: pharmacology and computer-assisted molecular modeling study. J Med Chem. 1989 Feb;32(2):305–309. doi: 10.1021/jm00122a006. [DOI] [PubMed] [Google Scholar]
  35. Sussman J. L., Harel M., Frolow F., Oefner C., Goldman A., Toker L., Silman I. Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein. Science. 1991 Aug 23;253(5022):872–879. doi: 10.1126/science.1678899. [DOI] [PubMed] [Google Scholar]
  36. Tomaselli G. F., McLaughlin J. T., Jurman M. E., Hawrot E., Yellen G. Mutations affecting agonist sensitivity of the nicotinic acetylcholine receptor. Biophys J. 1991 Sep;60(3):721–727. doi: 10.1016/S0006-3495(91)82102-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Van Duyne G. D., Standaert R. F., Karplus P. A., Schreiber S. L., Clardy J. Atomic structure of FKBP-FK506, an immunophilin-immunosuppressant complex. Science. 1991 May 10;252(5007):839–842. doi: 10.1126/science.1709302. [DOI] [PubMed] [Google Scholar]
  38. Wassermann N. H., Bartels E., Erlanger B. F. Conformational properties of the acetylcholine receptor as revealed by studies with constrained depolarizing ligands. Proc Natl Acad Sci U S A. 1979 Jan;76(1):256–259. doi: 10.1073/pnas.76.1.256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Waters J. A., Spivak C. E., Hermsmeier M., Yadav J. S., Liang R. F., Gund T. M. Synthesis, pharmacology, and molecular modeling studies of semirigid, nicotinic agonists. J Med Chem. 1988 Mar;31(3):545–554. doi: 10.1021/jm00398a010. [DOI] [PubMed] [Google Scholar]
  40. Weber M., David-Pfeuty T., Changeux J. P. Regulation of binding properties of the nicotinic receptor protein by cholinergic ligands in membrane fragments from Torpedo marmorata. Proc Natl Acad Sci U S A. 1975 Sep;72(9):3443–3447. doi: 10.1073/pnas.72.9.3443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Weiland G., Georgia B., Lappi S., Chignell C. F., Taylor P. Kinetics of agonist-mediated transitions in state of the cholinergic receptor. J Biol Chem. 1977 Nov 10;252(21):7648–7656. [PubMed] [Google Scholar]
  42. Weiland G., Georgia B., Wee V. T., Chignell C. F., Taylor P. Ligand interactions with cholinergic receptor-enriched membranes from Torpedo: influence of agonist exposure on receptor properties. Mol Pharmacol. 1976 Nov;12(6):1091–1105. [PubMed] [Google Scholar]
  43. Weiland G., Taylor P. Ligand specificity of state transitions in the cholinergic receptor: behavior of agonists and antagonists. Mol Pharmacol. 1979 Mar;15(2):197–212. [PubMed] [Google Scholar]
  44. Wüthrich K., von Freyberg B., Weber C., Wider G., Traber R., Widmer H., Braun W. Receptor-induced conformation change of the immunosuppressant cyclosporin A. Science. 1991 Nov 15;254(5034):953–954. doi: 10.1126/science.1948082. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES