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. 1980 Apr;77(4):2309–2313. doi: 10.1073/pnas.77.4.2309

Activation, inactivation, and desensitization of acetylcholine receptor channel complex detected by binding of perhydrohistrionicotoxin

Mohyee E Eldefrawi 1, Robert S Aronstam 1, Nabil M Bakry 1, Amira T Eldefrawi 1, Edson X Albuquerque 1
PMCID: PMC348704  PMID: 6246539

Abstract

The effects of receptor activation were studied on the interaction of perhydrohistrionicotoxin (H12-HTX) with the ionic channel of the nicotinic acetylcholine (AcCho) receptor in membranes from the electric organ of Torpedo ocellata and with the endplate region of the soleus muscle of the rat. In Torpedo membranes, the initial rate (i.e., within 30 sec) of [3H]H12-HTX bindings to the ionic channel of the AcCho receptor was accelerated 102- to 103-fold in the presence of carbamoylcholine (Carb). H12-HTX also inhibited Carb-activated 22Na+ influx, over 95% inhibition at 10 μM H12-HTX. At this concentration H12-HTX did not inhibit [3H]AcCho binding to the AcCho-receptor sites. There was good correspondence between the degree of acceleration of [3H]H12-HTX binding and the stimulation of 22Na+ influx over a wide range of Carb concentrations (up to 100 μM). Preincubation of Torpedo membranes with Carb decreased the initial rate of [3H]H12-HTX binding, as well as the rate of 22Na+ influx, which may reflect desensitization of the AcCho-receptor. d-Tubocurarine inhibited the agonist-mediated acceleration of [3H]H12-HTX binding and 22Na+ influx. In the soleus muscle endplate, H12-HTX inhibited the transient depolarization induced by microiontophoretic application of AcCho; the more receptors activated and channels opened, the stronger was the inhibition by H12-HTX. These findings suggest that H12-HTX binds to closed and open ionic channels, with a preference for the latter conformation. It is also suggested that the conformational changes associated with activation or desensitization of the receptor can be monitored by studying binding of [3H]H12-HTX to the ionic channel sites as well as by the AcCho-receptor-regulated 22Na+ influx.

Keywords: ionic channel, 22Na+ influx, electric organ, neuromuscular transmission, conformational changes

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Selected References

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

  1. Albuquerque E. X., Barnard E. A., Chiu T. H., Lapa A. J., Dolly J. O., Jansson S. E., Daly J., Witkop B. Acetylcholine receptor and ion conductance modulator sites at the murine neuromuscular junction: evidence from specific toxin reactions. Proc Natl Acad Sci U S A. 1973 Mar;70(3):949–953. doi: 10.1073/pnas.70.3.949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Burgermeister W., Catterall W. A., Witkop B. Histrionicotoxin enhances agonist-induced desensitization of acetylcholine receptor. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5754–5758. doi: 10.1073/pnas.74.12.5754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Eldefrawi A. T., Eldefrawi M. E., Albuquerque E. X., Oliveira A. C., Mansour N., Adler M., Daly J. W., Brown G. B., Burgermeister W., Witkop B. Perhydrohistrionicotoxin: a potential ligand for the ion conductance modulator of the acetylcholine receptor. Proc Natl Acad Sci U S A. 1977 May;74(5):2172–2176. doi: 10.1073/pnas.74.5.2172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Eldefrawi M. E., Eldefrawi A. T., Mansour N. A., Daly J. W., Witkop B., Albuquerque E. X. Acetylcholine receptor and ionic channel of Torpedo electroplax: binding of perhydrohistrionicotoxin to membrane and solubilized preparations. Biochemistry. 1978 Dec 12;17(25):5474–5484. doi: 10.1021/bi00618a023. [DOI] [PubMed] [Google Scholar]
  6. Elliott J., Raftery M. A. Binding of perhydrohistrionicotoxin to intact and detergent-solubilized membranes enriched in nicotinic acetylcholine receptor. Biochemistry. 1979 May 15;18(10):1868–1874. doi: 10.1021/bi00577a004. [DOI] [PubMed] [Google Scholar]
  7. Epstein M., Racker E. Reconstitution of carbamylcholine-dependent sodium ion flux and desensitization of the acetylcholine receptor from Torpedo californica. J Biol Chem. 1978 Oct 10;253(19):6660–6662. [PubMed] [Google Scholar]
  8. Heidmann T., Changeux J. P. Fast kinetic studies on the allosteric interactions between acetylcholine receptor and local anesthetic binding sites. Eur J Biochem. 1979 Feb 15;94(1):281–296. doi: 10.1111/j.1432-1033.1979.tb12894.x. [DOI] [PubMed] [Google Scholar]
  9. Heidmann T., Changeux J. P. Structural and functional properties of the acetylcholine receptor protein in its purified and membrane-bound states. Annu Rev Biochem. 1978;47:317–357. doi: 10.1146/annurev.bi.47.070178.001533. [DOI] [PubMed] [Google Scholar]
  10. Hess G. P., Lipkowitz S., Struve G. E. Acetylcholine-receptor-mediated ion flux in electroplax membrane microsacs (vesicles): change in mechanism produced by asymmetrical distribution of sodium and potassium ions. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1703–1707. doi: 10.1073/pnas.75.4.1703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Kato G., Changeux J. P. Studies on the effect of histrionicotoxin on the monocellular electroplax from Electrophorus electricus and on the binding of (3H)acetylcholine to membrane fragments from Torpedo marmorata. Mol Pharmacol. 1976 Jan;12(1):92–100. [PubMed] [Google Scholar]
  13. Katz B., Miledi R. A re-examination of curare action at the motor endplate. Proc R Soc Lond B Biol Sci. 1978 Dec 4;203(1151):119–133. doi: 10.1098/rspb.1978.0096. [DOI] [PubMed] [Google Scholar]
  14. Katz B., Miledi R. The statistical nature of the acetycholine potential and its molecular components. J Physiol. 1972 Aug;224(3):665–699. doi: 10.1113/jphysiol.1972.sp009918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  16. Lapa A. J., Albuquerque E. X., Sarvey J. M., Daly J., Witkop B. Effects of histrionicotoxin on the chemosensitive and electrical properties of skeletal muscle. Exp Neurol. 1975 Jun;47(3):558–580. doi: 10.1016/0014-4886(75)90088-6. [DOI] [PubMed] [Google Scholar]
  17. Masukawa L. M., Albuquerque E. X. Voltage- and time-dependent action of histrionicotoxin on the endplate current of the frog muscle. J Gen Physiol. 1978 Sep;72(3):351–367. doi: 10.1085/jgp.72.3.351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Miller D. L., Moore H. P., Hartig P. R., Raftery M. A. Fast cation flux from Torpedo californica membrane preparations: implications for a functional role for acetylcholine receptor dimers. Biochem Biophys Res Commun. 1978 Nov 29;85(2):632–640. doi: 10.1016/0006-291x(78)91209-3. [DOI] [PubMed] [Google Scholar]
  19. Schimerlik M. I., Quast U., Raftery M. A. Ligand-induced changes in membrane-bound acetylcholine receptor observed by ethidium fluorescence. 3. Stopped-flow studies with histrionicotoxin. Biochemistry. 1979 May 15;18(10):1902–1906. doi: 10.1021/bi00577a008. [DOI] [PubMed] [Google Scholar]
  20. Sugiyama H., Popot J. L., Changeux J. P. Studies on the electrogenic action of acetylcholine with Torpedo marmorata electric organ. III. Pharmocological desensitization in vitro of the receptor-rich membrane fragments by cholinergic agonists. J Mol Biol. 1976 Sep 25;106(3):485–496. doi: 10.1016/0022-2836(76)90248-5. [DOI] [PubMed] [Google Scholar]
  21. Tsai M. C., Mansour N. A., Eldefrawi A. T., Eldefrawi M. E., Albuquerque E. X. Mechanism of action of amantadine on neuromuscular transmission. Mol Pharmacol. 1978 Sep;14(5):787–803. [PubMed] [Google Scholar]
  22. Tsai M. C., Oliveira A. C., Albuquerque E. X., Eldefrawi M. E., Eldefrawi A. T. Mode of action of quinacrine on the acetylcholine receptor ionic channel complex. Mol Pharmacol. 1979 Sep;16(2):382–392. [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. 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]

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