Skip to main content
NCBI home page
The Journal of Physiology logoLink to The Journal of Physiology
. 1988 Jan;395:131–159. doi: 10.1113/jphysiol.1988.sp016912

Activation of ion channels in the frog end-plate by high concentrations of acetylcholine.

D Colquhoun 1, D C Ogden 1
PMCID: PMC1191987  PMID: 2457675

Abstract

1. The equilibrium relationship between acetylcholine (ACh) concentration and response (fraction of channels open), corrected for the effects of desensitization, has been estimated by single-ion-channel recording at the adult frog skeletal neuromuscular junction. At high ACh concentration channel openings occur in well-defined clusters separated by long desensitized intervals. The response, po, was estimated as the proportion of time for which a single channel was open during a cluster. 2. At negative membrane potential (-120 mV) po reached a maximum value of 0.9 at 100 microM-ACh and was half-maximum at 15 microM with a Hill slope of 1.6 at this point. At concentrations higher than 200 microM-ACh, po declined as a result of open-channel block by free ACh itself. 3. At positive membrane potentials (+100 mV) there was little channel block by ACh; po reached a maximum value of 0.41 at 500 microM-ACh, with half-maximum activation at 50 microM and Hill slope of 1.2 at this point. 4. Particular mechanisms for channel activation by ACh were fitted to the data by the method of least squares. Fits were fully determinate only if the two binding sites for ACh were assumed to be equivalent with no co-operativity in the ACh binding reactions. At negative potential the microscopic equilibrium constant for binding was K1 = K2 = 77 microM and the equilibrium constant for channel opening (opening/closing rates, beta/alpha) was 32. At positive potential the affinity was slightly higher, K = 32 microM, which confirms the view that the binding sites for ACh are outside the membrane electric field. The equilibrium constant for channel opening was reduced to 0.7 mainly as a result of the much shorter open lifetime (increased closing rate alpha) at positive potentials. 5. The data were also fitted well by very high values of beta/alpha together with a high degree of negative co-operativity or non-equivalence in ACh binding affinity (K2 much greater than K1). A good fit could also be obtained with moderate positive co-operativity combined with non-equivalence of the binding sites. 6. A mechanism that postulates a receptor with two independent gating subunits provided a poor fit to the data at negative potential. 7. The rate constants for channel opening and ACh dissociation were estimated by constraining the fitted parameters so that the burst length for channel opening was equal to its observed value at low concentrations of ACh.(ABSTRACT TRUNCATED AT 400 WORDS)

Full text

PDF
131

Selected References

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

  1. Adams D. J., Bevan S. Some properties of acetylcholine receptors in human cultured myotubes. Proc R Soc Lond B Biol Sci. 1985 Apr 22;224(1235):183–196. doi: 10.1098/rspb.1985.0028. [DOI] [PubMed] [Google Scholar]
  2. Adams D. J., Nonner W., Dwyer T. M., Hille B. Block of endplate channels by permeant cations in frog skeletal muscle. J Gen Physiol. 1981 Dec;78(6):593–615. doi: 10.1085/jgp.78.6.593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Adams P. R. Acetylcholine receptor kinetics. J Membr Biol. 1981 Feb 28;58(3):161–174. doi: 10.1007/BF01870902. [DOI] [PubMed] [Google Scholar]
  4. Adams P. R. An analysis of the dose-response curve at voltage-clamped frog-endplates. Pflugers Arch. 1975 Oct 28;360(2):145–153. doi: 10.1007/BF00580537. [DOI] [PubMed] [Google Scholar]
  5. Adams P. R. Relaxation experiments using bath-applied suberyldicholine. J Physiol. 1977 Jun;268(2):271–289. doi: 10.1113/jphysiol.1977.sp011857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Adams P. R. Voltage jump analysis of procaine action at frog end-plate. J Physiol. 1977 Jun;268(2):291–318. doi: 10.1113/jphysiol.1977.sp011858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Anderson C. R., Stevens C. F. Voltage clamp analysis of acetylcholine produced end-plate current fluctuations at frog neuromuscular junction. J Physiol. 1973 Dec;235(3):655–691. doi: 10.1113/jphysiol.1973.sp010410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Betz W., Sakmann B. Effects of proteolytic enzymes on function and structure of frog neuromuscular junctions. J Physiol. 1973 May;230(3):673–688. doi: 10.1113/jphysiol.1973.sp010211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Colquhoun D., Dreyer F., Sheridan R. E. The actions of tubocurarine at the frog neuromuscular junction. J Physiol. 1979 Aug;293:247–284. doi: 10.1113/jphysiol.1979.sp012888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Colquhoun D., Sakmann B. Fluctuations in the microsecond time range of the current through single acetylcholine receptor ion channels. Nature. 1981 Dec 3;294(5840):464–466. doi: 10.1038/294464a0. [DOI] [PubMed] [Google Scholar]
  12. DEL CASTILLO J., KATZ B. Interaction at end-plate receptors between different choline derivatives. Proc R Soc Lond B Biol Sci. 1957 May 7;146(924):369–381. doi: 10.1098/rspb.1957.0018. [DOI] [PubMed] [Google Scholar]
  13. Dionne V. E., Steinbach J. H., Stevens C. F. An analysis of the dose-response relationship at voltage-clamped frog neuromuscular junctions. J Physiol. 1978 Aug;281:421–444. doi: 10.1113/jphysiol.1978.sp012431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dreyer F., Peper K., Sterz R. Determination of dose-response curves by quantitative ionophoresis at the frog neuromuscular junction. J Physiol. 1978 Aug;281:395–419. doi: 10.1113/jphysiol.1978.sp012430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gardner P., Ogden D. C., Colquhoun D. Conductances of single ion channels opened by nicotinic agonists are indistinguishable. Nature. 1984 May 10;309(5964):160–162. doi: 10.1038/309160a0. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Hamm T. M., Reinking R. M., Roscoe D. D., Stuart D. G. Synchronous afferent discharge from a passive muscle of the cat: significance for interpreting spike-triggered averages. J Physiol. 1985 Aug;365:77–102. doi: 10.1113/jphysiol.1985.sp015760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Horn R., Vandenberg C. A. Statistical properties of single sodium channels. J Gen Physiol. 1984 Oct;84(4):505–534. doi: 10.1085/jgp.84.4.505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. JENKINSON D. H. The antagonism between tubocurarine and substances which depolarize the motor end-plate. J Physiol. 1960 Jul;152:309–324. doi: 10.1113/jphysiol.1960.sp006489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Lester H. A., Nerbonne J. M. Physiological and pharmacological manipulations with light flashes. Annu Rev Biophys Bioeng. 1982;11:151–175. doi: 10.1146/annurev.bb.11.060182.001055. [DOI] [PubMed] [Google Scholar]
  22. Magazanik L. G., Vyskocil F. Dependence of acetylcholine desensitization on the membrane potential of frog muscle fibre and on the ionic changes in the medium. J Physiol. 1970 Oct;210(3):507–518. doi: 10.1113/jphysiol.1970.sp009223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Magleby K. L., Pallotta B. S. A study of desensitization of acetylcholine receptors using nerve-released transmitter in the frog. J Physiol. 1981 Jul;316:225–250. doi: 10.1113/jphysiol.1981.sp013784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Magleby K. L., Stevens C. F. A quantitative description of end-plate currents. J Physiol. 1972 May;223(1):173–197. doi: 10.1113/jphysiol.1972.sp009840. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Magleby K. L., Terrar D. A. Factors affecting the time course of decay of end-plate currents: a possible cooperative action of acetylcholine on receptors at the frog neuromuscular junction. J Physiol. 1975 Jan;244(2):467–495. doi: 10.1113/jphysiol.1975.sp010808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Matthews-Bellinger J., Salpeter M. M. Distribution of acetylcholine receptors at frog neuromuscular junctions with a discussion of some physiological implications. J Physiol. 1978 Jun;279:197–213. doi: 10.1113/jphysiol.1978.sp012340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Neher E., Sakmann B. Voltage-dependence of drug-induced conductance in frog neuromuscular junction. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2140–2144. doi: 10.1073/pnas.72.6.2140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Neher E. The charge carried by single-channel currents of rat cultured muscle cells in the presence of local anaesthetics. J Physiol. 1983 Jun;339:663–678. doi: 10.1113/jphysiol.1983.sp014741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ogden D. C., Colquhoun D. Ion channel block by acetylcholine, carbachol and suberyldicholine at the frog neuromuscular junction. Proc R Soc Lond B Biol Sci. 1985 Sep 23;225(1240):329–355. doi: 10.1098/rspb.1985.0065. [DOI] [PubMed] [Google Scholar]
  31. Ogden D. C., Colquhoun D. The efficacy of agonists at the frog neuromuscular junction studied with single channel recording. Pflugers Arch. 1983 Nov;399(3):246–248. doi: 10.1007/BF00656725. [DOI] [PubMed] [Google Scholar]
  32. Pasquale E. B., Takeyasu K., Udgaonkar J. B., Cash D. J., Severski M. C., Hess G. P. Acetylcholine receptor: evidence for a regulatory binding site in investigations of suberyldicholine-induced transmembrane ion flux in Electrophorus electricus membrane vesicles. Biochemistry. 1983 Dec 6;22(25):5967–5973. doi: 10.1021/bi00294a041. [DOI] [PubMed] [Google Scholar]
  33. Rang H. P. Drug receptors and their function. Nature. 1971 May 14;231(5298):91–96. doi: 10.1038/231091a0. [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. Scubon-Mulieri B., Parsons R. L. Desensitization onset and recovery at the potassium-depolarized frog neuromuscular junction are voltage sensitive. J Gen Physiol. 1978 Mar;71(3):285–299. doi: 10.1085/jgp.71.3.285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Siegelbaum S. A., Trautmann A., Koenig J. Single acetylcholine-activated channel currents in developing muscle cells. Dev Biol. 1984 Aug;104(2):366–379. doi: 10.1016/0012-1606(84)90092-7. [DOI] [PubMed] [Google Scholar]
  37. Sigworth F. J. Open channel noise. I. Noise in acetylcholine receptor currents suggests conformational fluctuations. Biophys J. 1985 May;47(5):709–720. doi: 10.1016/S0006-3495(85)83968-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sine S. M., Steinbach J. H. Activation of a nicotinic acetylcholine receptor. Biophys J. 1984 Jan;45(1):175–185. doi: 10.1016/S0006-3495(84)84146-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. 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]
  41. Sine S. M., Steinbach J. H. Agonists block currents through acetylcholine receptor channels. Biophys J. 1984 Aug;46(2):277–283. doi: 10.1016/S0006-3495(84)84022-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sine S. M., Taylor P. Relationship between reversible antagonist occupancy and the functional capacity of the acetylcholine receptor. J Biol Chem. 1981 Jul 10;256(13):6692–6699. [PubMed] [Google Scholar]
  43. Takeyasu K., Udgaonkar J. B., Hess G. P. Acetylcholine receptor: evidence for a voltage-dependent regulatory site for acetylcholine. Chemical kinetic measurements in membrane vesicles using a voltage clamp. Biochemistry. 1983 Dec 6;22(25):5973–5978. doi: 10.1021/bi00294a042. [DOI] [PubMed] [Google Scholar]
  44. 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]

Articles from The Journal of Physiology are provided here courtesy of The Physiological Society

RESOURCES