Skip to main content
NCBI home page
The Journal of Physiology logoLink to The Journal of Physiology
. 1980 Aug;305:485–500. doi: 10.1113/jphysiol.1980.sp013377

Properties of toxin-resistant sodium channels produced by chemical modification in frog skeletal muscle.

B C Spalding
PMCID: PMC1282986  PMID: 6255148

Abstract

1. Single skeletal muscle fibres from the frog Rana pipiens were treated with the carboxyl group modifying reagent trimethyloxonium ion (TMO) and voltage clamped by the method of Hille & Campbell (1976). 2. TMO treatment reduced current through sodium channels to 0.33 +/- 0.03 that before treatment, but only 45 +/- 3% of this remaining current was blocked by 1 microM-tetrodotoxin (TTX) and only 37 +/- 5% by 100 nM-saxitoxin (STX). 3. This toxin resistance persisted in 90 microM-TTX, was not due to inactivation of toxin nor to components of the reaction solution other than TMO, but was prevented by the presence of 100 nM-STX during treatment with TMO. TMO-modified sodium channels can be blocked by the local anaesthetic lidocaine. 4. The permeabilities of TMO-modified channels to hydroxylammonium, ammonium, guanidinium, aminoguanidinium, methylammonium and tetramethylammonium ions relative to sodium were not significantly different from the permeabilities of untreated sodium channels. 5. Hydrogen ions blocked TMO-modified sodium channels, but the apparent pKa for block at +38 mV of 5.07 was significantly less than the corresponding value of 5.32 in untreated sodium channels. 6. It is suggested that TMO produces toxin resistance by esterifying an ionized carboxyl group which is an essential part of the toxin binding site. Such esterification would electrostatically reduce the local cation concentration, thus reducing the apparent pKa of hydrogen ion block and the single-channel conductance (Sigworth & Spalding, 1980). 7. It is concluded that the sodium channel contains a second acid group, near but distinct from an acid group previously hypothesized to be part of the selectivity filter and hydrogen ion binding site (Hille, 1971, 1972, 1975a).

Full text

PDF
485

Selected References

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

  1. Almers W., Levinson S. R. Tetrodotoxin binding to normal depolarized frog muscle and the conductance of a single sodium channel. J Physiol. 1975 May;247(2):483–509. doi: 10.1113/jphysiol.1975.sp010943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baker P. F., Rubinson K. A. Chemical modification of crab nerves can make them insensitive to the local anaesthetics tetrodotoxin and saxitoxin. Nature. 1975 Oct 2;257(5525):412–414. doi: 10.1038/257412a0. [DOI] [PubMed] [Google Scholar]
  3. Baker P. F., Rubinson K. A. TTX resistant action potentials in crab nerve after treatment with Meerwein's reagent (proceedings). J Physiol. 1977 Mar;266(1):3P–4P. [PubMed] [Google Scholar]
  4. Cahalan M. D., Almers W. Interactions between quaternary lidocaine, the sodium channel gates, and tetrodotoxin. Biophys J. 1979 Jul;27(1):39–55. doi: 10.1016/S0006-3495(79)85201-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Camougis G., Takman B. H., Tasse J. R. Potency difference between the zwitterion form and the cation forms of tetrodotoxin. Science. 1967 Jun 23;156(3782):1625–1627. doi: 10.1126/science.156.3782.1625. [DOI] [PubMed] [Google Scholar]
  6. Campbell D. T., Hille B. Kinetic and pharmacological properties of the sodium channel of frog skeletal muscle. J Gen Physiol. 1976 Mar;67(3):309–323. doi: 10.1085/jgp.67.3.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Campbell D. T. Ionic selectivity of the sodium channel of frog skeletal muscle. J Gen Physiol. 1976 Mar;67(3):295–307. doi: 10.1085/jgp.67.3.295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Catterall W. A., Morrow C. S. Binding to saxitoxin to electrically excitable neuroblastoma cells. Proc Natl Acad Sci U S A. 1978 Jan;75(1):218–222. doi: 10.1073/pnas.75.1.218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chandler W. K., Meves H. Voltage clamp experiments on internally perfused giant axons. J Physiol. 1965 Oct;180(4):788–820. doi: 10.1113/jphysiol.1965.sp007732. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chao Y., Vandlen R. L., Raftery M. A. Preferential chemical modification of a binding subsite on the acetylcholine receptor. Biochem Biophys Res Commun. 1975 Mar 3;63(1):300–307. doi: 10.1016/s0006-291x(75)80043-x. [DOI] [PubMed] [Google Scholar]
  11. Colquhoun D., Henderson R., Ritchie J. M. The binding of labelled tetrodotoxin to non-myelinated nerve fibres. J Physiol. 1972 Dec;227(1):95–126. doi: 10.1113/jphysiol.1972.sp010022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Drouin H., Neumcke B. Specific and unspecific charges at the sodium channels of the nerve membrane. Pflugers Arch. 1974;351(3):207–229. doi: 10.1007/BF00586919. [DOI] [PubMed] [Google Scholar]
  13. Ebert G. A., Goldman L. The permeability of the sodium channel in Myxicola to the alkali cations. J Gen Physiol. 1976 Sep;68(3):327–340. doi: 10.1085/jgp.68.3.327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Goldman D. E. POTENTIAL, IMPEDANCE, AND RECTIFICATION IN MEMBRANES. J Gen Physiol. 1943 Sep 20;27(1):37–60. doi: 10.1085/jgp.27.1.37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Henderson R., Ritchie J. M., Strichartz G. R. Evidence that tetrodotoxin and saxitoxin act at a metal cation binding site in the sodium channels of nerve membrane. Proc Natl Acad Sci U S A. 1974 Oct;71(10):3936–3940. doi: 10.1073/pnas.71.10.3936. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Henderson R., Ritchie J. M., Strichartz G. R. The binding of labelled saxitoxin to the sodium channels in nerve membranes. J Physiol. 1973 Dec;235(3):783–804. doi: 10.1113/jphysiol.1973.sp010417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hille B., Campbell D. T. An improved vaseline gap voltage clamp for skeletal muscle fibers. J Gen Physiol. 1976 Mar;67(3):265–293. doi: 10.1085/jgp.67.3.265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hille B. Charges and potentials at the nerve surface. Divalent ions and pH. J Gen Physiol. 1968 Feb;51(2):221–236. doi: 10.1085/jgp.51.2.221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hille B. Ionic selectivity, saturation, and block in sodium channels. A four-barrier model. J Gen Physiol. 1975 Nov;66(5):535–560. doi: 10.1085/jgp.66.5.535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hille B. Pharmacological modifications of the sodium channels of frog nerve. J Gen Physiol. 1968 Feb;51(2):199–219. doi: 10.1085/jgp.51.2.199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hille B. The permeability of the sodium channel to metal cations in myelinated nerve. J Gen Physiol. 1972 Jun;59(6):637–658. doi: 10.1085/jgp.59.6.637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hille B. The permeability of the sodium channel to organic cations in myelinated nerve. J Gen Physiol. 1971 Dec;58(6):599–619. doi: 10.1085/jgp.58.6.599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hille B. The receptor for tetrodotoxin and saxitoxin. A structural hypothesis. Biophys J. 1975 Jun;15(6):615–619. doi: 10.1016/S0006-3495(75)85842-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Huang L. Y., Catterall W. A., Ehrenstein G. Comparison of ionic selectivity of batrachotoxin-activated channels with different tetrodotoxin dissociation constants. J Gen Physiol. 1979 Jun;73(6):839–854. doi: 10.1085/jgp.73.6.839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kao C. Y., Nishiyama A. Actions of saxitoxin on peripheral neuromuscular systems. J Physiol. 1965 Sep;180(1):50–66. [PMC free article] [PubMed] [Google Scholar]
  26. Levy D. The esterification of insulin with triethyloxonium tetrafluoroborate. Biochim Biophys Acta. 1973 Jun 15;310(2):406–415. doi: 10.1016/0005-2795(73)90122-0. [DOI] [PubMed] [Google Scholar]
  27. Mozhayeva G. N., Naumov A. P., Negulyaev Y. A., Nosyreva E. D. The permeability of aconitine-modified sodium channels to univalent cations in myelinated nerve. Biochim Biophys Acta. 1977 May 2;466(3):461–473. doi: 10.1016/0005-2736(77)90339-x. [DOI] [PubMed] [Google Scholar]
  28. NARAHASHI T., DEGUCHI T., URAKAWA N., OHKUBO Y. Stabilization and rectification of muscle fiber membrane by tetrodotoxin. Am J Physiol. 1960 May;198:934–938. doi: 10.1152/ajplegacy.1960.198.5.934. [DOI] [PubMed] [Google Scholar]
  29. NARAHASHI T., MOORE J. W., SCOTT W. R. TETRODOTOXIN BLOCKAGE OF SODIUM CONDUCTANCE INCREASE IN LOBSTER GIANT AXONS. J Gen Physiol. 1964 May;47:965–974. doi: 10.1085/jgp.47.5.965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nakayama H., Tanizawa K., Kanaoka Y. Modification of carboxyl groups in the binding sites of trypsin with the Meerwein reagent. Biochem Biophys Res Commun. 1970 Aug 11;40(3):537–541. doi: 10.1016/0006-291x(70)90935-6. [DOI] [PubMed] [Google Scholar]
  31. Narahashi T., Moore J. W., Poston R. N. Tetrodotoxin derivatives: chemical structure and blockage of nerve membrane conductance. Science. 1967 May 19;156(3777):976–979. doi: 10.1126/science.156.3777.976. [DOI] [PubMed] [Google Scholar]
  32. Naumov A. P., Neguliaev Iu A., Nosyreva E. D. Izmenenie srodstva kinslotnoi gruppy natrievogo kanala k ionam vodoroda pri deistvii akonitina. Dokl Akad Nauk SSSR. 1979 Feb;244(1):229–232. [PubMed] [Google Scholar]
  33. Ogura Y., Mori Y. Mechanism of local anesthetic action of crystalline tetrodotoxin and its derivatives. Eur J Pharmacol. 1968 Apr;3(1):58–67. doi: 10.1016/0014-2999(68)90049-6. [DOI] [PubMed] [Google Scholar]
  34. Parsons S. M., Jao L., Dahlquist F. W., Borders C. L., Jr, Racs J., Groff T., Raftery M. A. The nature of amino acid side chains which are critical for the activity of lysozyme. Biochemistry. 1969 Feb;8(2):700–712. doi: 10.1021/bi00830a036. [DOI] [PubMed] [Google Scholar]
  35. Paterson A. K., Knowles J. R. The number of catalytically essential carboxyl groups in pepsin. Modification of the enzyme by trimethyloxonium fluoroborate. Eur J Biochem. 1972 Dec 18;31(3):510–517. doi: 10.1111/j.1432-1033.1972.tb02559.x. [DOI] [PubMed] [Google Scholar]
  36. Pooler J. P., Valenzeno D. P. Titration of sodium channel sites for hydrogen ion block and sensitized photochemical modification of lobster axons. Biochim Biophys Acta. 1979 Aug 7;555(2):307–315. doi: 10.1016/0005-2736(79)90170-6. [DOI] [PubMed] [Google Scholar]
  37. Rawn J. D., Lienhard G. E. The inactivation of acetylcholinesterase by trimethyloxonium ion, an active-site-directed methylating agent. Biochem Biophys Res Commun. 1974 Feb 4;56(3):654–660. doi: 10.1016/0006-291x(74)90655-x. [DOI] [PubMed] [Google Scholar]
  38. Reed J. K., Raftery M. A. Properties of the tetrodotoxin binding component in plasma membranes isolated from Electrophorus electricus. Biochemistry. 1976 Mar 9;15(5):944–953. doi: 10.1021/bi00650a002. [DOI] [PubMed] [Google Scholar]
  39. Schauf C. L., Davis F. A. Sensitivity of the sodium and potassium channels of Myxicola giant axons to changes in external pH. J Gen Physiol. 1976 Feb;67(2):185–195. doi: 10.1085/jgp.67.2.185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Schwarz W., Palade P. T., Hille B. Local anesthetics. Effect of pH on use-dependent block of sodium channels in frog muscle. Biophys J. 1977 Dec;20(3):343–368. doi: 10.1016/S0006-3495(77)85554-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Shrager P. Ionic conductance changes in voltage clamped crayfish axons at low pH. J Gen Physiol. 1974 Dec;64(6):666–690. doi: 10.1085/jgp.64.6.666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Shrager P., Profera C. Inhibition of the receptor for tetrodotoxin in nerve membranes by reagents modifying carboxyl groups. Biochim Biophys Acta. 1973 Aug 9;318(1):141–146. doi: 10.1016/0005-2736(73)90343-x. [DOI] [PubMed] [Google Scholar]
  43. Sigworth F. J., Spalding B. C. Chemical modification reduces the conductance of sodium channels in nerve. Nature. 1980 Jan 17;283(5744):293–295. doi: 10.1038/283293a0. [DOI] [PubMed] [Google Scholar]
  44. Strichartz G. R. The inhibition of sodium currents in myelinated nerve by quaternary derivatives of lidocaine. J Gen Physiol. 1973 Jul;62(1):37–57. doi: 10.1085/jgp.62.1.37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Ulbricht W., Wagner H. H. The influence of pH on equilibrium effects of tetrodotoxin on myelinated nerve fibres of Rana esculenta. J Physiol. 1975 Oct;252(1):159–184. doi: 10.1113/jphysiol.1975.sp011139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wagner H. H., Ulbricht W. Saxitoxin and procaine act independently on separate sites of the sodium channel. Pflugers Arch. 1976 Jun 29;364(1):65–70. doi: 10.1007/BF01062913. [DOI] [PubMed] [Google Scholar]
  47. Wagner H. H., Ulbricht W. The rates of saxitoxin action and of saxitoxin-tetrodotoxin interaction at the node of Ranvier. Pflugers Arch. 1975 Sep 29;359(4):297–315. doi: 10.1007/BF00581441. [DOI] [PubMed] [Google Scholar]
  48. Woodhull A. M. Ionic blockage of sodium channels in nerve. J Gen Physiol. 1973 Jun;61(6):687–708. doi: 10.1085/jgp.61.6.687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Yeh J. Z., Armstrong C. M. Immobilisation of gating charge by a substance that simulates inactivation. Nature. 1978 Jun 1;273(5661):387–389. doi: 10.1038/273387a0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES