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. 2000 Mar;78(3):1324–1334. doi: 10.1016/S0006-3495(00)76687-2

Electrostatic interactions regulate desensitization of the nicotinic acetylcholine receptor.

X Z Song 1, S E Pedersen 1
PMCID: PMC1300732  PMID: 10692319

Abstract

To determine the importance of electrostatic interactions for agonist binding to the nicotinic acetylcholine receptor (AChR), we examined the affinity of the fluorescent agonist dansyl-C6-choline for the AChR. Increasing ionic strength decreased the binding affinity in a noncompetitive manner and increased the Hill coefficient of binding. Small cations did not compete directly for dansyl-C6-choline binding. The sensitivity to ionic strength was reduced in the presence of proadifen, a noncompetitive antagonist that desensitizes the receptor. Moreover, at low ionic strength, the dansyl-C6-choline affinities were similar in the absence or presence of proadifen, a result consistent with the receptor being desensitized at low ionic strength. Similar ionic strength effects were observed for the binding of the noncompetitive antagonist [(3)H]ethidium when examined in the presence and absence of agonist to desensitize the AChR. Therefore, ionic strength modulates binding affinity through at least two mechanisms: by influencing the conformation of the AChR and by electrostatic effects at the binding sites. The results show that charge-charge interactions regulate the desensitization of the receptor. Analysis of dansyl-C6-choline binding to the desensitized conformation using the Debye-Hückel equation was consistent with the presence of five to nine negative charges within 20 A of the acetylcholine binding sites.

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Selected References

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  1. Addona G. H., Andrews S. H., Cafiso D. S. Estimating the electrostatic potential at the acetylcholine receptor agonist site using power saturation EPR. Biochim Biophys Acta. 1997 Oct 2;1329(1):74–84. doi: 10.1016/s0005-2736(97)00089-8. [DOI] [PubMed] [Google Scholar]
  2. Akk G., Auerbach A. Inorganic, monovalent cations compete with agonists for the transmitter binding site of nicotinic acetylcholine receptors. Biophys J. 1996 Jun;70(6):2652–2658. doi: 10.1016/S0006-3495(96)79834-X. [DOI] [PMC free article] [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. Boyd N. D., Cohen J. B. Desensitization of membrane-bound Torpedo acetylcholine receptor by amine noncompetitive antagonists and aliphatic alcohols: studies of [3H]acetylcholine binding and 22Na+ ion fluxes. Biochemistry. 1984 Aug 28;23(18):4023–4033. doi: 10.1021/bi00313a003. [DOI] [PubMed] [Google Scholar]
  5. Boyd N. D., Cohen J. B. Kinetics of binding of [3H]acetylcholine to Torpedo postsynaptic membranes: association and dissociation rate constants by rapid mixing and ultrafiltration. Biochemistry. 1980 Nov 11;19(23):5353–5358. doi: 10.1021/bi00564a032. [DOI] [PubMed] [Google Scholar]
  6. Changeux J. P., Devillers-Thiéry A., Chemouilli P. Acetylcholine receptor: an allosteric protein. Science. 1984 Sep 21;225(4668):1335–1345. doi: 10.1126/science.6382611. [DOI] [PubMed] [Google Scholar]
  7. Czajkowski C., Karlin A. Structure of the nicotinic receptor acetylcholine-binding site. Identification of acidic residues in the delta subunit within 0.9 nm of the 5 alpha subunit-binding. J Biol Chem. 1995 Feb 17;270(7):3160–3164. doi: 10.1074/jbc.270.7.3160. [DOI] [PubMed] [Google Scholar]
  8. Devillers-Thiéry A., Galzi J. L., Eiselé J. L., Bertrand S., Bertrand D., Changeux J. P. Functional architecture of the nicotinic acetylcholine receptor: a prototype of ligand-gated ion channels. J Membr Biol. 1993 Nov;136(2):97–112. doi: 10.1007/BF02505755. [DOI] [PubMed] [Google Scholar]
  9. Dougherty D. A. Cation-pi interactions in chemistry and biology: a new view of benzene, Phe, Tyr, and Trp. Science. 1996 Jan 12;271(5246):163–168. doi: 10.1126/science.271.5246.163. [DOI] [PubMed] [Google Scholar]
  10. Furois-Corbin S., Pullman A. A possible model for the inner wall of the acetylcholine receptor channel. Biochim Biophys Acta. 1989 Sep 18;984(3):339–350. doi: 10.1016/0005-2736(89)90301-5. [DOI] [PubMed] [Google Scholar]
  11. Heidmann T., Bernhardt J., Neumann E., Changeux J. P. Rapid kinetics of agonist binding and permeability response analyzed in parallel on acetylcholine receptor rich membranes from Torpedo marmorata. Biochemistry. 1983 Nov 8;22(23):5452–5459. doi: 10.1021/bi00292a029. [DOI] [PubMed] [Google Scholar]
  12. Herz J. M., Johnson D. A., Taylor P. Interaction of noncompetitive inhibitors with the acetylcholine receptor. The site specificity and spectroscopic properties of ethidium binding. J Biol Chem. 1987 May 25;262(15):7238–7247. [PubMed] [Google Scholar]
  13. Hucho F., Tsetlin V. I., Machold J. The emerging three-dimensional structure of a receptor. The nicotinic acetylcholine receptor. Eur J Biochem. 1996 Aug 1;239(3):539–557. doi: 10.1111/j.1432-1033.1996.0539u.x. [DOI] [PubMed] [Google Scholar]
  14. Imoto K., Busch C., Sakmann B., Mishina M., Konno T., Nakai J., Bujo H., Mori Y., Fukuda K., Numa S. Rings of negatively charged amino acids determine the acetylcholine receptor channel conductance. Nature. 1988 Oct 13;335(6191):645–648. doi: 10.1038/335645a0. [DOI] [PubMed] [Google Scholar]
  15. KATZ B., THESLEFF S. A study of the desensitization produced by acetylcholine at the motor end-plate. J Physiol. 1957 Aug 29;138(1):63–80. doi: 10.1113/jphysiol.1957.sp005838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Lurtz M. M., Hareland M. L., Pedersen S. E. Quinacrine and ethidium bromide bind the same locus on the nicotinic acetylcholine receptor from Torpedo californica. Biochemistry. 1997 Feb 25;36(8):2068–2075. doi: 10.1021/bi962547p. [DOI] [PubMed] [Google Scholar]
  18. Martin M., Czajkowski C., Karlin A. The contributions of aspartyl residues in the acetylcholine receptor gamma and delta subunits to the binding of agonists and competitive antagonists. J Biol Chem. 1996 Jun 7;271(23):13497–13503. doi: 10.1074/jbc.271.23.13497. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Nolte H. J., Rosenberry T. L., Neumann E. Effective charge on acetylcholinesterase active sites determined from the ionic strength dependence of association rate constants with cationic ligands. Biochemistry. 1980 Aug 5;19(16):3705–3711. doi: 10.1021/bi00557a011. [DOI] [PubMed] [Google Scholar]
  22. Ochoa E. L., Chattopadhyay A., McNamee M. G. Desensitization of the nicotinic acetylcholine receptor: molecular mechanisms and effect of modulators. Cell Mol Neurobiol. 1989 Jun;9(2):141–178. doi: 10.1007/BF00713026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Pedersen S. E., Dreyer E. B., Cohen J. B. Location of ligand-binding sites on the nicotinic acetylcholine receptor alpha-subunit. J Biol Chem. 1986 Oct 15;261(29):13735–13743. [PubMed] [Google Scholar]
  25. Pedersen S. E., Papineni R. V. Interaction of d-tubocurarine analogs with the Torpedo nicotinic acetylcholine receptor. Methylation and stereoisomerization affect site-selective competitive binding and binding to the noncompetitive site. J Biol Chem. 1995 Dec 29;270(52):31141–31150. doi: 10.1074/jbc.270.52.31141. [DOI] [PubMed] [Google Scholar]
  26. Pedersen S. E. Site-selective photoaffinity labeling of the Torpedo californica nicotinic acetylcholine receptor by azide derivatives of ethidium bromide. Mol Pharmacol. 1995 Jan;47(1):1–9. [PubMed] [Google Scholar]
  27. Prince R. J., Sine S. M. Molecular dissection of subunit interfaces in the acetylcholine receptor. Identification of residues that determine agonist selectivity. J Biol Chem. 1996 Oct 18;271(42):25770–25777. doi: 10.1074/jbc.271.42.25770. [DOI] [PubMed] [Google Scholar]
  28. Raines D. E., Krishnan N. S. Transient low-affinity agonist binding to Torpedo postsynaptic membranes resolved by using sequential mixing stopped-flow fluorescence spectroscopy. Biochemistry. 1998 Jan 20;37(3):956–964. doi: 10.1021/bi971689w. [DOI] [PubMed] [Google Scholar]
  29. Schmidt J., Raftery M. A. The cation sensitivity of the acetylcholine receptor from Torpedo californica. J Neurochem. 1974 Oct;23(4):617–623. doi: 10.1111/j.1471-4159.1974.tb04383.x. [DOI] [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., Kreienkamp H. J., Bren N., Maeda R., Taylor P. Molecular dissection of subunit interfaces in the acetylcholine receptor: identification of determinants of alpha-conotoxin M1 selectivity. Neuron. 1995 Jul;15(1):205–211. doi: 10.1016/0896-6273(95)90077-2. [DOI] [PubMed] [Google Scholar]
  32. Sobel A., Weber M., Changeux J. P. Large-scale purification of the acetylcholine-receptor protein in its membrane-bound and detergent-extracted forms from Torpedo marmorata electric organ. Eur J Biochem. 1977 Oct 17;80(1):215–224. doi: 10.1111/j.1432-1033.1977.tb11874.x. [DOI] [PubMed] [Google Scholar]
  33. Stauffer D. A., Karlin A. Electrostatic potential of the acetylcholine binding sites in the nicotinic receptor probed by reactions of binding-site cysteines with charged methanethiosulfonates. Biochemistry. 1994 Jun 7;33(22):6840–6849. doi: 10.1021/bi00188a013. [DOI] [PubMed] [Google Scholar]
  34. Sugiyama N., Boyd A. E., Taylor P. Anionic residue in the alpha-subunit of the nicotinic acetylcholine receptor contributing to subunit assembly and ligand binding. J Biol Chem. 1996 Oct 25;271(43):26575–26581. doi: 10.1074/jbc.271.43.26575. [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. Tan R. C., Truong T. N., McCammon J. A., Sussman J. L. Acetylcholinesterase: electrostatic steering increases the rate of ligand binding. Biochemistry. 1993 Jan 19;32(2):401–403. doi: 10.1021/bi00053a003. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Walker J. W., Takeyasu K., McNamee M. G. Activation and inactivation kinetics of Torpedo californica acetylcholine receptor in reconstituted membranes. Biochemistry. 1982 Oct 26;21(22):5384–5389. doi: 10.1021/bi00265a001. [DOI] [PubMed] [Google Scholar]
  39. White B. H., Cohen J. B. Agonist-induced changes in the structure of the acetylcholine receptor M2 regions revealed by photoincorporation of an uncharged nicotinic noncompetitive antagonist. J Biol Chem. 1992 Aug 5;267(22):15770–15783. [PubMed] [Google Scholar]
  40. Zhong W., Gallivan J. P., Zhang Y., Li L., Lester H. A., Dougherty D. A. From ab initio quantum mechanics to molecular neurobiology: a cation-pi binding site in the nicotinic receptor. Proc Natl Acad Sci U S A. 1998 Oct 13;95(21):12088–12093. doi: 10.1073/pnas.95.21.12088. [DOI] [PMC free article] [PubMed] [Google Scholar]

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