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. 1992 Aug;454:129–153. doi: 10.1113/jphysiol.1992.sp019257

A study of the bovine adrenal chromaffin nicotinic receptor using patch clamp and concentration-jump techniques.

D J Maconochie 1, D E Knight 1
PMCID: PMC1175598  PMID: 1282154

Abstract

1. Voltage clamp records have been obtained from bovine adrenal chromaffin cells in the outside-out and whole-cell configurations, in response to step changes of acetylcholine (ACh) concentration. The concentrations used ranged from 50 nM to 20 mM. 2. At high acetylcholine concentrations, the activation and desensitization kinetics of the nicotinic receptor, as observed in outside-out patches, may be described by a model incorporating a single, fast agonist binding step, and relatively slow isomerization to the open state. The affinity of the closed receptor for ACh is 310 microM, the channel opening rate constant is 460 s-1, and the closing rate constant is 29 s-1. 3. Single channel events, observed when nanomolar ACh concentrations are applied to whole cells, have two distinct channel lifetimes: 0.6 ms and 11-15 ms. The variation of the frequencies of the events with ACh concentration, suggests that the short lifetimes are openings of a singly liganded receptor and the longer lifetimes are openings of a doubly liganded receptor. 4. Only a single exponential associated with receptor desensitization is seen with outside-out patches, but two are seen with whole cells. It is postulated that there are two nicotinic receptor types present on adrenal chromaffin cells. 5. The rate of desensitization (9 s-1 and 26 s-1, whole cells; 24 s-1, patches), is fast enough to be significant in determining the open channel lifetime. 6. A sudden increase in current (rebound) is observed when a high concentration of ACh is abruptly removed from outside-out patches. This is evidence for a blocked state. The affinity of the blocking site for ACh is 1400 microM (outside-out patches). 7. The total number of activatable nicotinic channels per whole cell is estimated to be 2600.

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Selected References

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

  1. Adams P. R. A study of desensitization using voltage clamp. Pflugers Arch. 1975 Oct 28;360(2):135–144. doi: 10.1007/BF00580536. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Ascher P., Large W. A., Rang H. P. Studies on the mechanism of action of acetylcholine antagonists on rat parasympathetic ganglion cells. J Physiol. 1979 Oct;295:139–170. doi: 10.1113/jphysiol.1979.sp012958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Chiappinelli V. A. Actions of snake venom toxins on neuronal nicotinic receptors and other neuronal receptors. Pharmacol Ther. 1985;31(1-2):1–32. doi: 10.1016/0163-7258(85)90035-x. [DOI] [PubMed] [Google Scholar]
  6. Clapham D. E., Neher E. Substance P reduces acetylcholine-induced currents in isolated bovine chromaffin cells. J Physiol. 1984 Feb;347:255–277. doi: 10.1113/jphysiol.1984.sp015065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Colquhoun D., Hawkes A. G. Relaxation and fluctuations of membrane currents that flow through drug-operated channels. Proc R Soc Lond B Biol Sci. 1977 Nov 14;199(1135):231–262. doi: 10.1098/rspb.1977.0137. [DOI] [PubMed] [Google Scholar]
  8. Colquhoun D., Ogden D. C. Activation of ion channels in the frog end-plate by high concentrations of acetylcholine. J Physiol. 1988 Jan;395:131–159. doi: 10.1113/jphysiol.1988.sp016912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Conti-Tronconi B. M., Hunkapiller M. W., Raftery M. A. Molecular weight and structural nonequivalence of the mature alpha subunits of Torpedo californica acetylcholine receptor. Proc Natl Acad Sci U S A. 1984 May;81(9):2631–2634. doi: 10.1073/pnas.81.9.2631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cooper E., Couturier S., Ballivet M. Pentameric structure and subunit stoichiometry of a neuronal nicotinic acetylcholine receptor. Nature. 1991 Mar 21;350(6315):235–238. doi: 10.1038/350235a0. [DOI] [PubMed] [Google Scholar]
  12. Covarrubias M., Steinbach J. H. Excision of membrane patches reduces the mean open time of nicotinic acetylcholine receptors. Pflugers Arch. 1990 Jun;416(4):385–392. doi: 10.1007/BF00370744. [DOI] [PubMed] [Google Scholar]
  13. Derkach V. A., Selyanko A. A., Skok V. I. Acetylcholine-induced current fluctuations and fast excitatory post-synaptic currents in rabbit sympathetic neurones. J Physiol. 1983 Mar;336:511–526. doi: 10.1113/jphysiol.1983.sp014595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Devreotes P. N., Fambrough D. M. Acetylcholine receptor turnover in membranes of developing muscle fibers. J Cell Biol. 1975 May;65(2):335–358. doi: 10.1083/jcb.65.2.335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dilger J. P., Brett R. S. Direct measurement of the concentration- and time-dependent open probability of the nicotinic acetylcholine receptor channel. Biophys J. 1990 Apr;57(4):723–731. doi: 10.1016/S0006-3495(90)82593-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Fenwick E. M., Marty A., Neher E. A patch-clamp study of bovine chromaffin cells and of their sensitivity to acetylcholine. J Physiol. 1982 Oct;331:577–597. doi: 10.1113/jphysiol.1982.sp014393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Franke C., Hatt H., Dudel J. Steep concentration dependence and fast desensitization of nicotinic channel currents elicited by acetylcholine pulses, studied in adult vertebrate muscle. Pflugers Arch. 1991 Jan;417(5):509–516. doi: 10.1007/BF00370947. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Knight D. E., Baker P. F. Stimulus-secretion coupling in isolated bovine adrenal medullary cells. Q J Exp Physiol. 1983 Jan;68(1):123–143. doi: 10.1113/expphysiol.1983.sp002691. [DOI] [PubMed] [Google Scholar]
  22. Kuba K., Nishi S. Characteristics of fast excitatory postsynaptic current in bullfrog sympathetic ganglion cells. Effects of membrane potential, temperature and Ca ions. Pflugers Arch. 1979 Jan 31;378(3):205–212. doi: 10.1007/BF00592737. [DOI] [PubMed] [Google Scholar]
  23. Liu Y., Dilger J. P. Opening rate of acetylcholine receptor channels. Biophys J. 1991 Aug;60(2):424–432. doi: 10.1016/S0006-3495(91)82068-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. MacDermott A. B., Connor E. A., Dionne V. E., Parsons R. L. Voltage clamp study of fast excitatory synaptic currents in bullfrog sympathetic ganglion cells. J Gen Physiol. 1980 Jan;75(1):39–60. doi: 10.1085/jgp.75.1.39. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Maconochie D. J., Knight D. E. A method for making solution changes in the sub-millisecond range at the tip of a patch pipette. Pflugers Arch. 1989 Sep;414(5):589–596. doi: 10.1007/BF00580996. [DOI] [PubMed] [Google Scholar]
  26. Maconochie D. J., Knight D. E. Markov modelling of ensemble current relaxations: bovine adrenal nicotinic receptor currents analysed. J Physiol. 1992 Aug;454:155–182. doi: 10.1113/jphysiol.1992.sp019258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. 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]
  30. Ogden D. C., Gray P. T., Colquhoun D., Rang H. P. Kinetics of acetylcholine activated ion channels in chick ciliary ganglion neurones grown in tissue culture. Pflugers Arch. 1984 Jan;400(1):44–50. doi: 10.1007/BF00670535. [DOI] [PubMed] [Google Scholar]
  31. Oortgiesen M., Vijverberg H. P. Properties of neuronal type acetylcholine receptors in voltage clamped mouse neuroblastoma cells. Neuroscience. 1989;31(1):169–179. doi: 10.1016/0306-4522(89)90038-9. [DOI] [PubMed] [Google Scholar]
  32. PATON W. D. M., ZAIMIS E. J. Paralysis of autonomic ganglia by methonium salts. Br J Pharmacol Chemother. 1951 Mar;6(1):155–168. doi: 10.1111/j.1476-5381.1951.tb00631.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Prod'hom B., Pietrobon D., Hess P. Direct measurement of proton transfer rates to a group controlling the dihydropyridine-sensitive Ca2+ channel. Nature. 1987 Sep 17;329(6136):243–246. doi: 10.1038/329243a0. [DOI] [PubMed] [Google Scholar]
  34. Rang H. P., Colquhoun D., Rang H. P. The action of ganglionic blocking drugs on the synaptic responses of rat submandibular ganglion cells. Br J Pharmacol. 1982 Jan;75(1):151–168. doi: 10.1111/j.1476-5381.1982.tb08768.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rang H. P. The characteristics of synaptic currents and responses to acetylcholine of rat submandibular ganglion cells. J Physiol. 1981 Feb;311:23–55. doi: 10.1113/jphysiol.1981.sp013571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Robertson B. Actions of anaesthetics and avermectin on GABAA chloride channels in mammalian dorsal root ganglion neurones. Br J Pharmacol. 1989 Sep;98(1):167–176. doi: 10.1111/j.1476-5381.1989.tb16878.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Salpeter M. M., Elderfrawi M. E. Sizes of end plate compartments, densities of acetylcholine receptor and other quantitative aspects of neuromuscular transmission. J Histochem Cytochem. 1973 Sep;21(9):769–778. doi: 10.1177/21.9.769. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Sine S. M., Steinbach J. H. Acetylcholine receptor activation by a site-selective ligand: nature of brief open and closed states in BC3H-1 cells. J Physiol. 1986 Jan;370:357–379. doi: 10.1113/jphysiol.1986.sp015939. [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 high concentrations of agonist. J Physiol. 1987 Apr;385:325–359. doi: 10.1113/jphysiol.1987.sp016496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  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. 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]
  44. THESLEFT S. The mode of neuromuscular block caused by acetylcholine, nicotine, decamethonium and succinylcholine. Acta Physiol Scand. 1955 Oct 27;34(2-3):218–231. doi: 10.1111/j.1748-1716.1955.tb01242.x. [DOI] [PubMed] [Google Scholar]
  45. Weber M., Changeux J. P. Binding of Naja nigricollis (3H)alpha-toxin to membrane fragments from Electrophorus and Torpedo electric organs. 3. Effects of local anaesthetics on the binding of the tritiated alpha-neurotoxin. Mol Pharmacol. 1974 Jan;10(1):35–40. [PubMed] [Google Scholar]

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