Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 1999 Jan;76(1 Pt 1):207–218. doi: 10.1016/S0006-3495(99)77190-0

A mutational analysis of the acetylcholine receptor channel transmitter binding site.

G Akk 1, M Zhou 1, A Auerbach 1
PMCID: PMC1302512  PMID: 9876135

Abstract

Mutagenesis and single-channel kinetic analysis were used to investigate the roles of four acetylcholine receptor channel (AChR) residues that are candidates for interacting directly with the agonist. The EC50 of the ACh dose-response curve was increased following alpha-subunit mutations Y93F and Y198F and epsilon-subunit mutations D175N and E184Q. Single-channel kinetic modeling indicates that the increase was caused mainly by a reduced gating equilibrium constant (Theta) in alphaY198F and epsilonD175N, by an increase in the equilibrium dissociation constant for ACh (KD) and a reduction in Theta in alphaY93F, and only by a reduction in KD in epsilonE184Q. This mutation altered the affinity of only one of the two binding sites and was the only mutation that reduced competition by extracellular K+. Additional mutations of epsilonE184 showed that K+ competition was unaltered in epsilonE184D and was virtually eliminated in epsilonE184K, but that neither of these mutations altered the intrinsic affinity for ACh. Thus there is an apparent electrostatic interaction between the epsilonE184 side chain and K+ ( approximately 1.7kBT), but not ACh+. The results are discussed in terms of multisite and induced-fit models of ligand binding to the AChR.

Full Text

The Full Text of this article is available as a PDF (148.5 KB).

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. 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. Akk G., Sine S., Auerbach A. Binding sites contribute unequally to the gating of mouse nicotinic alpha D200N acetylcholine receptors. J Physiol. 1996 Oct 1;496(Pt 1):185–196. doi: 10.1113/jphysiol.1996.sp021676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Auerbach A. A statistical analysis of acetylcholine receptor activation in Xenopus myocytes: stepwise versus concerted models of gating. J Physiol. 1993 Feb;461:339–378. doi: 10.1113/jphysiol.1993.sp019517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Auerbach A., Akk G. Desensitization of mouse nicotinic acetylcholine receptor channels. A two-gate mechanism. J Gen Physiol. 1998 Aug;112(2):181–197. doi: 10.1085/jgp.112.2.181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Auerbach A., Sigurdson W., Chen J., Akk G. Voltage dependence of mouse acetylcholine receptor gating: different charge movements in di-, mono- and unliganded receptors. J Physiol. 1996 Jul 1;494(Pt 1):155–170. doi: 10.1113/jphysiol.1996.sp021482. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bennett W. S., Jr, Steitz T. A. Structure of a complex between yeast hexokinase A and glucose. II. Detailed comparisons of conformation and active site configuration with the native hexokinase B monomer and dimer. J Mol Biol. 1980 Jun 25;140(2):211–230. doi: 10.1016/0022-2836(80)90103-5. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Charnet P., Labarca C., Lester H. A. Structure of the gamma-less nicotinic acetylcholine receptor: learning from omission. Mol Pharmacol. 1992 Apr;41(4):708–717. [PubMed] [Google Scholar]
  10. Chen J., Zhang Y., Akk G., Sine S., Auerbach A. Activation kinetics of recombinant mouse nicotinic acetylcholine receptors: mutations of alpha-subunit tyrosine 190 affect both binding and gating. Biophys J. 1995 Sep;69(3):849–859. doi: 10.1016/S0006-3495(95)79959-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Czajkowski C., Karlin A. Agonist binding site of Torpedo electric tissue nicotinic acetylcholine receptor. A negatively charged region of the delta subunit within 0.9 nm of the alpha subunit binding site disulfide. J Biol Chem. 1991 Nov 25;266(33):22603–22612. [PubMed] [Google Scholar]
  12. Czajkowski C., Kaufmann C., Karlin A. Negatively charged amino acid residues in the nicotinic receptor delta subunit that contribute to the binding of acetylcholine. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6285–6289. doi: 10.1073/pnas.90.13.6285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Fersht A. R., Knill-Jones J. W., Bedouelle H., Winter G. Reconstruction by site-directed mutagenesis of the transition state for the activation of tyrosine by the tyrosyl-tRNA synthetase: a mobile loop envelopes the transition state in an induced-fit mechanism. Biochemistry. 1988 Mar 8;27(5):1581–1587. doi: 10.1021/bi00405a028. [DOI] [PubMed] [Google Scholar]
  16. Filatov G. N., White M. M. The role of conserved leucines in the M2 domain of the acetylcholine receptor in channel gating. Mol Pharmacol. 1995 Sep;48(3):379–384. [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Jones M. V., Sahara Y., Dzubay J. A., Westbrook G. L. Defining affinity with the GABAA receptor. J Neurosci. 1998 Nov 1;18(21):8590–8604. doi: 10.1523/JNEUROSCI.18-21-08590.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Karlin A., Akabas M. H. Toward a structural basis for the function of nicotinic acetylcholine receptors and their cousins. Neuron. 1995 Dec;15(6):1231–1244. doi: 10.1016/0896-6273(95)90004-7. [DOI] [PubMed] [Google Scholar]
  21. Labarca C., Nowak M. W., Zhang H., Tang L., Deshpande P., Lester H. A. Channel gating governed symmetrically by conserved leucine residues in the M2 domain of nicotinic receptors. Nature. 1995 Aug 10;376(6540):514–516. doi: 10.1038/376514a0. [DOI] [PubMed] [Google Scholar]
  22. Laberge M. Intrinsic protein electric fields: basic non-covalent interactions and relationship to protein-induced Stark effects. Biochim Biophys Acta. 1998 Aug 18;1386(2):305–330. doi: 10.1016/s0167-4838(98)00100-9. [DOI] [PubMed] [Google Scholar]
  23. Loewenthal R., Sancho J., Reinikainen T., Fersht A. R. Long-range surface charge-charge interactions in proteins. Comparison of experimental results with calculations from a theoretical method. J Mol Biol. 1993 Jul 20;232(2):574–583. doi: 10.1006/jmbi.1993.1412. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Middleton R. E., Cohen J. B. Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: [3H]nicotine as an agonist photoaffinity label. Biochemistry. 1991 Jul 16;30(28):6987–6997. doi: 10.1021/bi00242a026. [DOI] [PubMed] [Google Scholar]
  26. Nowak M. W., Kearney P. C., Sampson J. R., Saks M. E., Labarca C. G., Silverman S. K., Zhong W., Thorson J., Abelson J. N., Davidson N. Nicotinic receptor binding site probed with unnatural amino acid incorporation in intact cells. Science. 1995 Apr 21;268(5209):439–442. doi: 10.1126/science.7716551. [DOI] [PubMed] [Google Scholar]
  27. O'Leary M. E., White M. M. Mutational analysis of ligand-induced activation of the Torpedo acetylcholine receptor. J Biol Chem. 1992 Apr 25;267(12):8360–8365. [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. Post C. B., Ray W. J., Jr Reexamination of induced fit as a determinant of substrate specificity in enzymatic reactions. Biochemistry. 1995 Dec 12;34(49):15881–15885. doi: 10.1021/bi00049a001. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Qin F., Auerbach A., Sachs F. Estimating single-channel kinetic parameters from idealized patch-clamp data containing missed events. Biophys J. 1996 Jan;70(1):264–280. doi: 10.1016/S0006-3495(96)79568-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Radić Z., Duran R., Vellom D. C., Li Y., Cervenansky C., Taylor P. Site of fasciculin interaction with acetylcholinesterase. J Biol Chem. 1994 Apr 15;269(15):11233–11239. [PubMed] [Google Scholar]
  33. Radić Z., Kirchhoff P. D., Quinn D. M., McCammon J. A., Taylor P. Electrostatic influence on the kinetics of ligand binding to acetylcholinesterase. Distinctions between active center ligands and fasciculin. J Biol Chem. 1997 Sep 12;272(37):23265–23277. doi: 10.1074/jbc.272.37.23265. [DOI] [PubMed] [Google Scholar]
  34. Sakmann B., Patlak J., Neher E. Single acetylcholine-activated channels show burst-kinetics in presence of desensitizing concentrations of agonist. Nature. 1980 Jul 3;286(5768):71–73. doi: 10.1038/286071a0. [DOI] [PubMed] [Google Scholar]
  35. Sine S. M., Claudio T. Gamma- and delta-subunits regulate the affinity and the cooperativity of ligand binding to the acetylcholine receptor. J Biol Chem. 1991 Oct 15;266(29):19369–19377. [PubMed] [Google Scholar]
  36. Sine S. M. Molecular dissection of subunit interfaces in the acetylcholine receptor: identification of residues that determine curare selectivity. Proc Natl Acad Sci U S A. 1993 Oct 15;90(20):9436–9440. doi: 10.1073/pnas.90.20.9436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sine S. M., Ohno K., Bouzat C., Auerbach A., Milone M., Pruitt J. N., Engel A. G. Mutation of the acetylcholine receptor alpha subunit causes a slow-channel myasthenic syndrome by enhancing agonist binding affinity. Neuron. 1995 Jul;15(1):229–239. doi: 10.1016/0896-6273(95)90080-2. [DOI] [PubMed] [Google Scholar]
  38. Sine S. M., Quiram P., Papanikolaou F., Kreienkamp H. J., Taylor P. Conserved tyrosines in the alpha subunit of the nicotinic acetylcholine receptor stabilize quaternary ammonium groups of agonists and curariform antagonists. J Biol Chem. 1994 Mar 25;269(12):8808–8816. [PubMed] [Google Scholar]
  39. Sine S. M., Taylor P. The relationship between agonist occupation and the permeability response of the cholinergic receptor revealed by bound cobra alpha-toxin. J Biol Chem. 1980 Nov 10;255(21):10144–10156. [PubMed] [Google Scholar]
  40. 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]
  41. 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]
  42. Tsigelny I., Sugiyama N., Sine S. M., Taylor P. A model of the nicotinic receptor extracellular domain based on sequence identity and residue location. Biophys J. 1997 Jul;73(1):52–66. doi: 10.1016/S0006-3495(97)78047-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Unwin N. Acetylcholine receptor channel imaged in the open state. Nature. 1995 Jan 5;373(6509):37–43. doi: 10.1038/373037a0. [DOI] [PubMed] [Google Scholar]
  44. Unwin N. Nicotinic acetylcholine receptor at 9 A resolution. J Mol Biol. 1993 Feb 20;229(4):1101–1124. doi: 10.1006/jmbi.1993.1107. [DOI] [PubMed] [Google Scholar]
  45. Unwin N. Projection structure of the nicotinic acetylcholine receptor: distinct conformations of the alpha subunits. J Mol Biol. 1996 Apr 5;257(3):586–596. doi: 10.1006/jmbi.1996.0187. [DOI] [PubMed] [Google Scholar]
  46. Valenzuela C. F., Weign P., Yguerabide J., Johnson D. A. Transverse distance between the membrane and the agonist binding sites on the Torpedo acetylcholine receptor: a fluorescence study. Biophys J. 1994 Mar;66(3 Pt 1):674–682. doi: 10.1016/s0006-3495(94)80841-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wang H. L., Auerbach A., Bren N., Ohno K., Engel A. G., Sine S. M. Mutation in the M1 domain of the acetylcholine receptor alpha subunit decreases the rate of agonist dissociation. J Gen Physiol. 1997 Jun;109(6):757–766. doi: 10.1085/jgp.109.6.757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zhang Y., Chen J., Auerbach A. Activation of recombinant mouse acetylcholine receptors by acetylcholine, carbamylcholine and tetramethylammonium. J Physiol. 1995 Jul 1;486(Pt 1):189–206. doi: 10.1113/jphysiol.1995.sp020802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Zhou H. X., Wlodek S. T., McCammon J. A. Conformation gating as a mechanism for enzyme specificity. Proc Natl Acad Sci U S A. 1998 Aug 4;95(16):9280–9283. doi: 10.1073/pnas.95.16.9280. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES